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aLANGEmedicalbookHarper’sIllustratedBiochemistrytwenty-sixtheditionRobertK.Murray,MD,PhDProfessor(Emeritus)ofBiochemistryUniversityofTorontoToronto,OntarioDarylK.Granner,MDJoeC.DavisProfessorofBiomedicalScienceDirector,VanderbiltDiabetesCenterProfessorofMolecularPhysiologyandBiophysicsandofMedicineVanderbiltUniversityNashville,TennesseePeterA.Mayes,PhD,DScEmeritusProfessorofVeterinaryBiochemistryRoyalVeterinaryCollegeUniversityofLondonLondonVictorW.Rodwell,PhDProfessorofBiochemistryPurdueUniversityWestLafayette,IndianaLangeMedicalBooks/McGraw-HillMedicalPublishingDivisionNewYorkChicagoSanFranciscoLisbonLondonMadridMexicoCityMilanNewDelhiSanJuanSeoulSingaporeSydneyToronto
1Harper’sIllustratedBiochemistry,Twenty-SixthEditionCopyright©2003byTheMcGraw-HillCompanies,Inc.Allrightsreserved.PrintedintheUnitedStatesofAmerica.ExceptaspermittedundertheUnitedStatesCopyrightActof1976,nopartofthispublicationmaybereproducedordistributedinanyformorbyanymeans,orstoredinadatabaseorretrievalsystem,withoutthepriorwrittenpermissionofthepublisher.Previouseditionscopyright©2000,1996,1993,1990byAppleton&Lange;copyright©1988byLangeMedicalPublications.234567890DOC/DOC09876543ISBN0-07-138901-6ISSN1043-9811NoticeMedicineisanever-changingscience.Asnewresearchandclinicalexperiencebroadenourknowledge,changesintreat-mentanddrugtherapyarerequired.Theauthorsandthepublisherofthisworkhavecheckedwithsourcesbelievedtobereliableintheireffortstoprovideinformationthatiscompleteandgenerallyinaccordwiththestandardsacceptedatthetimeofpublication.However,inviewofthepossibilityofhumanerrororchangesinmedicalsciences,neithertheauthorsnorthepublishernoranyotherpartywhohasbeeninvolvedinthepreparationorpublicationofthisworkwarrantsthattheinformationcontainedhereinisineveryrespectaccurateorcomplete,andtheydisclaimallresponsibilityforanyer-rorsoromissionsorfortheresultsobtainedfromuseoftheinformationcontainedinthiswork.Readersareencouragedtoconfirmtheinformationcontainedhereinwithothersources.Forexampleandinparticular,readersareadvisedtochecktheproductinformationsheetincludedinthepackageofeachdrugtheyplantoadministertobecertainthattheinformationcontainedinthisworkisaccurateandthatchangeshavenotbeenmadeintherecommendeddoseorinthecontraindicationsforadministration.Thisrecommendationisofparticularimportanceinconnectionwithneworinfre-quentlyuseddrugs.ThisbookwassetinGaramondbyPineTreeCompositionTheeditorswereJanetFoltin,JimRansom,andJaneneMatragranoOransky.TheproductionsupervisorwasPhilGalea.TheillustrationmanagerwasCharissaBaker.ThetextdesignerwasEveSiegel.ThecoverdesignerwasMaryMcKeon.TheindexwaspreparedbyKathyPitcoff.RRDonnelleywasprinterandbinder.Thisbookisprintedonacid-freepaper.ISBN-0-07-121766-5(InternationalEdition)Copyright©2003.ExclusiverightsbytheMcGraw-HillCompanies,Inc.,formanufactureandexport.Thisbookcannotbere-exportedfromthecountrytowhichitisconsignedbyMcGraw-Hill.TheInternationalEditionisnotavailableinNorthAmerica.
2AuthorsDavidA.Bender,PhDPeterA.Mayes,PhD,DScSub-DeanRoyalFreeandUniversityCollegeMedicalEmeritusProfessorofVeterinaryBiochemistry,RoyalSchool,AssistantFacultyTutorandTutortoMed-VeterinaryCollege,UniversityofLondonicalStudents,SeniorLecturerinBiochemistry,De-partmentofBiochemistryandMolecularBiology,UniversityCollegeLondonRobertK.Murray,MD,PhDProfessor(Emeritus)ofBiochemistry,UniversityofKathleenM.Botham,PhD,DScTorontoReaderinBiochemistry,RoyalVeterinaryCollege,UniversityofLondonMargaretL.Rand,PhDScientist,ResearchInstitute,HospitalforSickChil-DarylK.Granner,MDdren,Toronto,andAssociateProfessor,Depart-mentsofLaboratoryMedicineandPathobiologyJoeC.DavisProfessorofBiomedicalScience,Director,andDepartmentofBiochemistry,UniversityofVanderbiltDiabetesCenter,ProfessorofMolecularTorontoPhysiologyandBiophysicsandofMedicine,Vander-biltUniversity,Nashville,TennesseeVictorW.Rodwell,PhDFrederickW.Keeley,PhDProfessorofBiochemistry,PurdueUniversity,WestLafayette,IndianaAssociateDirectorandSeniorScientist,ResearchInsti-tute,HospitalforSickChildren,Toronto,andPro-fessor,DepartmentofBiochemistry,UniversityofP.AnthonyWeil,PhDTorontoProfessorofMolecularPhysiologyandBiophysics,VanderbiltUniversitySchoolofMedicine,Nash-PeterJ.Kennelly,PhDville,TennesseeProfessorofBiochemistry,VirginiaPolytechnicInsti-tuteandStateUniversity,Blacksburg,Virginiavii
3PrefaceTheauthorsandpublisherarepleasedtopresentthetwenty-sixtheditionofHarper’sIllustratedBiochemistry.ReviewofPhysiologicalChemistrywasfirstpublishedin1939andrevisedin1944,anditquicklygainedawidereadership.In1951,thethirdeditionappearedwithHaroldA.Harper,UniversityofCaliforniaSchoolofMedicineatSanFran-cisco,asauthor.Dr.Harperremainedthesoleauthoruntilthenintheditionandco-authoredeightsubsequentedi-tions.PeterMayesandVictorRodwellhavebeenauthorssincethetenthedition,DarylGrannersincethetwentiethedition,andRobMurraysincethetwenty-firstedition.Becauseoftheincreasingcomplexityofbiochemicalknowl-edge,theyhaveaddedco-authorsinrecenteditions.FredKeeleyandMargaretRandhaveeachco-authoredonechapterwithRobMurrayforthisandpreviousedi-tions.PeterKennellyjoinedasaco-authorinthetwenty-fifthedition,andinthepresenteditionhasco-authoredwithVictorRodwellallofthechaptersdealingwiththestructureandfunctionofproteinsandenzymes.Thefollow-ingadditionalco-authorsareverywarmlywelcomedinthisedition:KathleenBothamhasco-authored,withPeterMayes,thechaptersonbioenergetics,biologicoxidation,oxidativephosphorylation,andlipidmetabolism.DavidBenderhasco-authored,alsowithPeterMayes,thechaptersdealingwithcarbohydratemetabolism,nutrition,diges-tion,andvitaminsandminerals.P.AnthonyWeilhasco-authoredchaptersdealingwithvariousaspectsofDNA,ofRNA,andofgeneexpressionwithDarylGranner.Weareallverygratefultoourco-authorsforbringingtheirex-pertiseandfreshperspectivestothetext.CHANGESINTHETWENTY-SIXTHEDITIONAmajorgoaloftheauthorscontinuestobetoprovidebothmedicalandotherstudentsofthehealthscienceswithabookthatbothdescribesthebasicsofbiochemistryandisuser-friendlyandinteresting.Asecondmajorongoinggoalistoreflectthemostsignificantadvancesinbiochemistrythatareimportanttomedicine.However,athirdmajorgoalofthiseditionwastoachieveasubstantialreductioninsize,asfeedbackindicatedthatmanyreaderspre-fershortertexts.Toachievethisgoal,allofthechapterswererigorouslyedited,involvingtheiramalgamation,division,ordele-tion,andmanywerereducedtoapproximatelyone-halftotwo-thirdsoftheirprevioussize.Thishasbeeneffectedwithoutlossofcrucialinformationbutwithgaininconcisenessandclarity.Despitethereductioninsize,therearemanynewfeaturesinthetwenty-sixthedition.Theseinclude:•Anewchapteronaminoacidsandpeptides,whichemphasizesthemannerinwhichthepropertiesofbiologicpeptidesderivefromtheindividualaminoacidsofwhichtheyarecomprised.•Anewchapterontheprimarystructureofproteins,whichprovidescoverageofbothclassicandnewlyemerging“proteomic”and“genomic”methodsforidentifyingproteins.Anewsectionontheapplicationofmassspectrometrytotheanalysisofproteinstructurehasbeenadded,includingcommentsontheidentificationofcovalentmodifica-tions.•Thechapteronthemechanismsofactionofenzymeshasbeenrevisedtoprovideacomprehensivedescriptionofthevariousphysicalmechanismsbywhichenzymescarryouttheircatalyticfunctions.•Thechaptersonintegrationofmetabolism,nutrition,digestionandabsorption,andvitaminsandmineralshavebeencompletelyre-written.•Amongimportantadditionstothevariouschaptersonmetabolismarethefollowing:updateoftheinformationonoxidativephosphorylation,includingadescriptionoftherotaryATPsynthase;newinsightsintotheroleofGTPingluconeogenesis;additionalinformationontheregulationofacetyl-CoAcarboxylase;newinformationonreceptorsinvolvedinlipoproteinmetabolismandreversecholesteroltransport;discussionoftheroleofleptininfatstorage;andnewinformationonbileacidregulation,includingtheroleofthefarnesoidXreceptor(FXR).•Thechapteronmembranebiochemistryinthepreviouseditionhasbeensplitintotwo,yieldingtwonewchaptersonthestructureandfunctionofmembranesandintracellulartrafficandsortingofproteins.•ConsiderablenewmaterialhasbeenaddedonRNAsynthesis,proteinsynthesis,generegulation,andvariousas-pectsofmoleculargenetics.•Muchofthematerialonindividualendocrineglandspresentinthetwenty-fiftheditionhasbeenreplacedwithnewchaptersdealingwiththediversityoftheendocrinesystem,withmolecularmechanismsofhormoneaction,andwithsignaltransduction.ix
4x/PREFACE•Thechapteronplasmaproteins,immunoglobulins,andbloodcoagulationinthepreviouseditionhasbeensplitintotwonewchaptersonplasmaproteinsandimmunoglobulinsandonhemostasisandthrombosis.•Newinformationhasbeenaddedinappropriatechaptersonlipidraftsandcaveolae,aquaporins,connexins,dis-ordersduetomutationsingenesencodingproteinsinvolvedinintracellularmembranetransport,absorptionofiron,andconformationaldiseasesandpharmacogenomics.•Anewandfinalchapteron“TheHumanGenomeProject”(HGP)hasbeenadded,whichbuildsonthematerialcoveredinChapters35through40.BecauseoftheimpactoftheresultsoftheHGPonthefutureofbiologyandmedicine,itappearedappropriatetoconcludethetextwithasummaryofitsmajorfindingsandtheirimplica-tionsforfuturework.•Asinitiatedinthepreviousedition,referencestousefulWebsiteshavebeenincludedinabriefAppendixattheendofthetext.ORGANIZATIONOFTHEBOOKThetextisdividedintotwointroductorychapters(“Biochemistry&Medicine”and“Water&pH”)followedbysixmainsections.SectionIdealswiththestructuresandfunctionsofproteinsandenzymes,theworkhorsesofthebody.Becausealmostallofthereactionsincellsarecatalyzedbyenzymes,itisvitaltounderstandthepropertiesofenzymesbeforeconsideringothertopics.SectionIIexplainshowvariouscellularreactionseitherutilizeorreleaseenergy,andittracesthepathwaysbywhichcarbohydratesandlipidsaresynthesizedanddegraded.Italsodescribesthemanyfunctionsofthesetwoclassesofmolecules.SectionIIIdealswiththeaminoacidsandtheirmanyfatesandalsodescribescertainkeyfeaturesofproteinca-tabolism.SectionIVdescribesthestructuresandfunctionsofthenucleotidesandnucleicacids,andcoversmanymajortopicssuchasDNAreplicationandrepair,RNAsynthesisandmodification,andproteinsynthesis.ItalsodiscussesnewfindingsonhowgenesareregulatedandpresentstheprinciplesofrecombinantDNAtechnology.SectionVdealswithaspectsofextracellularandintracellularcommunication.Topicscoveredincludemembranestructureandfunction,themolecularbasesoftheactionsofhormones,andthekeyfieldofsignaltransduction.SectionVIconsistsofdiscussionsofelevenspecialtopics:nutrition,digestion,andabsorption;vitaminsandminerals;intracellulartrafficandsortingofproteins;glycoproteins;theextracellularmatrix;muscleandthecy-toskeleton;plasmaproteinsandimmunoglobulins;hemostasisandthrombosis;redandwhitebloodcells;theme-tabolismofxenobiotics;andtheHumanGenomeProject.ACKNOWLEDGMENTSTheauthorsthankJanetFoltinforherthoroughlyprofessionalapproach.Herconstantinterestandinputhavehadasignificantimpactonthefinalstructureofthistext.WeareagainimmenselygratefultoJimRansomforhisexcel-lenteditorialwork;ithasbeenapleasuretoworkwithanindividualwhoconstantlyofferedwiseandinformedalter-nativestothesometimesprimitivetexttransmittedbytheauthors.ThesuperbeditorialskillsofJaneneMatragranoOranskyandHarrietLebowitzarewarmlyacknowledged,asistheexcellentartworkofCharissaBakerandhercol-leagues.TheauthorsareverygratefultoKathyPitcoffforherthoughtfulandmeticulousworkinpreparingtheIndex.Suggestionsfromstudentsandcolleaguesaroundtheworldhavebeenmosthelpfulintheformulationofthisedition.Welookforwardtoreceivingsimilarinputinthefuture.RobertK.Murray,MD,PhDDarylK.Granner,MDPeterA.Mayes,PhD,DScVictorW.Rodwell,PhDToronto,OntarioNashville,TennesseeLondonWestLafayette,IndianaMarch2003
5ContentsAuthors.............................................................................viiPreface..............................................................................ix1.Biochemistry&MedicineRobertK.Murray,MD,PhD...........................................................12.Water&pHVictorW.Rodwell,PhD,&PeterJ.Kennelly,PhD...........................................5SECTIONI.STRUCTURES&FUNCTIONSOFPROTEINS&ENZYMES...................143.AminoAcids&PeptidesVictorW.Rodwell,PhD,&PeterJ.Kennelly,PhD..........................................144.Proteins:DeterminationofPrimaryStructureVictorW.Rodwell,PhD,&PeterJ.Kennelly,PhD..........................................215.Proteins:HigherOrdersofStructureVictorW.Rodwell,PhD,&PeterJ.Kennelly,PhD..........................................306.Proteins:Myoglobin&HemoglobinVictorW.Rodwell,PhD,&PeterJ.Kennelly,PhD..........................................407.Enzymes:MechanismofActionVictorW.Rodwell,PhD,&PeterJ.Kennelly,PhD..........................................498.Enzymes:KineticsVictorW.Rodwell,PhD,&PeterJ.Kennelly,PhD..........................................609.Enzymes:RegulationofActivitiesVictorW.Rodwell,PhD,&PeterJ.Kennelly,PhD..........................................72SECTIONII.BIOENERGETICS&THEMETABOLISMOFCARBOHYDRATES&LIPIDS.......................................................................8010.Bioenergetics:TheRoleofATPPeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DSc.................................8011.BiologicOxidationPeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DSc.................................8612.TheRespiratoryChain&OxidativePhosphorylationPeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DSc.................................9213.CarbohydratesofPhysiologicSignificancePeterA.Mayes,PhD,DSc,&DavidA.Bender,PhD.......................................102iii
6iv/CONTENTS14.LipidsofPhysiologicSignificancePeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DSc................................11115.OverviewofMetabolismPeterA.Mayes,PhD,DSc,&DavidA.Bender,PhD.......................................12216.TheCitricAcidCycle:TheCatabolismofAcetyl-CoAPeterA.Mayes,PhD,DSc,&DavidA.Bender,PhD.......................................13017.Glycolysis&theOxidationofPyruvatePeterA.Mayes,PhD,DSc,&DavidA.Bender,PhD.......................................13618.MetabolismofGlycogenPeterA.Mayes,PhD,DSc,&DavidA.Bender,PhD.......................................14519.Gluconeogenesis&ControloftheBloodGlucosePeterA.Mayes,PhD,DSc,&DavidA.Bender,PhD.......................................15320.ThePentosePhosphatePathway&OtherPathwaysofHexoseMetabolismPeterA.Mayes,PhD,DSc,&DavidA.Bender,PhD.......................................16321.BiosynthesisofFattyAcidsPeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DSc................................17322.OxidationofFattyAcids:KetogenesisPeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DSc................................18023.MetabolismofUnsaturatedFattyAcids&EicosanoidsPeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DSc................................19024.MetabolismofAcylglycerols&SphingolipidsPeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DSc................................19725.LipidTransport&StoragePeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DSc................................20526.CholesterolSynthesis,Transport,&ExcretionPeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DSc................................21927.IntegrationofMetabolism—theProvisionofMetabolicFuelsDavidA.Bender,PhD,&PeterA.Mayes,PhD,DSc.......................................231SECTIONIII.METABOLISMOFPROTEINS&AMINOACIDS.........................23728.BiosynthesisoftheNutritionallyNonessentialAminoAcidsVictorW.Rodwell,PhD.............................................................23729.CatabolismofProteins&ofAminoAcidNitrogenVictorW.Rodwell,PhD.............................................................242
7CONTENTS/v30.CatabolismoftheCarbonSkeletonsofAminoAcidsVictorW.Rodwell,PhD.............................................................24931.ConversionofAminoAcidstoSpecializedProductsVictorW.Rodwell,PhD.............................................................26432.Porphyrins&BilePigmentsRobertK.Murray,MD,PhD.........................................................270SECTIONIV.STRUCTURE,FUNCTION,&REPLICATIONOFINFORMATIONALMACROMOLECULES........................................28633.NucleotidesVictorW.Rodwell,PhD.............................................................28634.MetabolismofPurine&PyrimidineNucleotidesVictorW.Rodwell,PhD.............................................................29335.NucleicAcidStructure&FunctionDarylK.Granner,MD.............................................................30336.DNAOrganization,Replication,&RepairDarylK.Granner,MD,&P.AnthonyWeil,PhD.........................................31437.RNASynthesis,Processing,&ModificationDarylK.Granner,MD,&P.AnthonyWeil,PhD.........................................34138.ProteinSynthesis&theGeneticCodeDarylK.Granner,MD.............................................................35839.RegulationofGeneExpressionDarylK.Granner,MD,&P.AnthonyWeil,PhD.........................................37440.MolecularGenetics,RecombinantDNA,&GenomicTechnologyDarylK.Granner,MD,&P.AnthonyWeil,PhD.........................................396SECTIONV.BIOCHEMISTRYOFEXTRACELLULAR&INTRACELLULARCOMMUNICATION...........................................41541.Membranes:Structure&FunctionRobertK.Murray,MD,PhD,&DarylK.Granner,MD....................................41542.TheDiversityoftheEndocrineSystemDarylK.Granner,MD.............................................................43443.HormoneAction&SignalTransductionDarylK.Granner,MD.............................................................456
8vi/CONTENTSSECTIONVI.SPECIALTOPICS....................................................47444.Nutrition,Digestion,&AbsorptionDavidA.Bender,PhD,&PeterA.Mayes,PhD,DSc.......................................47445.Vitamins&MineralsDavidA.Bender,PhD,&PeterA.Mayes,PhD,DSc.......................................48146.IntracellularTraffic&SortingofProteinsRobertK.Murray,MD,PhD.........................................................49847.GlycoproteinsRobertK.Murray,MD,PhD.........................................................51448.TheExtracellularMatrixRobertK.Murray,MD,PhD,&FrederickW.Keeley,PhD...................................53549.Muscle&theCytoskeletonRobertK.Murray,MD,PhD.........................................................55650.PlasmaProteins&ImmunoglobulinsRobertK.Murray,MD,PhD.........................................................58051.Hemostasis&ThrombosisMargaretL.Rand,PhD,&RobertK.Murray,MD,PhD....................................59852.Red&WhiteBloodCellsRobertK.Murray,MD,PhD.........................................................60953.MetabolismofXenobioticsRobertK.Murray,MD,PhD.........................................................62654.TheHumanGenomeProjectRobertK.Murray,MD,PhD.........................................................633Appendix..........................................................................639Index..............................................................................643
9Biochemistry&Medicine1RobertK.Murray,MD,PhDINTRODUCTIONbiochemistryisincreasinglybecomingtheircommonlanguage.Biochemistrycanbedefinedasthescienceconcernedwiththechemicalbasisoflife(Gkbios“life”).Thecellisthestructuralunitoflivingsystems.Thus,biochem-AReciprocalRelationshipBetweenistrycanalsobedescribedasthescienceconcernedwithBiochemistry&MedicineHasStimulatedthechemicalconstituentsoflivingcellsandwiththereac-MutualAdvancestionsandprocessestheyundergo.Bythisdefinition,bio-chemistryencompasseslargeareasofcellbiology,ofThetwomajorconcernsforworkersinthehealthsci-molecularbiology,andofmoleculargenetics.ences—andparticularlyphysicians—aretheunderstand-ingandmaintenanceofhealthandtheunderstandingTheAimofBiochemistryIstoDescribe&andeffectivetreatmentofdiseases.Biochemistryim-pactsenormouslyonbothofthesefundamentalcon-Explain,inMolecularTerms,AllChemicalcernsofmedicine.Infact,theinterrelationshipofbio-ProcessesofLivingCellschemistryandmedicineisawide,two-waystreet.ThemajorobjectiveofbiochemistryisthecompleteBiochemicalstudieshaveilluminatedmanyaspectsofunderstanding,atthemolecularlevel,ofallofthehealthanddisease,andconversely,thestudyofvariouschemicalprocessesassociatedwithlivingcells.Toaspectsofhealthanddiseasehasopenedupnewareasachievethisobjective,biochemistshavesoughttoiso-ofbiochemistry.Someexamplesofthistwo-waystreetlatethenumerousmoleculesfoundincells,determineareshowninFigure1–1.Forinstance,aknowledgeoftheirstructures,andanalyzehowtheyfunction.Manyproteinstructureandfunctionwasnecessarytoeluci-techniqueshavebeenusedforthesepurposes;someofdatethesinglebiochemicaldifferencebetweennormalthemaresummarizedinTable1–1.hemoglobinandsicklecellhemoglobin.Ontheotherhand,analysisofsicklecellhemoglobinhascontributedAKnowledgeofBiochemistryIsEssentialsignificantlytoourunderstandingofthestructureandtoAllLifeSciencesfunctionofbothnormalhemoglobinandotherpro-teins.AnalogousexamplesofreciprocalbenefitbetweenThebiochemistryofthenucleicacidsliesattheheartofbiochemistryandmedicinecouldbecitedfortheothergenetics;inturn,theuseofgeneticapproacheshasbeenpaireditemsshowninFigure1–1.Anotherexampleiscriticalforelucidatingmanyareasofbiochemistry.thepioneeringworkofArchibaldGarrod,aphysicianPhysiology,thestudyofbodyfunction,overlapswithinEnglandduringtheearly1900s.Hestudiedpatientsbiochemistryalmostcompletely.Immunologyemployswithanumberofrelativelyraredisorders(alkap-numerousbiochemicaltechniques,andmanyimmuno-tonuria,albinism,cystinuria,andpentosuria;thesearelogicapproacheshavefoundwideusebybiochemists.describedinlaterchapters)andestablishedthatthesePharmacologyandpharmacyrestonasoundknowl-conditionsweregeneticallydetermined.Garroddesig-edgeofbiochemistryandphysiology;inparticular,natedtheseconditionsasinbornerrorsofmetabo-mostdrugsaremetabolizedbyenzyme-catalyzedreac-lism.Hisinsightsprovidedamajorfoundationforthetions.Poisonsactonbiochemicalreactionsorprocesses;developmentofthefieldofhumanbiochemicalgenet-thisisthesubjectmatteroftoxicology.Biochemicalap-ics.Morerecenteffortstounderstandthebasisoftheproachesarebeingusedincreasinglytostudybasicas-geneticdiseaseknownasfamilialhypercholesterol-pectsofpathology(thestudyofdisease),suchasin-emia,whichresultsinsevereatherosclerosisatanearlyflammation,cellinjury,andcancer.Manyworkersinage,haveledtodramaticprogressinunderstandingofmicrobiology,zoology,andbotanyemploybiochemicalcellreceptorsandofmechanismsofuptakeofcholes-approachesalmostexclusively.Theserelationshipsareterolintocells.Studiesofoncogenesincancercellsnotsurprising,becauselifeasweknowitdependsonhavedirectedattentiontothemolecularmechanismsbiochemicalreactionsandprocesses.Infact,theoldinvolvedinthecontrolofnormalcellgrowth.Thesebarriersamongthelifesciencesarebreakingdown,andandmanyotherexamplesemphasizehowthestudyof1
102/CHAPTER1Table1–1.TheprincipalmethodsandNORMALBIOCHEMICALPROCESSESAREpreparationsusedinbiochemicallaboratories.THEBASISOFHEALTHTheWorldHealthOrganization(WHO)defines1MethodsforSeparatingandPurifyingBiomoleculeshealthasastateof“completephysical,mentalandso-Saltfractionation(eg,precipitationofproteinswithammo-cialwell-beingandnotmerelytheabsenceofdiseaseniumsulfate)andinfirmity.”Fromastrictlybiochemicalviewpoint,Chromatography:Paper;ionexchange;affinity;thin-layer;healthmaybeconsideredthatsituationinwhichallofgas-liquid;high-pressureliquid;gelfiltrationthemanythousandsofintra-andextracellularreactionsElectrophoresis:Paper;high-voltage;agarose;cellulosethatoccurinthebodyareproceedingatratescommen-acetate;starchgel;polyacrylamidegel;SDS-polyacryl-amidegelsuratewiththeorganism’smaximalsurvivalintheUltracentrifugationphysiologicstate.However,thisisanextremelyreduc-MethodsforDeterminingBiomolecularStructurestionistview,anditshouldbeapparentthatcaringforElementalanalysisthehealthofpatientsrequiresnotonlyawideknowl-UV,visible,infrared,andNMRspectroscopyedgeofbiologicprinciplesbutalsoofpsychologicandUseofacidoralkalinehydrolysistodegradethebiomole-socialprinciples.culeunderstudyintoitsbasicconstituentsUseofabatteryofenzymesofknownspecificitytode-BiochemicalResearchHasImpactongradethebiomoleculeunderstudy(eg,proteases,nucle-ases,glycosidases)Nutrition&PreventiveMedicineMassspectrometryOnemajorprerequisiteforthemaintenanceofhealthisSpecificsequencingmethods(eg,forproteinsandnucleicthattherebeoptimaldietaryintakeofanumberofacids)chemicals;thechiefofthesearevitamins,certainX-raycrystallographyaminoacids,certainfattyacids,variousminerals,andPreparationsforStudyingBiochemicalProcesseswater.Becausemuchofthesubjectmatterofbothbio-Wholeanimal(includestransgenicanimalsandanimalschemistryandnutritionisconcernedwiththestudyofwithgeneknockouts)variousaspectsofthesechemicals,thereisacloserela-IsolatedperfusedorganTissueslicetionshipbetweenthesetwosciences.Moreover,moreWholecellsemphasisisbeingplacedonsystematicattemptstoHomogenatemaintainhealthandforestalldisease,ie,onpreventiveIsolatedcellorganellesmedicine.Thus,nutritionalapproachesto—forexam-Subfractionationoforganellesple—thepreventionofatherosclerosisandcancerarePurifiedmetabolitesandenzymesreceivingincreasedemphasis.UnderstandingnutritionIsolatedgenes(includingpolymerasechainreactionanddependstoagreatextentonaknowledgeofbiochem-site-directedmutagenesis)istry.1Mostofthesemethodsaresuitableforanalyzingthecompo-nentspresentincellhomogenatesandotherbiochemicalprepa-Most&PerhapsAllDiseaseHasrations.ThesequentialuseofseveraltechniqueswillgenerallyaBiochemicalBasispermitpurificationofmostbiomolecules.Thereaderisreferredtotextsonmethodsofbiochemicalresearchfordetails.Webelievethatmostifnotalldiseasesaremanifesta-tionsofabnormalitiesofmolecules,chemicalreactions,orbiochemicalprocesses.Themajorfactorsresponsiblediseasecanopenupareasofcellfunctionforbasicbio-forcausingdiseasesinanimalsandhumansarelistedinchemicalresearch.Table1–2.AllofthemaffectoneormorecriticalTherelationshipbetweenmedicineandbiochem-chemicalreactionsormoleculesinthebody.Numerousistryhasimportantimplicationsfortheformer.Aslongexamplesofthebiochemicalbasesofdiseaseswillbeen-asmedicaltreatmentisfirmlygroundedinaknowledgecounteredinthistext;themajorityofthemareduetoofbiochemistryandotherbasicsciences,thepracticeofcauses5,7,and8.Inmostoftheseconditions,bio-medicinewillhavearationalbasisthatcanbeadaptedchemicalstudiescontributetoboththediagnosisandtoaccommodatenewknowledge.Thiscontrastswithtreatment.Somemajorusesofbiochemicalinvestiga-unorthodoxhealthcultsandatleastsome“alternativetionsandoflaboratorytestsinrelationtodiseasesaremedicine”practices,whichareoftenfoundedonlittlesummarizedinTable1–3.morethanmythandwishfulthinkingandgenerallyAdditionalexamplesofmanyoftheseusesarepre-lackanyintellectualbasis.sentedinvarioussectionsofthistext.
11BIOCHEMISTRY&MEDICINE/3BIOCHEMISTRYNucleicacidsProteinsLipidsCarbohydratesGeneticSicklecellAthero-DiabetesdiseasesanemiasclerosismellitusMEDICINEFigure1–1.Examplesofthetwo-waystreetconnectingbiochemistryandmedicine.Knowledgeofthebiochemicalmoleculesshowninthetoppartofthediagramhasclarifiedourunderstandingofthediseasesshowninthebottomhalf—andconversely,analysesofthediseasesshownbelowhavecastlightonmanyareasofbiochemistry.Notethatsicklecellanemiaisageneticdiseaseandthatbothatherosclerosisanddiabetesmellitushavegeneticcomponents.ImpactoftheHumanGenomeProject(HGP)onBiochemistry&MedicineTable1–3.SomeusesofbiochemicalRemarkableprogresswasmadeinthelate1990sinse-investigationsandlaboratorytestsinquencingthehumangenome.ThisculminatedinJuly2000,whenleadersofthetwogroupsinvolvedinthisrelationtodiseases.effort(theInternationalHumanGenomeSequencingConsortiumandCeleraGenomics,aprivatecompany)UseExampleannouncedthatover90%ofthegenomehadbeense-1.Torevealthefunda-Demonstrationofthena-quenced.Draftversionsofthesequencewerepublishedmentalcausesandtureofthegeneticde-mechanismsofdiseasesfectsincysticfibrosis.2.Tosuggestrationaltreat-AdietlowinphenylalanineTable1–2.Themajorcausesofdiseases.Allofmentsofdiseasesbasedfortreatmentofphenyl-thecauseslistedactbyinfluencingthevariouson(1)aboveketonuria.3.ToassistinthediagnosisUseoftheplasmaenzymebiochemicalmechanismsinthecellorinthe1ofspecificdiseasescreatinekinaseMBbody.(CK-MB)inthediagnosisofmyocardialinfarction.1.Physicalagents:Mechanicaltrauma,extremesoftemper-4.ToactasscreeningtestsUseofmeasurementofature,suddenchangesinatmosphericpressure,radia-fortheearlydiagnosisbloodthyroxineortion,electricshock.ofcertaindiseasesthyroid-stimulatinghor-2.Chemicalagents,includingdrugs:Certaintoxiccom-mone(TSH)intheneo-pounds,therapeuticdrugs,etc.nataldiagnosisofcon-3.Biologicagents:Viruses,bacteria,fungi,higherformsofgenitalhypothyroidism.parasites.5.ToassistinmonitoringUseoftheplasmaenzyme4.Oxygenlack:Lossofbloodsupply,depletionofthetheprogress(eg,re-alanineaminotransferaseoxygen-carryingcapacityoftheblood,poisoningofcovery,worsening,re-(ALT)inmonitoringthetheoxidativeenzymes.mission,orrelapse)ofprogressofinfectious5.Geneticdisorders:Congenital,molecular.certaindiseaseshepatitis.6.Immunologicreactions:Anaphylaxis,autoimmune6.ToassistinassessingUseofmeasurementofdisease.theresponseofdis-bloodcarcinoembryonic7.Nutritionalimbalances:Deficiencies,excesses.easestotherapyantigen(CEA)incertain8.Endocrineimbalances:Hormonaldeficiencies,excesses.patientswhohavebeen1treatedforcanceroftheAdapted,withpermission,fromRobbinsSL,CotramRS,KumarV:colon.ThePathologicBasisofDisease,3rded.Saunders,1984.
124/CHAPTER1inearly2001.Itisanticipatedthattheentiresequence•Thejudicioususeofvariousbiochemicallaboratorywillbecompletedby2003.Theimplicationsofthistestsisanintegralcomponentofdiagnosisandmoni-workforbiochemistry,allofbiology,andformedicinetoringoftreatment.aretremendous,andonlyafewpointsarementioned•Asoundknowledgeofbiochemistryandofotherre-here.Manypreviouslyunknowngeneshavebeenre-latedbasicdisciplinesisessentialfortherationalvealed;theirproteinproductsawaitcharacterization.practiceofmedicalandrelatedhealthsciences.Newlighthasbeenthrownonhumanevolution,andproceduresfortrackingdiseasegeneshavebeengreatlyrefined.TheresultsarehavingmajoreffectsonareasREFERENCESsuchasproteomics,bioinformatics,biotechnology,andFrutonJS:Proteins,Enzymes,Genes:TheInterplayofChemistryandpharmacogenomics.ReferencetothehumangenomeBiology.YaleUnivPress,1999.(Providesthehistoricalback-willbemadeinvarioussectionsofthistext.Thegroundformuchoftoday’sbiochemicalresearch.)HumanGenomeProjectisdiscussedinmoredetailinGarrodAE:Inbornerrorsofmetabolism.(CroonianLectures.)Chapter54.Lancet1908;2:1,73,142,214.InternationalHumanGenomeSequencingConsortium.Initialse-SUMMARYquencingandanalysisofthehumangenome.Nature2001:409;860.(Theissue[15February]consistsofarticles•Biochemistryisthescienceconcernedwithstudyingdedicatedtoanalysesofthehumangenome.)thevariousmoleculesthatoccurinlivingcellsandKornbergA:Basicresearch:Thelifelineofmedicine.FASEBJorganismsandwiththeirchemicalreactions.Because1992;6:3143.lifedependsonbiochemicalreactions,biochemistryKornbergA:Centenaryofthebirthofmodernbiochemistry.hasbecomethebasiclanguageofallbiologicsci-FASEBJ1997;11:1209.ences.McKusickVA:MendelianInheritanceinMan.CatalogsofHumanGenesandGeneticDisorders,12thed.JohnsHopkinsUniv•BiochemistryisconcernedwiththeentirespectrumPress,1998.[AbbreviatedMIM]oflifeforms,fromrelativelysimplevirusesandbacte-OnlineMendelianInheritanceinMan(OMIM):CenterforMed-riatocomplexhumanbeings.icalGenetics,JohnsHopkinsUniversityandNationalCenter•Biochemistryandmedicineareintimatelyrelated.forBiotechnologyInformation,NationalLibraryofMedi-Healthdependsonaharmoniousbalanceofbio-cine,1997.http://www.ncbi.nlm.nih.gov/omim/chemicalreactionsoccurringinthebody,anddisease(ThenumbersassignedtotheentriesinMIMandOMIMwillbereflectsabnormalitiesinbiomolecules,biochemicalcitedinselectedchaptersofthiswork.Consultingthisexten-sivecollectionofdiseasesandotherrelevantentries—specificreactions,orbiochemicalprocesses.proteins,enzymes,etc—willgreatlyexpandthereader’s•Advancesinbiochemicalknowledgehaveillumi-knowledgeandunderstandingofvarioustopicsreferredtonatedmanyareasofmedicine.Conversely,thestudyanddiscussedinthistext.Theonlineversionisupdatedal-ofdiseaseshasoftenrevealedpreviouslyunsuspectedmostdaily.)aspectsofbiochemistry.Thedeterminationofthese-ScriverCRetal(editors):TheMetabolicandMolecularBasesofIn-quenceofthehumangenome,nearlycomplete,willheritedDisease,8thed.McGraw-Hill,2001.haveagreatimpactonallareasofbiology,includingVenterJCetal:TheSequenceoftheHumanGenome.Science2001;291:1304.(Theissue[16February]containstheCelerabiochemistry,bioinformatics,andbiotechnology.draftversionandotherarticlesdedicatedtoanalysesofthe•Biochemicalapproachesareoftenfundamentalinil-humangenome.)luminatingthecausesofdiseasesandindesigningWilliamsDL,MarksV:ScientificFoundationsofBiochemistryinappropriatetherapies.ClinicalPractice,2nded.Butterworth-Heinemann,1994.
13Water&pH2VictorW.Rodwell,PhD,&PeterJ.Kennelly,PhDBIOMEDICALIMPORTANCEoxygenatompullselectronsawayfromthehydrogennuclei,leavingthemwithapartialpositivecharge,Wateristhepredominantchemicalcomponentofliv-whileitstwounsharedelectronpairsconstitutearegioningorganisms.Itsuniquephysicalproperties,whichin-oflocalnegativecharge.cludetheabilitytosolvateawiderangeoforganicandWater,astrongdipole,hasahighdielectriccon-inorganicmolecules,derivefromwater’sdipolarstruc-stant.AsdescribedquantitativelybyCoulomb’slaw,tureandexceptionalcapacityforforminghydrogenthestrengthofinteractionFbetweenoppositelybonds.Themannerinwhichwaterinteractswithasol-chargedparticlesisinverselyproportionatetothedi-vatedbiomoleculeinfluencesthestructureofeach.Anelectricconstantεofthesurroundingmedium.Thedi-excellentnucleophile,waterisareactantorproductinelectricconstantforavacuumisunity;forhexaneitismanymetabolicreactions.Waterhasaslightpropensity1.9;forethanolitis24.3;andforwateritis78.5.todissociateintohydroxideionsandprotons.TheWaterthereforegreatlydecreasestheforceofattractionacidityofaqueoussolutionsisgenerallyreportedusingbetweenchargedandpolarspeciesrelativetowater-freethelogarithmicpHscale.Bicarbonateandotherbuffersenvironmentswithlowerdielectricconstants.ItsstrongnormallymaintainthepHofextracellularfluidbe-dipoleandhighdielectricconstantenablewatertodis-tween7.35and7.45.Suspecteddisturbancesofacid-solvelargequantitiesofchargedcompoundssuchasbasebalanceareverifiedbymeasuringthepHofarter-salts.ialbloodandtheCO2contentofvenousblood.Causesofacidosis(bloodpH<7.35)includediabeticketosisandlacticacidosis.Alkalosis(pH>7.45)may,forex-WaterMoleculesFormHydrogenBondsample,followvomitingofacidicgastriccontents.Regu-Anunshieldedhydrogennucleuscovalentlyboundtolationofwaterbalancedependsuponhypothalamicanelectron-withdrawingoxygenornitrogenatomcanmechanismsthatcontrolthirst,onantidiuretichor-interactwithanunsharedelectronpaironanotheroxy-mone(ADH),onretentionorexcretionofwaterbythegenornitrogenatomtoformahydrogenbond.Sincekidneys,andonevaporativeloss.Nephrogenicdiabeteswatermoleculescontainbothofthesefeatures,hydro-insipidus,whichinvolvestheinabilitytoconcentrategenbondingfavorstheself-associationofwatermole-urineoradjusttosubtlechangesinextracellularfluidculesintoorderedarrays(Figure2–2).Hydrogenbond-osmolarity,resultsfromtheunresponsivenessofrenalingprofoundlyinfluencesthephysicalpropertiesoftubularosmoreceptorstoADH.waterandaccountsforitsexceptionallyhighviscosity,surfacetension,andboilingpoint.Onaverage,eachmoleculeinliquidwaterassociatesthroughhydrogenWATERISANIDEALBIOLOGICSOLVENTbondswith3.5others.Thesebondsarebothrelativelyweakandtransient,withahalf-lifeofaboutonemi-WaterMoleculesFormDipolescrosecond.RuptureofahydrogenbondinliquidwaterAwatermoleculeisanirregular,slightlyskewedtetra-requiresonlyabout4.5kcal/mol,lessthan5%ofthehedronwithoxygenatitscenter(Figure2–1).ThetwoenergyrequiredtoruptureacovalentO⎯Hbond.hydrogensandtheunsharedelectronsoftheremainingHydrogenbondingenableswatertodissolvemany3twosp-hybridizedorbitalsoccupythecornersoftheorganicbiomoleculesthatcontainfunctionalgroupstetrahedron.The105-degreeanglebetweenthehydro-whichcanparticipateinhydrogenbonding.Theoxy-gensdiffersslightlyfromtheidealtetrahedralangle,genatomsofaldehydes,ketones,andamidesprovide109.5degrees.Ammoniaisalsotetrahedral,witha107-pairsofelectronsthatcanserveashydrogenacceptors.degreeanglebetweenitshydrogens.Waterisadipole,Alcoholsandaminescanservebothashydrogenaccep-amoleculewithelectricalchargedistributedasymmetri-torsandasdonorsofunshieldedhydrogenatomsforcallyaboutitsstructure.Thestronglyelectronegativeformationofhydrogenbonds(Figure2–3).5
146/CHAPTER2HCH3CH2OHO2eH2eHH105°CH3CH2OHOHCH2CH3Figure2–1.ThewatermoleculehastetrahedralRRIIgeometry.COHNRIIIIRINTERACTIONWITHWATERINFLUENCESTHESTRUCTUREOFBIOMOLECULESFigure2–3.Additionalpolargroupsparticipateinhydrogenbonding.ShownarehydrogenbondsformedCovalent&NoncovalentBondsStabilizebetweenanalcoholandwater,betweentwomoleculesBiologicMoleculesofethanol,andbetweenthepeptidecarbonyloxygenThecovalentbondisthestrongestforcethatholdsandthepeptidenitrogenhydrogenofanadjacentmoleculestogether(Table2–1).Noncovalentforces,aminoacid.whileoflessermagnitude,makesignificantcontribu-tionstothestructure,stability,andfunctionalcompe-phosphatidylserineorphosphatidylethanolaminecon-tenceofmacromoleculesinlivingcells.Theseforces,tactwaterwhiletheirhydrophobicfattyacylsidechainswhichcanbeeitherattractiveorrepulsive,involvein-clustertogether,excludingwater.Thispatternmaxi-teractionsbothwithinthebiomoleculeandbetweenitmizestheopportunitiesfortheformationofenergeti-andthewaterthatformstheprincipalcomponentofcallyfavorablecharge-dipole,dipole-dipole,andhydro-thesurroundingenvironment.genbondinginteractionsbetweenpolargroupsonthebiomoleculeandwater.ItalsominimizesenergeticallyBiomoleculesFoldtoPositionPolar&unfavorablecontactbetweenwaterandhydrophobicChargedGroupsonTheirSurfacesgroups.Mostbiomoleculesareamphipathic;thatis,theypos-HydrophobicInteractionssessregionsrichinchargedorpolarfunctionalgroupsaswellasregionswithhydrophobiccharacter.ProteinsHydrophobicinteractionreferstothetendencyofnon-tendtofoldwiththeR-groupsofaminoacidswithhy-polarcompoundstoself-associateinanaqueousenvi-drophobicsidechainsintheinterior.Aminoacidswithronment.Thisself-associationisdrivenneitherbymu-chargedorpolaraminoacidsidechains(eg,arginine,tualattractionnorbywhataresometimesincorrectlyglutamate,serine)generallyarepresentonthesurfacereferredtoas“hydrophobicbonds.”Self-associationincontactwithwater.Asimilarpatternprevailsinaarisesfromtheneedtominimizeenergeticallyunfavor-phospholipidbilayer,wherethechargedheadgroupsofableinteractionsbetweennonpolargroupsandwater.Table2–1.BondenergiesforatomsofbiologicHHHHOOsignificance.HHHHOOHOBondEnergyBondEnergyHOHHType(kcal/mol)Type(kcal/mol)HOHO—O34O==O96S—S51C—H99Figure2–2.Left:AssociationoftwodipolarwaterC—N70C==S108S—H81O—H110moleculesbyahydrogenbond(dottedline).Right:C—C82C==C147Hydrogen-bondedclusteroffourwatermolecules.C—O84C==N147Notethatwatercanservesimultaneouslybothasahy-N—H94C==O164drogendonorandasahydrogenacceptor.
15WATER&pH/7Whilethehydrogensofnonpolargroupssuchasthethebackbonetowaterwhileburyingtherelativelyhy-methylenegroupsofhydrocarbonsdonotformhydro-drophobicnucleotidebasesinside.Theextendedback-genbonds,theydoaffectthestructureofthewaterthatbonemaximizesthedistancebetweennegativelysurroundsthem.Watermoleculesadjacenttoahy-chargedbackbonephosphates,minimizingunfavorabledrophobicgrouparerestrictedinthenumberoforien-electrostaticinteractions.tations(degreesoffreedom)thatpermitthemtopar-ticipateinthemaximumnumberofenergeticallyWATERISANEXCELLENTNUCLEOPHILEfavorablehydrogenbonds.Maximalformationofmul-tiplehydrogenbondscanbemaintainedonlybyin-Metabolicreactionsofteninvolvetheattackbylonecreasingtheorderoftheadjacentwatermolecules,withpairsofelectronsonelectron-richmoleculestermedacorrespondingdecreaseinentropy.nucleophilesonelectron-pooratomscalledelec-Itfollowsfromthesecondlawofthermodynamicstrophiles.Nucleophilesandelectrophilesdonotneces-thattheoptimalfreeenergyofahydrocarbon-watersarilypossessaformalnegativeorpositivecharge.3mixtureisafunctionofbothmaximalenthalpy(fromWater,whosetwolonepairsofspelectronsbearapar-hydrogenbonding)andminimumentropy(maximumtialnegativecharge,isanexcellentnucleophile.Otherdegreesoffreedom).Thus,nonpolarmoleculestendtonucleophilesofbiologicimportanceincludetheoxygenformdropletswithminimalexposedsurfacearea,re-atomsofphosphates,alcohols,andcarboxylicacids;theducingthenumberofwatermoleculesaffected.Forthesulfurofthiols;thenitrogenofamines;andtheimid-samereason,intheaqueousenvironmentofthelivingazoleringofhistidine.Commonelectrophilesincludecellthehydrophobicportionsofbiopolymerstendtothecarbonylcarbonsinamides,esters,aldehydes,andbeburiedinsidethestructureofthemolecule,orwithinketonesandthephosphorusatomsofphosphoesters.alipidbilayer,minimizingcontactwithwater.Nucleophilicattackbywatergenerallyresultsinthecleavageoftheamide,glycoside,oresterbondsthatholdbiopolymerstogether.Thisprocessistermedhy-ElectrostaticInteractionsdrolysis.Conversely,whenmonomerunitsarejoinedInteractionsbetweenchargedgroupsshapebiomolecu-togethertoformbiopolymerssuchasproteinsorglyco-larstructure.Electrostaticinteractionsbetweenoppo-gen,waterisaproduct,asshownbelowfortheforma-sitelychargedgroupswithinorbetweenbiomoleculestionofapeptidebondbetweentwoaminoacids.aretermedsaltbridges.Saltbridgesarecomparableinstrengthtohydrogenbondsbutactoverlargerdis-O+HNtances.Theythusoftenfacilitatethebindingofcharged3moleculesandionstoproteinsandnucleicacids.OH+HNHO–AlanineVanderWaalsForcesOVanderWaalsforcesarisefromattractionsbetweenValinetransientdipolesgeneratedbytherapidmovementofelectronsonallneutralatoms.Significantlyweakerthanhydrogenbondsbutpotentiallyextremelynumer-H2Oous,vanderWaalsforcesdecreaseasthesixthpowerofthedistanceseparatingatoms.Thus,theyactoververyOshortdistances,typically2–4Å.+HN3NHO–MultipleForcesStabilizeBiomoleculesTheDNAdoublehelixillustratesthecontributionofOmultipleforcestothestructureofbiomolecules.WhileeachindividualDNAstrandisheldtogetherbycova-Whilehydrolysisisathermodynamicallyfavoredre-lentbonds,thetwostrandsofthehelixareheldto-action,theamideandphosphoesterbondsofpolypep-getherexclusivelybynoncovalentinteractions.Thesetidesandoligonucleotidesarestableintheaqueousen-noncovalentinteractionsincludehydrogenbondsbe-vironmentofthecell.Thisseeminglyparadoxictweennucleotidebases(Watson-Crickbasepairing)behaviorreflectsthefactthatthethermodynamicsgov-andvanderWaalsinteractionsbetweenthestackederningtheequilibriumofareactiondonotdeterminepurineandpyrimidinebases.Thehelixpresentsthetherateatwhichitwilltakeplace.Inthecell,proteinchargedphosphategroupsandpolarribosesugarsofcatalystscalledenzymesareusedtoacceleratetherate
168/CHAPTER2ofhydrolyticreactionswhenneeded.ProteasescatalyzeH7O3+.Theprotonisneverthelessroutinelyrepre-thehydrolysisofproteinsintotheircomponentaminosentedasH+,eventhoughitisinfacthighlyhydrated.acids,whilenucleasescatalyzethehydrolysisoftheSincehydroniumandhydroxideionscontinuouslyphosphoesterbondsinDNAandRNA.Carefulcontrolrecombinetoformwatermolecules,anindividualhy-oftheactivitiesoftheseenzymesisrequiredtoensuredrogenoroxygencannotbestatedtobepresentasanthattheyactonlyonappropriatetargetmolecules.ionoraspartofawatermolecule.Atoneinstantitisanion.Aninstantlateritispartofamolecule.Individ-ManyMetabolicReactionsInvolveualionsormoleculesarethereforenotconsidered.WeGroupTransferreferinsteadtotheprobabilitythatatanyinstantintimeahydrogenwillbepresentasanionoraspartofaIngrouptransferreactions,agroupGistransferred22watermolecule.Since1gofwatercontains3.46×10fromadonorDtoanacceptorA,forminganacceptormolecules,theionizationofwatercanbedescribedsta-groupcomplexA–G:tistically.Tostatethattheprobabilitythatahydrogenexistsasanionis0.01meansthatahydrogenatomhasDGAAGD−=+−+onechancein100ofbeinganionand99chancesoutof100ofbeingpartofawatermolecule.TheactualThehydrolysisandphosphorolysisofglycogenrepre-probabilityofahydrogenatominpurewaterexistingassentgrouptransferreactionsinwhichglucosylgroups−9ahydrogenionisapproximately1.8×10.Theproba-aretransferredtowaterortoorthophosphate.Thebilityofitsbeingpartofamoleculethusisalmostequilibriumconstantforthehydrolysisofcovalentunity.Statedanotherway,foreveryhydrogenionandbondsstronglyfavorstheformationofsplitproducts.hydroxylioninpurewaterthereare1.8billionor1.8×Thebiosynthesisofmacromoleculesalsoinvolvesgroup910watermolecules.Hydrogenionsandhydroxylionstransferreactionsinwhichthethermodynamicallyun-neverthelesscontributesignificantlytothepropertiesoffavoredsynthesisofcovalentbondsiscoupledtofa-water.voredreactionssothattheoverallchangeinfreeenergyFordissociationofwater,favorsbiopolymersynthesis.Giventhenucleophiliccharacterofwateranditshighconcentrationincells,whyarebiopolymerssuchasproteinsandDNArela-[]HO+[H−]K=tivelystable?Andhowcansynthesisofbiopolymers[HO2]occurinanapparentlyaqueousenvironment?Centraltobothquestionsarethepropertiesofenzymes.Inthewherebracketsrepresentmolarconcentrations(strictlyabsenceofenzymiccatalysis,eventhermodynamicallyspeaking,molaractivities)andKisthedissociationhighlyfavoredreactionsdonotnecessarilytakeplaceconstant.Sinceonemole(mol)ofwaterweighs18g,rapidly.Preciseanddifferentialcontrolofenzymeac-oneliter(L)(1000g)ofwatercontains1000×18=tivityandthesequestrationofenzymesinspecificor-55.56mol.Purewaterthusis55.56molar.Sincetheganellesdetermineunderwhatphysiologicconditionsaprobabilitythatahydrogeninpurewaterwillexistasagivenbiopolymerwillbesynthesizedordegraded.−9hydrogenionis1.8×10,themolarconcentrationofNewlysynthesizedpolymersarenotimmediatelyhy-H+ions(orofOH−ions)inpurewateristheproductdrolyzed,inpartbecausetheactivesitesofbiosynthetic−9oftheprobability,1.8×10,timesthemolarconcen-enzymessequestersubstratesinanenvironmentfrom−7trationofwater,55.56mol/L.Theresultis1.0×10whichwatercanbeexcluded.mol/L.WecannowcalculateKforwater:WaterMoleculesExhibitaSlightbutImportantTendencytoDissociate+−−77−[]HO[]H[]10[]10K==Theabilityofwatertoionize,whileslight,isofcentral[]HO2[.]5556importanceforlife.Sincewatercanactbothasanacid−−1416=×=001810../1810×molLandasabase,itsionizationmayberepresentedasanintermolecularprotontransferthatformsahydroniumion(HO+)andahydroxideion(OH−):Themolarconcentrationofwater,55.56mol/L,is3toogreattobesignificantlyaffectedbydissociation.ItHO++HOHO=+OH−thereforeisconsideredtobeessentiallyconstant.This223constantmaythenbeincorporatedintothedissociationThetransferredprotonisactuallyassociatedwithaconstantKtoprovideausefulnewconstantKwtermedclusterofwatermolecules.Protonsexistinsolutionnottheionproductforwater.TherelationshipbetweenonlyasH3O+,butalsoasmultimerssuchasH5O2+andKwandKisshownbelow:
17WATER&pH/9+−termediates,whosephosphorylgroupcontainstwodis-[]HO[]H−16K==1810./×molLsociableprotons,thefirstofwhichisstronglyacidic.[]HO2ThefollowingexamplesillustratehowtocalculateKK==()[HO][][HOH+−]thepHofacidicandbasicsolutions.w2Example1:WhatisthepHofasolutionwhosehy-−16=×(.1810molL/)(.5556molL/)drogenionconcentrationis3.2×10−4mol/L?=×10010.(−14molL/)2pH=−log[H+]NotethatthedimensionsofKaremolesperliterand=×−log(.3210−4)22thoseofKwaremolesperliter.Asitsnamesuggests,−4=−−log(.)log(3210)theionproductKwisnumericallyequaltotheproductofthemolarconcentrationsofH+andOH−:=+−0540..=35.K=[]HO+[]H−wExample2:WhatisthepHofasolutionwhosehy-At25°C,K=(10−7)2,or10−14(mol/L)2.Attempera-droxideionconcentrationis4.0×10−4mol/L?Wefirstwturesbelow25°C,Kissomewhatlessthan10−14;and−wdefineaquantitypOHthatisequalto−log[OH]andattemperaturesabove25°CitissomewhatgreaterthanthatmaybederivedfromthedefinitionofKw:−1410.Withinthestatedlimitationsoftheeffectoftem--142perature,Kwequals10(mol/L)forallaqueousso-K==[]HO+[]H−−1014wlutions,evensolutionsofacidsorbases.WeshalluseKwtocalculatethepHofacidicandbasicsolutions.Therefore:log[HO+−]log[+=H]log10−14pHISTHENEGATIVELOGOFTHEHYDROGENIONCONCENTRATIONorThetermpHwasintroducedin1909bySörensen,whodefinedpHasthenegativelogofthehydrogenionpHpOH+=14concentration:Tosolvetheproblembythisapproach:pH=−log[H+][].OH−−=×40104Thisdefinition,whilenotrigorous,sufficesformanypOH=−log[OH−]biochemicalpurposes.TocalculatethepHofasolution:1.Calculatehydrogenionconcentration[H+].=×−log(.4010−4)2.Calculatethebase10logarithmof[H+].=−−log(.)log40(10−4)3.pHisthenegativeofthevaluefoundinstep2.=−.+.06040Forexample,forpurewaterat25°C,=.34pH===−−−log[logH+]10−7(−7)=7.0Now:LowpHvaluescorrespondtohighconcentrationsofpH==14−−pOH1434.H+andhighpHvaluescorrespondtolowconcentra-=106.tionsofH+.Acidsareprotondonorsandbasesareprotonac-−2Example3:WhatarethepHvaluesof(a)2.0×10ceptors.Strongacids(eg,HClorHSO)completely−624mol/LKOHandof(b)2.0×10mol/LKOH?Thedissociateintoanionsandcationseveninstronglyacidic−OHarisesfromtwosources,KOHandwater.Sincesolutions(lowpH).WeakacidsdissociateonlypartiallypHisdeterminedbythetotal[H+](andpOHbytheinacidicsolutions.Similarly,strongbases(eg,KOHor−total[OH]),bothsourcesmustbeconsidered.IntheNaOH)—butnotweakbases(eg,Ca[OH]2)—arefirstcase(a),thecontributionofwatertothetotalcompletelydissociatedathighpH.Manybiochemicals−[OH]isnegligible.Thesamecannotbesaidfortheareweakacids.Exceptionsincludephosphorylatedin-secondcase(b):
1810/CHAPTER2belowaretheexpressionsforthedissociationconstantConcentration(mol/L)(Ka)fortworepresentativeweakacids,R⎯COOHand(a)(b)R⎯NH3+.−2−6MolarityofKOH2.0×102.0×10−−2−6R——COOHR=COO−+H+[OH]fromKOH2.0×102.0×10−−7−7[OH]fromwater1.0×101.0×10−+−−2−6[—RCOO][H]Total[OH]2.00001×102.1×10Ka=[—RCOOH]++R——NH32=RNH+HOnceadecisionhasbeenreachedaboutthesignificance+ofthecontributionbywater,pHmaybecalculatedas[—RNHH2][]Ka=above.[—RNH+]3TheaboveexamplesassumethatthestrongbaseKOHiscompletelydissociatedinsolutionandthattheSincethenumericvaluesofKaforweakacidsarenega-−concentrationofOHionswasthusequaltothatofthetiveexponentialnumbers,weexpressKaaspKa,whereKOH.Thisassumptionisvalidfordilutesolutionsofstrongbasesoracidsbutnotforweakbasesoracids.pKKa=−logSinceweakelectrolytesdissociateonlyslightlyinsolu-tion,wemustusethedissociationconst−anttocalcu-NotethatpKaisrelatedtoKaaspHisto[H+].Thelatetheconcentrationof[H+](or[OH])producedbystrongertheacid,theloweritspKavalue.agivenmolarityofaweakacid(orbase)beforecalcu-+−pKaisusedtoexpresstherelativestrengthsofbothlatingtotal[H](ortotal[OH])andsubsequentlypH.acidsandbases.Foranyweakacid,itsconjugateisastrongbase.Similarly,theconjugateofastrongbaseisaweakacid.Therelativestrengthsofbasesareex-FunctionalGroupsThatAreWeakAcidspressedintermsofthepKaoftheirconjugateacids.ForHaveGreatPhysiologicSignificancepolyproteiccompoundscontainingmorethanonedis-Manybiochemicalspossessfunctionalgroupsthataresociableproton,anumericalsubscriptisassignedtoweakacidsorbases.Carboxylgroups,aminogroups,eachinorderofrelativeacidity.Foradissociationofandthesecondphosphatedissociationofphosphatees-thetypetersarepresentinproteinsandnucleicacids,mostcoenzymes,andmostintermediarymetabolites.Knowl-+RN—H3→R—NH2edgeofthedissociationofweakacidsandbasesthusisbasictounderstandingtheinfluenceofintracellularpHthepKaisthepHatwhichtheconcentrationoftheonstructureandbiologicactivity.Charge-basedsepara-acidR⎯NH3+equalsthatofthebaseR⎯NH2.tionssuchaselectrophoresisandionexchangechro-FromtheaboveequationsthatrelateKato[H+]andmatographyalsoarebestunderstoodintermsofthetotheconcentrationsofundissociatedacidanditscon-dissociationbehavioroffunctionalgroups.jugatebase,whenWetermtheprotonatedspecies(eg,HAorR⎯NH3+)theacidandtheunprotonatedspecies(eg,−−[]R——COO=[RCOOH]AorR⎯NH2)itsconjugatebase.Similarly,wemay−refertoabase(eg,AorR⎯NH2)anditsconjugateacid(eg,HAorR⎯NH3+).Representativeweakacidsorwhen(left),theirconjugatebases(center),andthepKavalues+(right)includethefollowing:[]RN——HRN23=[]HR——CHCOOHR—CH—COO−pK=45−22athen+R—CH22——NH32R—CHNHpKa=910−+Ka=[]H−HCO23HCOp3Ka=64.−−2Thus,whentheassociated(protonated)anddissociatedHPO24HPO4pKa=72.(conjugatebase)speciesarepresentatequalconcentra-tions,theprevailinghydrogenionconcentration[H+]Weexpresstherelativestrengthsofweakacidsandisnumericallyequaltothedissociationconstant,Ka.Ifbasesintermsoftheirdissociationconstants.Shownthelogarithmsofbothsidesoftheaboveequationare
19WATER&pH/11takenandbothsidesaremultipliedby−1,theexpres-SubstitutepHandpKafor−log[H+]and−logKa,re-sionswouldbeasfollows:spectively;then:K=[]H+a[]HA+pH=pKa−log−−−loglogKa=[H][]ASince−logKaisdefinedaspKa,and−log[H+]de-InversionofthelasttermremovestheminussignfinespH,theequationmayberewrittenasandgivestheHenderson-Hasselbalchequation:ppKa=H[]A−pH=+pKalog[]HAie,thepKaofanacidgroupisthepHatwhichthepro-tonatedandunprotonatedspeciesarepresentatequalTheHenderson-Hasselbalchequationhasgreatpre-concentrations.ThepKaforanacidmaybedetermineddictivevalueinprotonicequilibria.Forexample,byadding0.5equivalentofalkaliperequivalentofacid.TheresultingpHwillbethepKoftheacid.−a(1)Whenanacidisexactlyhalf-neutralized,[A]=[HA].Undertheseconditions,TheHenderson-HasselbalchEquation−[]A1DescribestheBehaviorpH=+pKKaalog=+=+pplogKa0[]HA1ofWeakAcids&BuffersTheHenderson-Hasselbalchequationisderivedbelow.Therefore,athalf-neutralization,pH=pKa.Aweakacid,HA,ionizesasfollows:−(2)Whentheratio[A]/[HA]=100:1,HA=H++A−[]A−pH=+pKalogTheequilibriumconstantforthisdissociationis[]HA+−pH=+pKKaalog100/1=p+2[]HA[]Ka=[]HA−(3)Whentheratio[A]/[HA]=1:10,Cross-multiplicationgivespH=+pKKaalog1/10=p+(−1)[]HA+[][]−=KHAaIftheequationisevaluatedatratiosof[A−]/[HA]3−3−rangingfrom10to10andthecalculatedpHvaluesDividebothsidesby[A]:areplotted,theresultinggraphdescribesthetitrationcurveforaweakacid(Figure2–4).+[]HA[]H=Ka[]A−SolutionsofWeakAcids&TheirSaltsTakethelogofbothsides:BufferChangesinpH+⎛[]HA⎞Solutionsofweakacidsorbasesandtheirconjugateslog[H]=log⎜Ka−⎟exhibitbuffering,theabilitytoresistachangeinpH⎝[]A⎠followingadditionofstrongacidorbase.Sincemany[]HAmetabolicreactionsareaccompaniedbythereleaseor=+logKalog−uptakeofprotons,mostintracellularreactionsare[]Abuffered.OxidativemetabolismproducesCO2,thean-hydrideofcarbonicacid,whichifnotbufferedwouldMultiplythroughby−1:producesevereacidosis.MaintenanceofaconstantpHinvolvesbufferingbyphosphate,bicarbonate,andpro-−−log[H+]=logK−log[]HAteins,whichacceptorreleaseprotonstoresistachangea[]A−
2012/CHAPTER21.01.0tothepKa.AsolutionofaweakacidanditsconjugatebasebuffersmosteffectivelyinthepHrangepKa±1.00.80.8pHunit.Figure2–4alsoillustratesthenetchargeonone0.60.6moleculeoftheacidasafunctionofpH.Afractionalchargeof−0.5doesnotmeanthatanindividualmole-0.40.4culebearsafractionalcharge,buttheprobabilitythataNetchargegivenmoleculehasaunitnegativechargeis0.5.Con-0.20.2siderationofthenetchargeonmacromoleculesasafunctionofpHprovidesthebasisforseparatorytech-meqofalkaliaddedpermeqofacid00niquessuchasionexchangechromatographyandelec-2345678trophoresis.pHFigure2–4.TitrationcurveforanacidofthetypeAcidStrengthDependsonHA.TheheavydotinthecenterofthecurveindicatesMolecularStructurethepKa5.0.Manyacidsofbiologicinterestpossessmorethanonedissociatinggroup.ThepresenceofadjacentnegativechargehindersthereleaseofaprotonfromanearbyinpH.Forexperimentsusingtissueextractsoren-group,raisingitspKa.ThisisapparentfromthepKazymes,constantpHismaintainedbytheadditionofvaluesforthethreedissociatinggroupsofphosphoricbufferssuchasMES([2-N-morpholino]ethanesulfonicacidandcitricacid(Table2–2).Theeffectofadjacentacid,pKa6.1),inorganicorthophosphate(pKa27.2),chargedecreaseswithdistance.ThesecondpKaforsuc-HEPES(N-hydroxyethylpiperazine-N9-2-ethanesulfoniccinicacid,whichhastwomethylenegroupsbetweenitsacid,pKa6.8),orTris(tris[hydroxymethyl]amino-carboxylgroups,is5.6,whereasthesecondpKaforglu-methane,pKa8.3).ThevalueofpKarelativetothede-siredpHisthemajordeterminantofwhichbufferisse-lected.BufferingcanbeobservedbyusingapHmeterTable2–2.Relativestrengthsofselectedacidsofwhiletitratingaweakacidorbase(Figure2–4).WecanalsocalculatethepHshiftthataccompaniesaddi-biologicsignificance.TabulatedvaluesarethepKationofacidorbasetoabufferedsolution.Intheexam-values(−logofthedissociationconstant)ofple,thebufferedsolution(aweakacid,pKa=5.0,andselectedmonoprotic,diprotic,andtriproticacids.itsconjugatebase)isinitiallyatoneoffourpHvalues.WewillcalculatethepHshiftthatresultswhen0.1MonoproticAcidsmeqofKOHisaddedto1meqofeachsolution:FormicpK3.75LacticpK3.86AceticpK4.76InitialpH5.005.375.605.86AmmoniumionpK9.25−[A]initial0.500.700.800.88[HA]initial0.500.300.200.12DiproticAcids−([A]/[HA])initial1.002.334.007.33CarbonicpK16.37Additionof0.1meqofKOHproducespK210.25[A−]0.600.800.900.98SuccinicpK14.21final[HA]final0.400.200.100.02pK25.64([A−]/[HA])1.504.009.0049.0GlutaricpK14.34finallog([A−]/[HA])0.1760.6020.951.69pK25.41finalFinalpH5.185.605.956.69TriproticAcidsΔpH0.180.600.951.69PhosphoricpK12.15pK26.82pK312.38NoticethatthechangeinpHpermilliequivalentofCitricpK13.08OH−addeddependsontheinitialpH.Thesolutionre-pK24.74sistschangesinpHmosteffectivelyatpHvaluesclosepK35.40
21WATER&pH/13taricacid,whichhasoneadditionalmethylenegroup,•Macromoleculesexchangeinternalsurfacehydrogenis5.4.bondsforhydrogenbondstowater.EntropicforcesdictatethatmacromoleculesexposepolarregionstopKaValuesDependonthePropertiesanaqueousinterfaceandburynonpolarregions.oftheMedium•Saltbonds,hydrophobicinteractions,andvanderWaalsforcesparticipateinmaintainingmolecularThepKaofafunctionalgroupisalsoprofoundlyinflu-structure.encedbythesurroundingmedium.Themediummay+•pHisthenegativelogof[H].AlowpHcharacter-eitherraiseorlowerthepKadependingonwhethertheizesanacidicsolution,andahighpHdenotesabasicundissociatedacidoritsconjugatebaseisthechargedsolution.species.TheeffectofdielectricconstantonpKamaybeobservedbyaddingethanoltowater.ThepKaofacar-•ThestrengthofweakacidsisexpressedbypKa,theboxylicacidincreases,whereasthatofanaminedecreasesnegativelogoftheaciddissociationconstant.StrongbecauseethanoldecreasestheabilityofwatertosolvateacidshavelowpKavaluesandweakacidshavehighachargedspecies.ThepKavaluesofdissociatinggroupspKavalues.intheinteriorsofproteinsthusareprofoundlyaffected•BuffersresistachangeinpHwhenprotonsarepro-bytheirlocalenvironment,includingthepresenceorducedorconsumed.Maximumbufferingcapacityabsenceofwater.occurs±1pHunitoneithersideofpKa.Physiologicbuffersincludebicarbonate,orthophosphate,andproteins.SUMMARYREFERENCES•Waterformshydrogen-bondedclusterswithitselfandwithotherprotondonorsoracceptors.HydrogenSegelIM:BiochemicalCalculations.Wiley,1968.bondsaccountforthesurfacetension,viscosity,liquidWigginsPM:Roleofwaterinsomebiologicalprocesses.Microbiolstateatroomtemperature,andsolventpowerofwater.Rev1990;54:432.•CompoundsthatcontainO,N,orScanserveashy-drogenbonddonorsoracceptors.
22SECTIONIStructures&FunctionsofProteins&EnzymesAminoAcids&Peptides3VictorW.Rodwell,PhD,&PeterJ.Kennelly,PhDBIOMEDICALIMPORTANCEmorethan20aminoacids,itsredundancylimitstheavailablecodonstothe20L-α-aminoacidslistedinInadditiontoprovidingthemonomerunitsfromwhichTable3–1,classifiedaccordingtothepolarityoftheirRthelongpolypeptidechainsofproteinsaresynthesized,groups.Bothone-andthree-letterabbreviationsforeachtheL-α-aminoacidsandtheirderivativesparticipateinaminoacidcanbeusedtorepresenttheaminoacidsincellularfunctionsasdiverseasnervetransmissionandpeptides(Table3–1).Someproteinscontainadditionalthebiosynthesisofporphyrins,purines,pyrimidines,aminoacidsthatarisebymodificationofanaminoacidandurea.Shortpolymersofaminoacidscalledpeptidesalreadypresentinapeptide.Examplesincludeconver-performprominentrolesintheneuroendocrinesystemsionofpeptidylprolineandlysineto4-hydroxyprolineashormones,hormone-releasingfactors,neuromodula-and5-hydroxylysine;theconversionofpeptidylgluta-tors,orneurotransmitters.Whileproteinscontainonlymatetoγ-carboxyglutamate;andthemethylation,L-α-aminoacids,microorganismselaboratepeptidesformylation,acetylation,prenylation,andphosphoryla-thatcontainbothD-andL-α-aminoacids.Severaloftionofcertainaminoacylresidues.Thesemodificationsthesepeptidesareoftherapeuticvalue,includingthean-extendthebiologicdiversityofproteinsbyalteringtheirtibioticsbacitracinandgramicidinAandtheantitumorsolubility,stability,andinteractionwithotherproteins.agentbleomycin.Certainothermicrobialpeptidesaretoxic.Thecyanobacterialpeptidesmicrocystinandnodularinarelethalinlargedoses,whilesmallquantitiesOnlyL--AminoAcidsOccurinProteinspromotetheformationofhepatictumors.Neitherhu-Withthesoleexceptionofglycine,theα-carbonofmansnoranyotherhigheranimalscansynthesize10ofaminoacidsischiral.Althoughsomeproteinaminothe20commonL-α-aminoacidsinamountsadequateacidsaredextrorotatoryandsomelevorotatory,allsharetosupportinfantgrowthortomaintainhealthinadults.theabsoluteconfigurationofL-glyceraldehydeandthusConsequently,thehumandietmustcontainadequateareL-α-aminoacids.SeveralfreeL-α-aminoacidsfulfillquantitiesofthesenutritionallyessentialaminoacids.importantrolesinmetabolicprocesses.Examplesin-cludeornithine,citrulline,andargininosuccinatethatPROPERTIESOFAMINOACIDSparticipateinureasynthesis;tyrosineinformationofTheGeneticCodeSpecifiesthyroidhormones;andglutamateinneurotransmitterbiosynthesis.D-Aminoacidsthatoccurnaturallyin-20L--AminoAcidscludefreeD-serineandD-aspartateinbraintissue,Oftheover300naturallyoccurringaminoacids,20con-D-alanineandD-glutamateinthecellwallsofgram-stitutethemonomerunitsofproteins.Whileanonre-positivebacteria,andD-aminoacidsinsomenonmam-dundantthree-lettergeneticcodecouldaccommodatemalianpeptidesandcertainantibiotics.14
23Table3–1.L-α-Aminoacidspresentinproteins.NameSymbolStructuralFormulapK1pK2pK3WithAliphaticSideChains-COOH-NH3+RGroupGlycineGly[G]–2.49.8HCHCOO+NH3AlanineAla[A]–2.49.9CH3CHCOO+NH3H3C–CHCHCOOValineVal[V]2.29.7HCNH+33H3C–CHCH2CHCOOLeucineLeu[L]2.39.7HCNH+33CH3CH2IsoleucineIle[I]CHCHCOO–2.39.8CH+3NH3WithSideChainsContainingHydroxylic(OH)GroupsSerineSer[S]–2.29.2about13CH2CHCOO+OHNH3ThreonineThr[T]–2.19.1about13CH3CHCHCOO+OHNH3TyrosineTyr[Y]Seebelow.WithSideChainsContainingSulfurAtomsCysteineCys[C]–1.910.88.3CH2CHCOO+SHNH3MethionineMet[M]–2.19.3CH2CH2CHCOO+SCH3NH3WithSideChainsContainingAcidicGroupsorTheirAmidesAsparticacidAsp[D]––2.09.93.9OOCCH2CHCOO+NH3AsparagineAsn[N]–2.18.8H2NCCH2CHCOO+ONH3––OOCCH2CH2CHCOOGlutamicacidGlu[E]2.19.54.1+NH3–H2NCCH2CH2CHCOOGlutamineGln[Q]2.29.1+ONH3(continued)15
2416/CHAPTER3Table3–1.L-α-Aminoacidspresentinproteins.(continued)NameSymbolStructuralFormulapK1pK2pK3WithSideChainsContainingBasicGroups-COOH-NH3+RGroupArginineArg[R]–1.89.012.5HNCH2CH2CH2CHCOO++CNH2NH3NH2–CH2CH2CH2CH2CHCOOLysineLys[K]2.29.210.8++NH3NH3–CH2CHCOOHistidineHis[H]1.89.36.0HNNNH+3ContainingAromaticRingsHistidineHis[H]Seeabove.–PhenylalaninePhe[F]CH2CHCOO2.29.2+NH3TyrosineTyr[Y]2.29.110.1–HOCH2CHCOO+NH3TryptophanTrp[W]2.49.4–CH2CHCOO+NH3NHIminoAcidProlinePro[P]2.010.6+–NCOOH2AminoAcidsMayHavePositive,Negative,Moleculesthatcontainanequalnumberofioniz-orZeroNetChargeablegroupsofoppositechargeandthatthereforebearnonetchargearetermedzwitterions.AminoacidsinChargedandunchargedformsoftheionizablebloodandmosttissuesthusshouldberepresentedasin⎯COOHand⎯NH3+weakacidgroupsexistinsolu-A,below.tioninprotonicequilibrium:NH+NH32R——COOH=RCOO−+H+O–OH++RN—H32=R—NHH+RROOWhilebothR⎯COOHandR⎯NH3+areweakacids,ABR⎯COOHisafarstrongeracidthanR⎯NH3+.AtphysiologicpH(pH7.4),carboxylgroupsexistalmostStructureBcannotexistinaqueoussolutionbecauseat−entirelyasR⎯COOandaminogroupspredomi-anypHlowenoughtoprotonatethecarboxylgroupnantlyasR⎯NH3+.Figure3–1illustratestheeffectoftheaminogroupwouldalsobeprotonated.Similarly,pHonthechargedstateofasparticacid.atanypHsufficientlyhighforanunchargedamino
25AMINOACIDS&PEPTIDES/17OH+OH+OH+OO––OHOHO+pK1=2.09+pK2=3.86+pK3=9.82NH3(α-COOH)NH3(β-COOH)NH3(—NH+)NH23HO–O–O–OOOOOABCDInstrongacidAroundpH3;AroundpH6–8;Instrongalkali(belowpH1);netcharge=0netcharge=–1(abovepH11);netcharge=+1netcharge=–2Figure3–1.Protonicequilibriaofasparticacid.grouptopredominate,acarboxylgroupwillbepresentAtItsIsoelectricpH(pI),anAminoAcid−asR⎯COO.TheunchargedrepresentationB(above)BearsNoNetChargeis,however,oftenusedforreactionsthatdonotinvolveprotonicequilibria.Theisoelectricspeciesistheformofamoleculethathasanequalnumberofpositiveandnegativechargesandthusiselectricallyneutral.TheisoelectricpH,alsopKaValuesExpresstheStrengthscalledthepI,isthepHmidwaybetweenpKavaluesonofWeakAcidseithersideoftheisoelectricspecies.ForanaminoacidTheacidstrengthsofweakacidsareexpressedastheirsuchasalaninethathasonlytwodissociatinggroups,pKa(Table3–1).Theimidazolegroupofhistidineandthereisnoambiguity.ThefirstpKa(R⎯COOH)istheguanidinogroupofarginineexistasresonancehy-2.35andthesecondpKa(R⎯NH3+)is9.69.Theiso-bridswithpositivechargedistributedbetweenbothni-electricpH(pI)ofalaninethusistrogens(histidine)orallthreenitrogens(arginine)(Fig-ure3–2).Thenetchargeonanaminoacid—theppKK12+235969..+pl===602.algebraicsumofallthepositivelyandnegatively22chargedgroupspresent—dependsuponthepKavaluesofitsfunctionalgroupsandonthepHofthesurround-Forpolyfunctionalacids,pIisalsothepHmidwaybe-ingmedium.AlteringthechargeonaminoacidsandtweenthepKavaluesoneithersideoftheisoionictheirderivativesbyvaryingthepHfacilitatesthephysi-species.Forexample,thepIforasparticacidiscalseparationofaminoacids,peptides,andproteins(seeChapter4).ppKK12+209396..+pl===302.22Forlysine,pIiscalculatedfrom:RRppKK23+NHNHpl=2NNHHSimilarconsiderationsapplytoallpolyproticacids(eg,proteins),regardlessofthenumberofdissociatinggroupspresent.Intheclinicallaboratory,knowledgeofRRRthepIguidesselectionofconditionsforelectrophoreticNHNHNHseparations.Forexample,electrophoresisatpH7.0willCNH2CNH2CNH2separatetwomoleculeswithpIvaluesof6.0and8.0becauseatpH8.0themoleculewithapIof6.0willNH2NH2NH2haveanetpositivecharge,andthatwithpIof8.0anetnegativecharge.Similarconsiderationsapplytounder-Figure3–2.Resonancehybridsoftheprotonatedstandingchromatographicseparationsonionicsup-formsoftheRgroupsofhistidineandarginine.portssuchasDEAEcellulose(seeChapter4).
2618/CHAPTER3pKaValuesVaryWiththeEnvironmentTHE-RGROUPSDETERMINETHETheenvironmentofadissociablegroupaffectsitspKa.PROPERTIESOFAMINOACIDSThepKavaluesoftheRgroupsoffreeaminoacidsinSinceglycine,thesmallestaminoacid,canbeaccommo-aqueoussolution(Table3–1)thusprovideonlyanap-datedinplacesinaccessibletootheraminoacids,itoftenproximateguidetothepKavaluesofthesameaminooccurswherepeptidesbendsharply.ThehydrophobicRacidswhenpresentinproteins.Apolarenvironment−+groupsofalanine,valine,leucine,andisoleucineandthefavorsthechargedform(R⎯COOorR⎯NH3),aromaticRgroupsofphenylalanine,tyrosine,andtryp-andanonpolarenvironmentfavorstheunchargedformtophantypicallyoccurprimarilyintheinteriorofcy-(R⎯COOHorR⎯NH2).Anonpolarenvironmenttosolicproteins.ThechargedRgroupsofbasicandthusraisesthepKaofacarboxylgroup(makingitaacidicaminoacidsstabilizespecificproteinconforma-weakeracid)butlowersthatofanaminogroup(makingtionsviaionicinteractions,orsaltbonds.Thesebondsitastrongeracid).Thepresenceofadjacentchargedalsofunctionin“chargerelay”systemsduringenzymaticgroupscanreinforceorcounteractsolventeffects.Thecatalysisandelectrontransportinrespiringmitochon-pKaofafunctionalgroupthuswilldependuponitslo-dria.Histidineplaysuniquerolesinenzymaticcatalysis.cationwithinagivenprotein.VariationsinpKacanen-ThepKaofitsimidazoleprotonpermitsittofunctionatcompasswholepHunits(Table3–2).pKavaluesthatneutralpHaseitherabaseoranacidcatalyst.Thepri-divergefromthoselistedbyasmuchasthreepHunitsmaryalcoholgroupofserineandtheprimarythioalco-arecommonattheactivesitesofenzymes.Anextremehol(⎯SH)groupofcysteineareexcellentnucleophilesexample,aburiedasparticacidofthioredoxin,hasaandcanfunctionassuchduringenzymaticcatalysis.pKaabove9—ashiftofoversixpHunits!However,thesecondaryalcoholgroupofthreonine,whileagoodnucleophile,doesnotfulfillananalogousTheSolubilityandMeltingPointsroleincatalysis.The⎯OHgroupsofserine,tyrosine,ofAminoAcidsReflectandthreoninealsoparticipateinregulationoftheactiv-TheirIonicCharacterityofenzymeswhosecatalyticactivitydependsonthephosphorylationstateoftheseresidues.Thechargedfunctionalgroupsofaminoacidsensurethattheyarereadilysolvatedby—andthussolublein—polarsolventssuchaswaterandethanolbutinsolubleFUNCTIONALGROUPSDICTATETHEinnonpolarsolventssuchasbenzene,hexane,orether.CHEMICALREACTIONSOFAMINOACIDSSimilarly,thehighamountofenergyrequiredtodis-Eachfunctionalgroupofanaminoacidexhibitsallofrupttheionicforcesthatstabilizethecrystallatticeitscharacteristicchemicalreactions.Forcarboxylicacidaccountforthehighmeltingpointsofaminoacidsgroups,thesereactionsincludetheformationofesters,(>200°C).amides,andacidanhydrides;foraminogroups,acyla-Aminoacidsdonotabsorbvisiblelightandthusaretion,amidation,andesterification;andfor⎯OHandcolorless.However,tyrosine,phenylalanine,andespe-⎯SHgroups,oxidationandesterification.Themostciallytryptophanabsorbhigh-wavelength(250–290importantreactionofaminoacidsistheformationofanm)ultravioletlight.Tryptophanthereforemakesthepeptidebond(shadedblue).majorcontributiontotheabilityofmostproteinstoabsorblightintheregionof280nm.+HNO3HNO–NTable3–2.TypicalrangeofpKvaluesforHaOOionizablegroupsinproteins.SHAlanylCysteinylValineDissociatingGrouppKaRangeα-Carboxyl3.5–4.0AminoAcidSequenceDeterminesNon-αCOOHofAsporGlu4.0–4.8PrimaryStructureImidazoleofHis6.5–7.4SHofCys8.5–9.0ThenumberandorderofalloftheaminoacidresiduesOHofTyr9.5–10.5inapolypeptideconstituteitsprimarystructure.α-Amino8.0–9.0Aminoacidspresentinpeptidesarecalledaminoacylε-AminoofLys9.8–10.4residuesandarenamedbyreplacingthe-ateor-inesuf-GuanidiniumofArg~12.0fixesoffreeaminoacidswith-yl(eg,alanyl,aspartyl,ty-
27AMINOACIDS&PEPTIDES/19rosyl).PeptidesarethennamedasderivativesoftheSHcarboxylterminalaminoacylresidue.Forexample,Lys-Leu-Tyr-Glniscalledlysyl-leucyl-tyrosyl-glutamine.OCH2HThe-ineendingonglutamineindicatesthatitsα-car-CCHNboxylgroupisnotinvolvedinpeptidebondformation.CHNCCH22HOCOO–PeptideStructuresAreEasytoDrawCH2HCNH+Prefixesliketri-orocta-denotepeptideswiththreeor3eightresidues,respectively,notthosewiththreeorCOO–eightpeptidebonds.Byconvention,peptidesarewrit-tenwiththeresiduethatbearsthefreeα-aminogroupFigure3–3.Glutathione(γ-glutamyl-cysteinyl-attheleft.Todrawapeptide,useazigzagtorepresentglycine).Notethenon-αpeptidebondthatlinksthemainchainorbackbone.Addthemainchainatoms,GlutoCys.whichoccurintherepeatingorder:α-nitrogen,α-car-bon,carbonylcarbon.Nowaddahydrogenatomtoeachα-carbonandtoeachpeptidenitrogen,andanreleasinghormone(TRH)iscyclizedtopyroglutamicoxygentothecarbonylcarbon.Finally,addtheappro-acid,andthecarboxylgroupofthecarboxylterminalpriateRgroups(shaded)toeachα-carbonatom.prolylresidueisamidated.Peptideselaboratedbyfungi,bacteria,andloweranimalscancontainnonproteinNNCCαCaminoacids.TheantibioticstyrocidinandgramicidinSCαNCCαarecyclicpolypeptidesthatcontainD-phenylalanineandornithine.TheheptapeptideopioidsdermorphinOHCH3anddeltophorinintheskinofSouthAmericantreeH+HNCCNCOO–3frogscontainD-tyrosineandD-alanine.CNCCHHHCH2CH2OPeptidesArePolyelectrolytes–OOCOHThepeptidebondisunchargedatanypHofphysiologicinterest.FormationofpeptidesfromaminoacidsisThree-letterabbreviationslinkedbystraightlinesthereforeaccompaniedbyanetlossofonepositiveandrepresentanunambiguousprimarystructure.Linesareonenegativechargeperpeptidebondformed.Peptidesomittedforsingle-letterabbreviations.neverthelessarechargedatphysiologicpHowingtotheirGlu-Ala-Lys-Gly-Tyr-Alacarboxylandaminoterminalgroupsand,wherepresent,theiracidicorbasicRgroups.Asforaminoacids,thenetEAKGYAchargeonapeptidedependsonthepHofitsenviron-WherethereisuncertaintyabouttheorderofaportionmentandonthepKavaluesofitsdissociatinggroups.ofapolypeptide,thequestionableresiduesareenclosedinbracketsandseparatedbycommas.ThePeptideBondHasPartialGluLysAlaGlyTyrHisAla--(,,)--Double-BondCharacterAlthoughpeptidesarewrittenasifasinglebondlinkedSomePeptidesContainUnusualtheα-carboxylandα-nitrogenatoms,thisbondinfactexhibitspartialdouble-bondcharacter:AminoAcidsOO–Inmammals,peptidehormonestypicallycontainonlytheα-aminoacidsofproteinslinkedbystandardpep-CC+tidebonds.Otherpeptidesmay,however,containnon-NNproteinaminoacids,derivativesoftheproteinaminoHHacids,oraminoacidslinkedbyanatypicalpeptidebond.Forexample,theaminoterminalglutamateofTherethusisnofreedomofrotationaboutthebondglutathione,whichparticipatesinproteinfoldingandthatconnectsthecarbonylcarbonandthenitrogenofainthemetabolismofxenobiotics(Chapter53),ispeptidebond.Consequently,allfourofthecoloredlinkedtocysteinebyanon-αpeptidebond(FigureatomsofFigure3–4arecoplanar.Theimposedsemi-3–3).Theaminoterminalglutamateofthyrotropin-rigidityofthepeptidebondhasimportantconse-
2820/CHAPTER3mixtureoffreeaminoacidsisthentreatedwith6-amino-OR′HHON-hydroxysuccinimidylcarbamate,whichreactswith0.123nmtheirα-aminogroups,formingfluorescentderivativesthatarethenseparatedandidentifiedusinghigh-pressure121°122°0.132nmCCNCliquidchromatography(seeChapter5).Ninhydrin,also120°0.147nmwidelyusedfordetectingaminoacids,formsapurple117°N110°CCN120°120°0.153nmproductwithα-aminoacidsandayellowadductwith0.1nmtheiminegroupsofprolineandhydroxyproline.HHOHR′′SUMMARY0.36nm•BothD-aminoacidsandnon-α-aminoacidsoccurinnature,butonlyL-α-aminoacidsarepresentinFigure3–4.Dimensionsofafullyextendedpoly-proteins.peptidechain.Thefouratomsofthepeptidebond•Allaminoacidspossessatleasttwoweaklyacidic(coloredblue)arecoplanar.Theunshadedatomsare+functionalgroups,R⎯NH3andR⎯COOH.theα-carbonatom,theα-hydrogenatom,andtheα-RManyalsopossessadditionalweaklyacidicfunctionalgroupoftheparticularaminoacid.Freerotationcangroupssuchas⎯OH,⎯SH,guanidino,orimid-occuraboutthebondsthatconnecttheα-carbonwithazolegroups.theα-nitrogenandwiththeα-carbonylcarbon(blue•ThepKavaluesofallfunctionalgroupsofanaminoarrows).Theextendedpolypeptidechainisthusasemi-aciddictateitsnetchargeatagivenpH.pIisthepHrigidstructurewithtwo-thirdsoftheatomsoftheback-atwhichanaminoacidbearsnonetchargeandthusboneheldinafixedplanarrelationshiponetoanother.doesnotmoveinadirectcurrentelectricalfield.Thedistancebetweenadjacentα-carbonatomsis0.36•Ofthebiochemicalreactionsofaminoacids,thenm(3.6Å).Theinteratomicdistancesandbondangles,mostimportantistheformationofpeptidebonds.whicharenotequivalent,arealsoshown.(Redrawnand•TheRgroupsofaminoacidsdeterminetheiruniquereproduced,withpermission,fromPaulingL,CoreyLP,biochemicalfunctions.AminoacidsareclassifiedasBransonHR:Thestructureofproteins:Twohydrogen-basic,acidic,aromatic,aliphatic,orsulfur-containingbondedhelicalconfigurationsofthepolypeptidechain.basedonthepropertiesoftheirRgroups.ProcNatlAcadSciUSA1951;37:205.)•Peptidesarenamedforthenumberofaminoacidresiduespresent,andasderivativesofthecarboxylquencesforhigherordersofproteinstructure.Encir-terminalresidue.Theprimarystructureofapeptideclingarrows(Figure3–4)indicatefreerotationaboutisitsaminoacidsequence,startingfromtheamino-theremainingbondsofthepolypeptidebackbone.terminalresidue.•Thepartialdouble-bondcharacterofthebondthatNoncovalentForcesConstrainPeptidelinksthecarbonylcarbonandthenitrogenofapep-Conformationstiderendersfouratomsofthepeptidebondcoplanarandrestrictsthenumberofpossiblepeptideconfor-Foldingofapeptideprobablyoccurscoincidentwithmations.itsbiosynthesis(seeChapter38).Thephysiologicallyactiveconformationreflectstheaminoacidsequence,REFERENCESsterichindrance,andnoncovalentinteractions(eg,hy-drogenbonding,hydrophobicinteractions)betweenDoolittleRF:Reconstructinghistorywithaminoacidsequences.residues.Commonconformationsincludeα-helicesProteinSci1992;1:191.andβ-pleatedsheets(seeChapter5).KreilG:D-Aminoacidsinanimalpeptides.AnnuRevBiochem1997;66:337.NokiharaK,GerhardtJ:DevelopmentofanimprovedautomatedANALYSISOFTHEAMINOACIDgas-chromatographicchiralanalysissystem:applicationtoCONTENTOFBIOLOGICMATERIALSnon-naturalaminoacidsandnaturalproteinhydrolysates.Chirality2001;13:431.InordertodeterminetheidentityandquantityofeachSangerF:Sequences,sequences,andsequences.AnnuRevBiochemaminoacidinasampleofbiologicmaterial,itisfirstnec-1988;57:1.essarytohydrolyzethepeptidebondsthatlinktheaminoWilsonNAetal:Asparticacid26inreducedEscherichiacolithiore-acidstogetherbytreatmentwithhotHCl.TheresultingdoxinhasapKagreaterthan9.Biochemistry1995;34:8931.
29Proteins:DeterminationofPrimaryStructure4VictorW.Rodwell,PhD,&PeterJ.Kennelly,PhDBIOMEDICALIMPORTANCEColumnChromatographyProteinsperformmultiplecriticallyimportantroles.AnColumnchromatographyofproteinsemploysastheinternalproteinnetwork,thecytoskeleton(Chapterstationaryphaseacolumncontainingsmallspherical49),maintainscellularshapeandphysicalintegrity.beadsofmodifiedcellulose,acrylamide,orsilicawhoseActinandmyosinfilamentsformthecontractilema-surfacetypicallyhasbeencoatedwithchemicalfunc-chineryofmuscle(Chapter49).Hemoglobintrans-tionalgroups.Thesestationaryphasematricesinteractportsoxygen(Chapter6),whilecirculatingantibodieswithproteinsbasedontheircharge,hydrophobicity,searchoutforeigninvaders(Chapter50).Enzymescat-andligand-bindingproperties.Aproteinmixtureisap-alyzereactionsthatgenerateenergy,synthesizeandde-pliedtothecolumnandtheliquidmobilephaseisper-gradebiomolecules,replicateandtranscribegenes,colatedthroughit.SmallportionsofthemobilephaseprocessmRNAs,etc(Chapter7).Receptorsenablecellsoreluantarecollectedastheyemerge(Figure4–1).tosenseandrespondtohormonesandotherenviron-mentalcues(Chapters42and43).AnimportantgoalPartitionChromatographyofmolecularmedicineistheidentificationofproteinswhosepresence,absence,ordeficiencyisassociatedColumnchromatographicseparationsdependonthewithspecificphysiologicstatesordiseases.Theprimaryrelativeaffinityofdifferentproteinsforagivenstation-sequenceofaproteinprovidesbothamolecularfinger-aryphaseandforthemobilephase.Associationbe-printforitsidentificationandinformationthatcanbetweeneachproteinandthematrixisweakandtran-usedtoidentifyandclonethegeneorgenesthaten-sient.Proteinsthatinteractmorestronglywiththecodeit.stationaryphaseareretainedlonger.ThelengthoftimethataproteinisassociatedwiththestationaryphaseisafunctionofthecompositionofboththestationaryandPROTEINS&PEPTIDESMUSTBEmobilephases.Optimalseparationoftheproteinofin-terestfromotherproteinsthuscanbeachievedbycare-PURIFIEDPRIORTOANALYSISfulmanipulationofthecompositionofthetwophases.Highlypurifiedproteinisessentialfordeterminationofitsaminoacidsequence.Cellscontainthousandsofdif-SizeExclusionChromatographyferentproteins,eachinwidelyvaryingamounts.TheisolationofaspecificproteininquantitiessufficientforSizeexclusion—orgelfiltration—chromatographysep-analysisthuspresentsaformidablechallengethatmayaratesproteinsbasedontheirStokesradius,thediam-requiremultiplesuccessivepurificationtechniques.eterofthespheretheyoccupyastheytumbleinsolu-Classicapproachesexploitdifferencesinrelativesolu-tion.TheStokesradiusisafunctionofmolecularmassbilityofindividualproteinsasafunctionofpH(iso-andshape.Atumblingelongatedproteinoccupiesaelectricprecipitation),polarity(precipitationwithlargervolumethanasphericalproteinofthesamemass.ethanoloracetone),orsaltconcentration(saltingoutSizeexclusionchromatographyemploysporousbeadswithammoniumsulfate).Chromatographicseparations(Figure4–2).Theporesareanalogoustoindentationspartitionmoleculesbetweentwophases,onemobileinariverbank.Asobjectsmovedownstream,thosethatandtheotherstationary.Forseparationofaminoacidsenteranindentationareretardeduntiltheydriftbackorsugars,thestationaryphase,ormatrix,maybeaintothemaincurrent.Similarly,proteinswithStokessheetoffilterpaper(paperchromatography)orathinradiitoolargetoenterthepores(excludedproteins)re-layerofcellulose,silica,oralumina(thin-layerchro-mainintheflowingmobilephaseandemergebeforematography;TLC).proteinsthatcanenterthepores(includedproteins).21
3022/CHAPTER4RCFFigure4–1.Componentsofasimpleliquidchromatographyapparatus.R:Reser-voirofmobilephaseliquid,deliveredeitherbygravityorusingapump.C:Glassorplasticcolumncontainingstationaryphase.F:Fractioncollectorforcollectingpor-tions,calledfractions,oftheeluantliquidinseparatetesttubes.Proteinsthusemergefromagelfiltrationcolumninde-IonExchangeChromatographyscendingorderoftheirStokesradii.Inionexchangechromatography,proteinsinteractwiththestationaryphasebycharge-chargeinteractions.Pro-AbsorptionChromatographyteinswithanetpositivechargeatagivenpHadheretoForabsorptionchromatography,theproteinmixtureisbeadswithnegativelychargedfunctionalgroupssuchasappliedtoacolumnunderconditionswherethepro-carboxylatesorsulfates(cationexchangers).Similarly,teinofinterestassociateswiththestationaryphasesoproteinswithanetnegativechargeadheretobeadswithtightlythatitspartitioncoefficientisessentiallyunity.positivelychargedfunctionalgroups,typicallytertiaryorNonadheringmoleculesarefirstelutedanddiscarded.quaternaryamines(anionexchangers).Proteins,whichProteinsarethensequentiallyreleasedbydisruptingthearepolyanions,competeagainstmonovalentionsforforcesthatstabilizetheprotein-stationaryphasecom-bindingtothesupport—thustheterm“ionexchange.”plex,mostoftenbyusingagradientofincreasingsaltForexample,proteinsbindtodiethylaminoethylconcentration.Thecompositionofthemobilephaseis(DEAE)cellulosebyreplacingthecounter-ions(gener-−−alteredgraduallysothatmoleculesareselectivelyre-allyClorCH3COO)thatneutralizetheprotonatedleasedindescendingorderoftheiraffinityforthesta-amine.Boundproteinsareselectivelydisplacedbygrad-tionaryphase.uallyraisingtheconcentrationofmonovalentionsin
31PROTEINS:DETERMINATIONOFPRIMARYSTRUCTURE/23ABCFigure4–2.Size-exclusionchromatography.A:Amixtureoflargemolecules(diamonds)andsmallmolecules(circles)areappliedtothetopofagelfiltrationcolumn.B:Uponenteringthecolumn,thesmallmoleculesenterporesinthesta-tionaryphasematrixfromwhichthelargemoleculesareexcluded.C:Asthemo-bilephaseflowsdownthecolumn,thelarge,excludedmoleculesflowwithitwhilethesmallmolecules,whicharetemporarilyshelteredfromtheflowwhenin-sidethepores,lagfartherandfartherbehind.themobilephase.Proteinseluteininverseorderofthefiedbyaffinitychromatographyusingimmobilizedsub-strengthoftheirinteractionswiththestationaryphase.strates,products,coenzymes,orinhibitors.Intheory,SincethenetchargeonaproteinisdeterminedbyonlyproteinsthatinteractwiththeimmobilizedligandthepH(seeChapter3),sequentialelutionofproteinsadhere.Boundproteinsarethenelutedeitherbycompe-maybeachievedbychangingthepHofthemobiletitionwithsolubleligandor,lessselectively,bydisrupt-phase.Alternatively,aproteincanbesubjectedtocon-ingprotein-ligandinteractionsusingurea,guanidinesecutiveroundsofionexchangechromatography,eachhydrochloride,mildlyacidicpH,orhighsaltconcentra-atadifferentpH,suchthatproteinsthatco-eluteatonetions.StationaryphasematricesavailablecommerciallypHeluteatdifferentsaltconcentrationsatanotherpH.containligandssuchasNAD+orATPanalogs.Amongthemostpowerfulandwidelyapplicableaffinitymatri-HydrophobicInteractionChromatographycesarethoseusedforthepurificationofsuitablymodi-2+fiedrecombinantproteins.TheseincludeaNimatrixHydrophobicinteractionchromatographyseparatesthatbindsproteinswithanattachedpolyhistidine“tag”proteinsbasedontheirtendencytoassociatewithasta-andaglutathionematrixthatbindsarecombinantpro-tionaryphasematrixcoatedwithhydrophobicgroupsteinlinkedtoglutathioneS-transferase.(eg,phenylSepharose,octylSepharose).ProteinswithexposedhydrophobicsurfacesadheretothematrixviahydrophobicinteractionsthatareenhancedbyamobilePeptidesArePurifiedbyReversed-Phasephaseofhighionicstrength.NonadherentproteinsareHigh-PressureChromatographyfirstwashedaway.Thepolarityofthemobilephaseisthendecreasedbygraduallyloweringthesaltconcentra-Thestationaryphasematricesusedinclassiccolumntion.Iftheinteractionbetweenproteinandstationarychromatographyarespongymaterialswhosecompress-phaseisparticularlystrong,ethanolorglycerolmaybeibilitylimitsflowofthemobilephase.High-pressureliq-addedtothemobilephasetodecreaseitspolarityanduidchromatography(HPLC)employsincompressiblefurtherweakenhydrophobicinteractions.silicaoraluminamicrobeadsasthestationaryphaseandpressuresofuptoafewthousandpsi.Incompressiblematricespermitbothhighflowratesandenhancedreso-AffinityChromatographylution.HPLCcanresolvecomplexmixturesoflipidsorAffinitychromatographyexploitsthehighselectivityofpeptideswhosepropertiesdifferonlyslightly.Reversed-mostproteinsfortheirligands.Enzymesmaybepuri-phaseHPLCexploitsahydrophobicstationaryphaseof
3224/CHAPTER4aliphaticpolymers3–18carbonatomsinlength.Peptidethroughtheacrylamidematrixdeterminestherateofmixturesareelutedusingagradientofawater-misciblemigration.Sincelargecomplexesencountergreaterre-organicsolventsuchasacetonitrileormethanol.sistance,polypeptidesseparatebasedontheirrelativemolecularmass(Mr).IndividualpolypeptidestrappedProteinPurityIsAssessedbyintheacrylamidegelarevisualizedbystainingwithPolyacrylamideGelElectrophoresisdyessuchasCoomassieblue(Figure4–4).(PAGE)IsoelectricFocusing(IEF)Themostwidelyusedmethodfordeterminingthepu-rityofaproteinisSDS-PAGE—polyacrylamidegelIonicbufferscalledampholytesandanappliedelectricelectrophoresis(PAGE)inthepresenceoftheanionicfieldareusedtogenerateapHgradientwithinapoly-detergentsodiumdodecylsulfate(SDS).Electrophore-acrylamidematrix.AppliedproteinsmigrateuntiltheysisseparateschargedbiomoleculesbasedontheratesatreachtheregionofthematrixwherethepHmatcheswhichtheymigrateinanappliedelectricalfield.Fortheirisoelectricpoint(pI),thepHatwhichapeptide’sSDS-PAGE,acrylamideispolymerizedandcross-netchargeiszero.IEFisusedinconjunctionwithSDS-linkedtoformaporousmatrix.SDSdenaturesandPAGEfortwo-dimensionalelectrophoresis,whichsepa-bindstoproteinsataratioofonemoleculeofSDSperratespolypeptidesbasedonpIinonedimensionandtwopeptidebonds.Whenusedinconjunctionwith2-basedonMrinthesecond(Figure4–5).Two-dimen-mercaptoethanolordithiothreitoltoreduceandbreaksionalelectrophoresisisparticularlywellsuitedforsepa-disulfidebonds(Figure4–3),SDSseparatesthecom-ratingthecomponentsofcomplexmixturesofproteins.ponentpolypeptidesofmultimericproteins.ThelargenumberofanionicSDSmolecules,eachbearingaSANGERWASTHEFIRSTTODETERMINEchargeof−1,oneachpolypeptideoverwhelmstheTHESEQUENCEOFAPOLYPEPTIDEchargecontributionsoftheaminoacidfunctionalgroups.Sincethecharge-to-massratioofeachSDS-Matureinsulinconsistsofthe21-residueAchainandpolypeptidecomplexisapproximatelyequal,thephysi-the30-residueBchainlinkedbydisulfidebonds.Fred-calresistanceeachpeptideencountersasitmoveserickSangerreducedthedisulfidebonds(Figure4–3),NHOHNHSOHNSHONHOOSHHCOOHCH25OHNHOHHNSO−2OHNOHSHNHOFigure4–4.UseofSDS-PAGEtoobservesuccessivepurificationofarecombinantprotein.ThegelwasFigure4–3.Oxidativecleavageofadjacentpolypep-stainedwithCoomassieblue.Shownareproteinstan-tidechainslinkedbydisulfidebonds(shaded)byper-dards(laneS)oftheindicatedmass,crudecellextractformicacid(left)orreductivecleavagebyβ-mercap-(E),high-speedsupernatantliquid(H),andtheDEAE-toethanol(right)formstwopeptidesthatcontainSepharosefraction(D).Therecombinantproteinhasacysteicacidresiduesorcysteinylresidues,respectively.massofabout45kDa.
33PROTEINS:DETERMINATIONOFPRIMARYSTRUCTURE/25pH=3pH=10IEFSDSPAGEFigure4–5.Two-dimensionalIEF-SDS-PAGE.ThegelwasstainedwithCoomassieblue.Acrudebacter-ialextractwasfirstsubjectedtoisoelectricfocusing(IEF)inapH3–10gradient.TheIEFgelwasthenplacedhorizontallyonthetopofanSDSgel,andtheproteinsthenfurtherresolvedbySDS-PAGE.Noticethegreatlyimprovedresolutionofdistinctpolypep-tidesrelativetoordinarySDS-PAGEgel(Figure4–4).separatedtheAandBchains,andcleavedeachchainLargePolypeptidesAreFirstCleavedIntointosmallerpeptidesusingtrypsin,chymotrypsin,andSmallerSegmentspepsin.Theresultingpeptideswerethenisolatedandtreatedwithacidtohydrolyzepeptidebondsandgener-Whilethefirst20–30residuesofapeptidecanreadilyatepeptideswithasfewastwoorthreeaminoacids.bedeterminedbytheEdmanmethod,mostpolypep-Eachpeptidewasreactedwith1-fluoro-2,4-dinitroben-tidescontainseveralhundredaminoacids.Conse-zene(Sanger’sreagent),whichderivatizestheexposedquently,mostpolypeptidesmustfirstbecleavedintoα-aminogroupofaminoterminalresidues.TheaminosmallerpeptidespriortoEdmansequencing.Cleavageacidcontentofeachpeptidewasthendetermined.alsomaybenecessarytocircumventposttranslationalWhiletheε-aminogroupoflysinealsoreactswithmodificationsthatrenderaprotein’sα-aminogroupSanger’sreagent,amino-terminallysinescanbedistin-“blocked”,orunreactivewiththeEdmanreagent.guishedfromthoseatotherpositionsbecausetheyreactItusuallyisnecessarytogenerateseveralpeptideswith2molofSanger’sreagent.Workingbackwardstousingmorethanonemethodofcleavage.ThisreflectslargerfragmentsenabledSangertodeterminethecom-bothinconsistencyinthespacingofchemicallyorenzy-pletesequenceofinsulin,anaccomplishmentforwhichmaticallysusceptiblecleavagesitesandtheneedforsetshereceivedaNobelPrizein1958.ofpeptideswhosesequencesoverlapsoonecaninferthesequenceofthepolypeptidefromwhichtheyderive(Figure4–7).ReagentsforthechemicalorenzymaticTHEEDMANREACTIONENABLEScleavageofproteinsincludecyanogenbromide(CNBr),PEPTIDES&PROTEINStrypsin,andStaphylococcusaureusV8protease(TableTOBESEQUENCED4–1).Followingcleavage,theresultingpeptidesarepu-rifiedbyreversed-phaseHPLC—oroccasionallybyPehrEdmanintroducedphenylisothiocyanate(Edman’sSDS-PAGE—andsequenced.reagent)toselectivelylabeltheamino-terminalresidueofapeptide.IncontrasttoSanger’sreagent,thephenylthiohydantoin(PTH)derivativecanberemovedMOLECULARBIOLOGYHASundermildconditionstogenerateanewaminoterminalREVOLUTIONIZEDTHEDETERMINATIONresidue(Figure4–6).SuccessiveroundsofderivatizationOFPRIMARYSTRUCTUREwithEdman’sreagentcanthereforebeusedtosequencemanyresiduesofasinglesampleofpeptide.Edmanse-KnowledgeofDNAsequencespermitsdeductionofquencinghasbeenautomated,usingathinfilmorsolidtheprimarystructuresofpolypeptides.DNAsequenc-matrixtoimmobilizethepeptideandHPLCtoidentifyingrequiresonlyminuteamountsofDNAandcanPTHaminoacids.Moderngas-phasesequencerscanreadilyyieldthesequenceofhundredsofnucleotides.analyzeaslittleasafewpicomolesofpeptide.TocloneandsequencetheDNAthatencodesapartic-
3426/CHAPTER4SPeptideXPeptideYCPeptideZN+OHNH2CarboxylterminalAminoterminalNportionofportionofpeptideXpeptideYNRHOR′Figure4–7.TheoverlappingpeptideZisusedtode-Phenylisothiocyanate(Edmanreagent)ducethatpeptidesXandYarepresentintheoriginalandapeptideproteinintheorderX→Y,notY←X.sequencecanbedeterminedandthegeneticcodeusedtoinfertheprimarystructureoftheencodedpoly-Speptide.Thehybridapproachenhancesthespeedandeffi-NNHciencyofprimarystructureanalysisandtherangeofHproteinsthatcanbesequenced.Italsocircumventsob-OHstaclessuchasthepresenceofanamino-terminalblock-Ninggrouporthelackofakeyoverlappeptide.OnlyaNRfewsegmentsofprimarystructuremustbedeterminedHOR′byEdmananalysis.AphenylthiohydantoicacidDNAsequencingrevealstheorderinwhichaminoacidsareaddedtothenascentpolypeptidechainasitisH+,nitro-HOsynthesizedontheribosomes.However,itprovidesno2methaneinformationaboutposttranslationalmodificationssuchasproteolyticprocessing,methylation,glycosylation,SOphosphorylation,hydroxylationofprolineandlysine,NH2anddisulfidebondformationthataccompanymatura-NNH+Ntion.WhileEdmansequencingcandetectthepresenceHRofmostposttranslationalevents,technicallimitationsORoftenpreventidentificationofaspecificmodification.AphenylthiohydantoinandapeptideshorterbyoneresidueTable4–1.Methodsforcleavingpolypeptides.Figure4–6.TheEdmanreaction.Phenylisothio-cyanatederivatizestheamino-terminalresidueofaMethodBondCleavedpeptideasaphenylthiohydantoicacid.Treatmentwithacidinanonhydroxylicsolventreleasesaphenylthio-CNBrMet-Xhydantoin,whichissubsequentlyidentifiedbyitschro-TrypsinLys-XandArg-Xmatographicmobility,andapeptideoneresidueChymotrypsinHydrophobicaminoacid-Xshorter.Theprocessisthenrepeated.EndoproteinaseLys-CLys-XEndoproteinaseArg-CArg-Xularprotein,somemeansofidentifyingthecorrectclone—eg,knowledgeofaportionofitsnucleotidese-EndoproteinaseAsp-NX-Aspquence—isessential.AhybridapproachthushasV8proteaseGlu-X,particularlywhereXishydro-emerged.Edmansequencingisusedtoprovideapartialphobicaminoacidsequence.OligonucleotideprimersmodeledonthispartialsequencecanthenbeusedtoidentifyHydroxylamineAsn-Glyclonesortoamplifytheappropriategenebythepoly-o-IodosobenzeneTrp-Xmerasechainreaction(PCR)(seeChapter40).OnceanMildacidAsp-ProauthenticDNAcloneisobtained,itsoligonucleotide
35PROTEINS:DETERMINATIONOFPRIMARYSTRUCTURE/27MASSSPECTROMETRYDETECTSCOVALENTMODIFICATIONSMassspectrometry,whichdiscriminatesmoleculesbasedsolelyontheirmass,isidealfordetectingtheSAphosphate,hydroxyl,andothergroupsonposttransla-tionallymodifiedaminoacids.Eachaddsaspecificandreadilyidentifiedincrementofmasstothemodifiedaminoacid(Table4–2).Foranalysisbymassspec-trometry,asampleinavacuumisvaporizedunderEconditionswhereprotonationcanoccur,impartingDpositivecharge.AnelectricalfieldthenpropelsthecationsthroughamagneticfieldwhichdeflectsthemFigure4–8.Basiccomponentsofasimplemassatarightangletotheiroriginaldirectionofflightandspectrometer.Amixtureofmoleculesisvaporizedinanfocusesthemontoadetector(Figure4–8).Themag-ionizedstateinthesamplechamberS.Thesemole-neticforcerequiredtodeflectthepathofeachionicculesarethenaccelerateddowntheflighttubebyanspeciesontothedetector,measuredasthecurrentap-electricalpotentialappliedtoacceleratorgridA.Anad-pliedtotheelectromagnet,isrecorded.Forionsofjustableelectromagnet,E,appliesamagneticfieldthatidenticalnetcharge,thisforceisproportionatetotheirdeflectstheflightoftheindividualionsuntiltheystrikemass.Inatime-of-flightmassspectrometer,abrieflyappliedelectricfieldacceleratestheionstowardsade-thedetector,D.Thegreaterthemassoftheion,thetectorthatrecordsthetimeatwhicheachionarrives.higherthemagneticfieldrequiredtofocusitontotheFormoleculesofidenticalcharge,thevelocitytowhichdetector.theyareaccelerated—andhencethetimerequiredtoreachthedetector—willbeinverselyproportionatetotheirmass.ConventionalmassspectrometersgenerallyareusedphaseHPLCcolumnareintroduceddirectlyintothetodeterminethemassesofmoleculesof1000Daormassspectrometerforimmediatedeterminationofless,whereastime-of-flightmassspectrometersaretheirmasses.suitedfordeterminingthelargemassesofproteins.PeptidesinsidethemassspectrometerarebrokenTheanalysisofpeptidesandproteinsbymassspec-downintosmallerunitsbycollisionswithneutralhe-tometryinitiallywashinderedbydifficultiesinliumatoms(collision-induceddissociation),andthevolatilizinglargeorganicmolecules.However,matrix-massesoftheindividualfragmentsaredetermined.assistedlaser-desorption(MALDI)andelectrospraySincepeptidebondsaremuchmorelabilethancarbon-dispersion(eg,nanospray)permitthemassesofevencarbonbonds,themostabundantfragmentswilldifferlargepolypeptides(>100,000Da)tobedeterminedfromoneanotherbyunitsequivalenttooneortwowithextraordinaryaccuracy(±1Da).Usingelectro-aminoacids.Since—withtheexceptionofleucineandspraydispersion,peptideselutingfromareversed-isoleucine—themolecularmassofeachaminoacidisunique,thesequenceofthepeptidecanberecon-structedfromthemassesofitsfragments.Table4–2.Massincreasesresultingfromcommonposttranslationalmodifications.TandemMassSpectrometryComplexpeptidemixturescannowbeanalyzedwith-ModificationMassIncrease(Da)outpriorpurificationbytandemmassspectrometry,Phosphorylation80whichemploystheequivalentoftwomassspectrome-terslinkedinseries.Thefirstspectrometerseparatesin-Hydroxylation16dividualpeptidesbasedupontheirdifferencesinmass.Methylation14Byadjustingthefieldstrengthofthefirstmagnet,asin-glepeptidecanbedirectedintothesecondmassspec-Acetylation42trometer,wherefragmentsaregeneratedandtheirMyristylation210massesdetermined.AsthesensitivityandversatilityofPalmitoylation238massspectrometrycontinuetoincrease,itisdisplacingEdmansequencersforthedirectanalysisofproteinpri-Glycosylation162marystructure.
3628/CHAPTER4GENOMICSENABLESPROTEINSTOBEinthehemoglobintetramerundergochangepre-andIDENTIFIEDFROMSMALLAMOUNTSpostpartum.ManyproteinsundergoposttranslationalOFSEQUENCEDATAmodificationsduringmaturationintofunctionallycompetentformsorasameansofregulatingtheirprop-Primarystructureanalysishasbeenrevolutionizedbyerties.Knowledgeofthehumangenomethereforerep-genomics,theapplicationofautomatedoligonucleotideresentsonlythebeginningofthetaskofdescribingliv-sequencingandcomputerizeddataretrievalandanalysisingorganismsinmoleculardetailandunderstandingtosequenceanorganism’sentiregeneticcomplement.thedynamicsofprocessessuchasgrowth,aging,andThefirstgenomesequencedwasthatofHaemophilusdisease.Asthehumanbodycontainsthousandsofcellinfluenzae,in1995.Bymid2001,thecompletetypes,eachcontainingthousandsofproteins,thepro-genomesequencesforover50organismshadbeende-teome—thesetofalltheproteinsexpressedbyanindi-termined.Theseincludethehumangenomeandthosevidualcellataparticulartime—representsamovingofseveralbacterialpathogens;theresultsandsignifi-targetofformidabledimensions.canceoftheHumanGenomeProjectarediscussedinChapter54.Wheregenomesequenceisknown,theTwo-DimensionalElectrophoresis&taskofdeterminingaprotein’sDNA-derivedprimaryGeneArrayChipsAreUsedtoSurveysequenceismateriallysimplified.Inessence,thesecondProteinExpressionhalfofthehybridapproachhasalreadybeencom-pleted.Allthatremainsistoacquiresufficientinforma-Onegoalofproteomicsistheidentificationofproteinstiontopermittheopenreadingframe(ORF)thatwhoselevelsofexpressioncorrelatewithmedicallysig-encodestheproteintoberetrievedfromanInternet-nificantevents.Thepresumptionisthatproteinswhoseaccessiblegenomedatabaseandidentified.Insomeappearanceordisappearanceisassociatedwithaspecificcases,asegmentofaminoacidsequenceonlyfourorphysiologicconditionordiseasewillprovideinsightsfiveresiduesinlengthmaybesufficienttoidentifytheintorootcausesandmechanisms.DeterminationofthecorrectORF.proteomescharacteristicofeachcelltyperequirestheComputerizedsearchalgorithmsassisttheidentifi-utmostefficiencyintheisolationandidentificationofcationofthegeneencodingagivenproteinandclarifyindividualproteins.Thecontemporaryapproachuti-uncertaintiesthatarisefromEdmansequencingandlizesroboticautomationtospeedsamplepreparationmassspectrometry.Byexploitingcomputerstosolveandlargetwo-dimensionalgelstoresolvecellularpro-complexpuzzles,thespectrumofinformationsuitableteins.IndividualpolypeptidesarethenextractedandforidentificationoftheORFthatencodesaparticularanalyzedbyEdmansequencingormassspectroscopy.polypeptideisgreatlyexpanded.Inpeptidemassprofil-Whileonlyabout1000proteinscanberesolvedonaing,forexample,apeptidedigestisintroducedintothesinglegel,two-dimensionalelectrophoresishasamajormassspectrometerandthesizesofthepeptidesarede-advantageinthatitexaminestheproteinsthemselves.termined.AcomputeristhenusedtofindanORFAnalternativeandcomplementaryapproachemployswhosepredictedproteinproductwould,ifbrokengenearrays,sometimescalledDNAchips,todetectthedownintopeptidesbythecleavagemethodselected,expressionofthemRNAswhichencodeproteins.produceasetofpeptideswhosemassesmatchthoseob-WhilechangesintheexpressionofthemRNAencod-servedbymassspectrometry.ingaproteindonotnecessarilyreflectcomparablechangesinthelevelofthecorrespondingprotein,genePROTEOMICS&THEPROTEOMEarraysaremoresensitiveprobesthantwo-dimensionalgelsandthuscanexaminemoregeneproducts.TheGoalofProteomicsIstoIdentifytheEntireComplementofProteinsElaboratedBioinformaticsAssistsIdentificationbyaCellUnderDiverseConditionsofProteinFunctionsWhilethesequenceofthehumangenomeisknown,Thefunctionsofalargeproportionoftheproteinsen-thepictureprovidedbygenomicsaloneisbothstaticcodedbythehumangenomearepresentlyunknown.andincomplete.ProteomicsaimstoidentifytheentireRecentadvancesinbioinformaticspermitresearcherstocomplementofproteinselaboratedbyacellunderdi-compareaminoacidsequencestodiscovercluestopo-verseconditions.Asgenesareswitchedonandoff,pro-tentialproperties,physiologicroles,andmechanismsofteinsaresynthesizedinparticularcelltypesatspecificactionofproteins.Algorithmsexploitthetendencyoftimesofgrowthordifferentiationandinresponsetonaturetoemployvariationsofastructuralthemetoexternalstimuli.Musclecellsexpressproteinsnotex-performsimilarfunctionsinseveralproteins(eg,thepressedbyneuralcells,andthetypeofsubunitspresentRossmannnucleotidebindingfoldtobindNAD(P)H,
37PROTEINS:DETERMINATIONOFPRIMARYSTRUCTURE/29nucleartargetingsequences,andEFhandstobind•Scientistsarenowtryingtodeterminetheprimary2+Ca).Thesedomainsgenerallyaredetectedinthepri-sequenceandfunctionalroleofeveryproteinex-marystructurebyconservationofparticularaminopressedinalivingcell,knownasitsproteome.acidsatkeypositions.Insightsintothepropertiesand•Amajorgoalistheidentificationofproteinswhosephysiologicroleofanewlydiscoveredproteinthusmayappearanceordisappearancecorrelateswithphysio-beinferredbycomparingitsprimarystructurewithlogicphenomena,aging,orspecificdiseases.thatofknownproteins.REFERENCESSUMMARYDeutscherMP(editor):GuidetoProteinPurification.MethodsEn-•Longaminoacidpolymersorpolypeptidesconstitutezymol1990;182.(Entirevolume.)thebasicstructuralunitofproteins,andthestructureGeveartK,VandekerckhoveJ:Proteinidentificationmethodsinofaproteinprovidesinsightintohowitfulfillsitsproteomics.Electrophoresis2000;21:1145.functions.HelmuthL:Genomeresearch:mapofthehumangenome3.0.Sci-•TheEdmanreactionenabledaminoacidsequenceence2001;293:583.analysistobeautomated.Massspectrometrypro-KhanJetal:DNAmicroarraytechnology:theanticipatedimpactvidesasensitiveandversatiletoolfordeterminingonthestudyofhumandisease.BiochimBiophysActa1999;1423:M17.primarystructureandfortheidentificationofpost-translationalmodifications.McLaffertyFWetal:Biomoleculemassspectrometry.Science1999;284:1289.•DNAcloningandmolecularbiologycoupledwithPatnaikSK,BlumenfeldOO:Useofon-linetoolsanddatabasesforproteinchemistryprovideahybridapproachthatroutinesequenceanalyses.AnalBiochem2001;289:1.greatlyincreasesthespeedandefficiencyfordetermi-SchenaMetal:Quantitativemonitoringofgeneexpressionpat-nationofprimarystructuresofproteins.ternswithacomplementaryDNAmicroarray.Science•Genomics—theanalysisoftheentireoligonucleotide1995;270:467.sequenceofanorganism’scompletegeneticmater-SemsarianC,SeidmanCE:Molecularmedicineinthe21stcen-ial—hasprovidedfurtherenhancements.tury.InternMedJ2001;31:53.TempleLKetal:Essaysonscienceandsociety:definingdiseasein•Computeralgorithmsfacilitateidentificationofthethegenomicsera.Science2001;293:807.openreadingframesthatencodeagivenproteinbyWilkinsMRetal:High-throughputmassspectrometricdiscoveryusingpartialsequencesandpeptidemassprofilingtoofproteinpost-translationalmodifications.JMolBiolsearchsequencedatabases.1999;289:645.
38Proteins:HigherOrdersofStructure5VictorW.Rodwell,PhD,&PeterJ.Kennelly,PhDBIOMEDICALIMPORTANCEGlobularproteinsarecompact,areroughlysphericalorovoidinshape,andhaveaxialratios(theratioofProteinscatalyzemetabolicreactions,powercellulartheirshortesttolongestdimensions)ofnotover3.motion,andformmacromolecularrodsandcablesthatMostenzymesareglobularproteins,whoselargeinter-providestructuralintegritytohair,bones,tendons,andnalvolumeprovidesamplespaceinwhichtocon-teeth.Innature,formfollowsfunction.Thestructuralstructcavitiesofthespecificshape,charge,andhy-varietyofhumanproteinsthereforereflectsthesophis-drophobicityorhydrophilicityrequiredtobindticationanddiversityoftheirbiologicroles.Maturationsubstratesandpromotecatalysis.Bycontrast,manyofanewlysynthesizedpolypeptideintoabiologicallystructuralproteinsadopthighlyextendedconforma-functionalproteinrequiresthatitbefoldedintoaspe-tions.Thesefibrousproteinspossessaxialratiosof10cificthree-dimensionalarrangement,orconformation.ormore.Duringmaturation,posttranslationalmodificationsLipoproteinsandglycoproteinscontaincovalentlymayaddnewchemicalgroupsorremovetransientlyboundlipidandcarbohydrate,respectively.Myoglobin,neededpeptidesegments.Geneticornutritionaldefi-hemoglobin,cytochromes,andmanyotherproteinscienciesthatimpedeproteinmaturationaredeleteriouscontaintightlyassociatedmetalionsandaretermedtohealth.ExamplesoftheformerincludeCreutzfeldt-metalloproteins.Withthedevelopmentandapplica-Jakobdisease,scrapie,Alzheimer’sdisease,andbovinetionoftechniquesfordeterminingtheaminoacidse-spongiformencephalopathy(madcowdisease).Scurvyquencesofproteins(Chapter4),morepreciseclassifica-representsanutritionaldeficiencythatimpairsproteintionschemeshaveemergedbaseduponsimilarity,ormaturation.homology,inaminoacidsequenceandstructure.However,manyearlyclassificationtermsremaininCONFORMATIONVERSUScommonuse.CONFIGURATIONThetermsconfigurationandconformationareoftenconfused.Configurationreferstothegeometricrela-PROTEINSARECONSTRUCTEDUSINGtionshipbetweenagivensetofatoms,forexample,MODULARPRINCIPLESthosethatdistinguishL-fromD-aminoacids.Intercon-Proteinsperformcomplexphysicalandcatalyticfunc-versionofconfigurationalalternativesrequiresbreakingtionsbypositioningspecificchemicalgroupsinapre-covalentbonds.Conformationreferstothespatialre-cisethree-dimensionalarrangement.Thepolypeptidelationshipofeveryatominamolecule.Interconversionscaffoldcontainingthesegroupsmustadoptaconfor-betweenconformersoccurswithoutcovalentbondrup-mationthatisbothfunctionallyefficientandphys-ture,withretentionofconfiguration,andtypicallyviaicallystrong.Atfirstglance,thebiosynthesisofrotationaboutsinglebonds.polypeptidescomprisedoftensofthousandsofindi-vidualatomswouldappeartobeextremelychalleng-PROTEINSWEREINITIALLYCLASSIFIEDing.Whenoneconsidersthatatypicalpolypeptide50BYTHEIRGROSSCHARACTERISTICScanadopt≥10distinctconformations,foldingintotheconformationappropriatetotheirbiologicfunc-Scientistsinitiallyapproachedstructure-functionrela-tionwouldappeartobeevenmoredifficult.Asde-tionshipsinproteinsbyseparatingthemintoclassesscribedinChapters3and4,synthesisofthepolypep-baseduponpropertiessuchassolubility,shape,orthetidebackbonesofproteinsemploysasmallsetofpresenceofnonproteingroups.Forexample,thepro-commonbuildingblocksormodules,theaminoacids,teinsthatcanbeextractedfromcellsusingsolutionsatjoinedbyacommonlinkage,thepeptidebond.AphysiologicpHandionicstrengthareclassifiedassol-stepwisemodularpathwaysimplifiesthefoldinganduble.Extractionofintegralmembraneproteinsre-processingofnewlysynthesizedpolypeptidesintoma-quiresdissolutionofthemembranewithdetergents.tureproteins.30
39PROTEINS:HIGHERORDERSOFSTRUCTURE/31THEFOURORDERSOFPROTEINSTRUCTUREThemodularnatureofproteinsynthesisandfoldingareembodiedintheconceptofordersofproteinstruc-ture:primarystructure,thesequenceoftheamino90acidsinapolypeptidechain;secondarystructure,thefoldingofshort(3-to30-residue),contiguoussegmentsofpolypeptideintogeometricallyorderedunits;ter-tiarystructure,thethree-dimensionalassemblyofsec-ψ0ondarystructuralunitstoformlargerfunctionalunitssuchasthematurepolypeptideanditscomponentdo-mains;andquaternarystructure,thenumberandtypesofpolypeptideunitsofoligomericproteinsandtheirspatialarrangement.–90SECONDARYSTRUCTUREPeptideBondsRestrictPossibleSecondaryConformations–90090φFreerotationispossibleaboutonlytwoofthethreeco-valentbondsofthepolypeptidebackbone:theα-car-Figure5–1.Ramachandranplotofthemainchainbon(Cα)tothecarbonylcarbon(Co)bondandthephi(Φ)andpsi(Ψ)anglesforapproximately1000Cαtonitrogenbond(Figure3–4).Thepartialdouble-nonglycineresiduesineightproteinswhosestructuresbondcharacterofthepeptidebondthatlinksCototheweresolvedathighresolution.Thedotsrepresental-α-nitrogenrequiresthatthecarbonylcarbon,carbonyllowablecombinationsandthespacesprohibitedcom-oxygen,andα-nitrogenremaincoplanar,thusprevent-binationsofphiandpsiangles.(Reproduced,withper-ingrotation.TheangleabouttheCα⎯Nbondismission,fromRichardsonJS:Theanatomyandtaxonomytermedthephi(Φ)angle,andthatabouttheCo⎯Cαofproteinstructures.AdvProteinChem1981;34:167.)bondthepsi(Ψ)angle.Foraminoacidsotherthanglycine,mostcombinationsofphiandpsianglesaredisallowedbecauseofsterichindrance(Figure5–1).Theconformationsofprolineareevenmorerestrictedoccurinnature.Schematicdiagramsofproteinsrepre-duetotheabsenceoffreerotationoftheN⎯Cαbond.sentαhelicesascylinders.RegionsoforderedsecondarystructurearisewhenaThestabilityofanαhelixarisesprimarilyfromhy-seriesofaminoacylresiduesadoptsimilarphiandpsidrogenbondsformedbetweentheoxygenofthepep-angles.Extendedsegmentsofpolypeptide(eg,loops)tidebondcarbonylandthehydrogenatomofthepep-canpossessavarietyofsuchangles.Theanglesthatde-tidebondnitrogenofthefourthresiduedownthefinethetwomostcommontypesofsecondarystruc-polypeptidechain(Figure5–4).Theabilitytoformtheture,thehelixandthesheet,fallwithinthelowermaximumnumberofhydrogenbonds,supplementedandupperleft-handquadrantsofaRamachandranbyvanderWaalsinteractionsinthecoreofthistightlyplot,respectively(Figure5–1).packedstructure,providesthethermodynamicdrivingforcefortheformationofanαhelix.Sincethepeptidebondnitrogenofprolinelacksahydrogenatomtocon-TheAlphaHelixtributetoahydrogenbond,prolinecanonlybestablyThepolypeptidebackboneofanαhelixistwistedbyaccommodatedwithinthefirstturnofanαhelix.anequalamountabouteachα-carbonwithaphiangleWhenpresentelsewhere,prolinedisruptstheconfor-ofapproximately−57degreesandapsiangleofapprox-mationofthehelix,producingabend.Becauseofitsimately−47degrees.Acompleteturnofthehelixcon-smallsize,glycinealsoofteninducesbendsinαhelices.tainsanaverageof3.6aminoacylresidues,andthedis-ManyαheliceshavepredominantlyhydrophobicRtanceitrisesperturn(itspitch)is0.54nm(Figuregroupsononesideoftheaxisofthehelixandpredomi-5–2).TheRgroupsofeachaminoacylresidueinanαnantlyhydrophiliconesontheother.Theseamphi-helixfaceoutward(Figure5–3).Proteinscontainonlypathichelicesarewelladaptedtotheformationofin-L-aminoacids,forwhicharight-handedαhelixisbyterfacesbetweenpolarandnonpolarregionssuchasthefarthemorestable,andonlyright-handedαheliceshydrophobicinteriorofaproteinanditsaqueousenvi-
4032/CHAPTER5RRNCRCRNCCNCRCNCRRCNCCRNRCCFigure5–3.Viewdowntheaxisofanαhelix.TheNCsidechains(R)areontheoutsideofthehelix.ThevanCderWaalsradiioftheatomsarelargerthanshownhere;NChence,thereisalmostnofreespaceinsidethehelix.C(Slightlymodifiedandreproduced,withpermission,from0.54-nmpitchStryerL:Biochemistry,3rded.Freeman,1995.Copyright(3.6residues)NC©1995byW.H.FreemanandCo.)CNC0.15nmCpolypeptidechainproceedinthesamedirectionaminoNCtocarboxyl,oranantiparallelsheet,inwhichtheypro-ceedinoppositedirections(Figure5–5).Eitherconfig-urationpermitsthemaximumnumberofhydrogenbondsbetweensegments,orstrands,ofthesheet.MostFigure5–2.Orientationofthemainchainatomsofaβsheetsarenotperfectlyflatbuttendtohavearight-peptideabouttheaxisofanαhelix.handedtwist.Clustersoftwistedstrandsofβsheetformthecoreofmanyglobularproteins(Figure5–6).Schematicdiagramsrepresentβsheetsasarrowsthatronment.Clustersofamphipathichelicescancreateapointintheaminotocarboxylterminaldirection.channel,orpore,thatpermitsspecificpolarmoleculestopassthroughhydrophobiccellmembranes.Loops&BendsRoughlyhalfoftheresiduesina“typical”globularpro-TheBetaSheetteinresideinαhelicesandβsheetsandhalfinloops,Thesecond(hence“beta”)recognizableregularsec-turns,bends,andotherextendedconformationalfea-ondarystructureinproteinsistheβsheet.Theaminotures.Turnsandbendsrefertoshortsegmentsofacidresiduesofaβsheet,whenviewededge-on,formaaminoacidsthatjointwounitsofsecondarystructure,zigzagorpleatedpatterninwhichtheRgroupsofadja-suchastwoadjacentstrandsofanantiparallelβsheet.centresiduespointinoppositedirections.UnliketheAβturninvolvesfouraminoacylresidues,inwhichthecompactbackboneoftheαhelix,thepeptidebackbonefirstresidueishydrogen-bondedtothefourth,resultingoftheβsheetishighlyextended.Butliketheαhelix,inatight180-degreeturn(Figure5–7).Prolineandβsheetsderivemuchoftheirstabilityfromhydrogenglycineoftenarepresentinβturns.bondsbetweenthecarbonyloxygensandamidehydro-Loopsareregionsthatcontainresiduesbeyondthegensofpeptidebonds.However,incontrasttotheαminimumnumbernecessarytoconnectadjacentre-helix,thesebondsareformedwithadjacentsegmentsofgionsofsecondarystructure.Irregularinconformation,βsheet(Figure5–5).loopsneverthelessservekeybiologicroles.FormanyInteractingβsheetscanbearrangedeithertoformaenzymes,theloopsthatbridgedomainsresponsibleforparallelβsheet,inwhichtheadjacentsegmentsofthebindingsubstratesoftencontainaminoacylresidues
41PROTEINS:HIGHERORDERSOFSTRUCTURE/33NCCRNCRCNRCCNRCCNCRCNRCCONRCCNRCCNCRCNCRCNRCFigure5–5.Spacingandbondanglesofthehydro-Cgenbondsofantiparallelandparallelpleatedβsheets.Arrowsindicatethedirectionofeachstrand.Thehydro-gen-donatingα-nitrogenatomsareshownasbluecir-cles.Hydrogenbondsareindicatedbydottedlines.ForFigure5–4.Hydrogenbonds(dottedlines)formedclarityinpresentation,RgroupsandhydrogensarebetweenHandOatomsstabilizeapolypeptideinanomitted.Top:Antiparallelβsheet.Pairsofhydrogenα-helicalconformation.(Reprinted,withpermission,bondsalternatebetweenbeingclosetogetherandfromHaggisGHetal:IntroductiontoMolecularBiology.wideapartandareorientedapproximatelyperpendicu-Wiley,1964.)lartothepolypeptidebackbone.Bottom:Parallelβsheet.Thehydrogenbondsareevenlyspacedbutslantinalternatedirections.thatparticipateincatalysis.Helix-loop-helixmotifsprovidetheoligonucleotide-bindingportionofDNA-bindingproteinssuchasrepressorsandtranscriptionfactors.Structuralmotifssuchasthehelix-loop-helixderedregionsassumeanorderedconformationuponmotifthatareintermediatebetweensecondaryandter-bindingofaligand.Thisstructuralflexibilityenablestiarystructuresareoftentermedsupersecondarystruc-suchregionstoactasligand-controlledswitchesthataf-tures.Sincemanyloopsandbendsresideonthesurfacefectproteinstructureandfunction.ofproteinsandarethusexposedtosolvent,theyconsti-tutereadilyaccessiblesites,orepitopes,forrecognitionTertiary&QuaternaryStructureandbindingofantibodies.Whileloopslackapparentstructuralregularity,theyTheterm“tertiarystructure”referstotheentirethree-existinaspecificconformationstabilizedthroughhy-dimensionalconformationofapolypeptide.Itindicates,drogenbonding,saltbridges,andhydrophobicinterac-inthree-dimensionalspace,howsecondarystructuraltionswithotherportionsoftheprotein.However,notfeatures—helices,sheets,bends,turns,andloops—allportionsofproteinsarenecessarilyordered.Proteinsassembletoformdomainsandhowthesedomainsre-maycontain“disordered”regions,oftenattheextremelatespatiallytooneanother.Adomainisasectionofaminoorcarboxylterminal,characterizedbyhighcon-proteinstructuresufficienttoperformaparticularformationalflexibility.Inmanyinstances,thesedisor-chemicalorphysicaltasksuchasbindingofasubstrate
4234/CHAPTER5COOHHHCH2NHCαHCαCHNOCOCOHNCH3CHOH2CαCαHHFigure5–7.Aβ-turnthatlinkstwosegmentsofan-tiparallelβsheet.Thedottedlineindicatesthehydro-genbondbetweenthefirstandfourthaminoacidsofthefour-residuesegmentAla-Gly-Asp-Ser.3015orotherligand.Otherdomainsmayanchoraproteinto55amembraneorinteractwitharegulatorymoleculethatNmodulatesitsfunction.Asmallpolypeptidesuchas3458070triosephosphateisomerase(Figure5–6)ormyoglobin50280(Chapter6)mayconsistofasingledomain.Bycontrast,33090proteinkinasescontaintwodomains.Proteinkinases150185350catalyzethetransferofaphosphorylgroupfromATPtoapeptideorprotein.Theaminoterminalportionofthe145C230377polypeptide,whichisrichinβsheet,bindsATP,while245320310thecarboxylterminaldomain,whichisrichinαhelix,110220260bindsthepeptideorproteinsubstrate(Figure5–8).Thegroupsthatcatalyzephosphoryltransferresideinaloop300258positionedattheinterfaceofthetwodomains.120Insomecases,proteinsareassembledfrommore205thanonepolypeptide,orprotomer.Quaternarystruc-170turedefinesthepolypeptidecompositionofaprotein125and,foranoligomericprotein,thespatialrelationshipsFigure5–6.Examplesoftertiarystructureofpro-betweenitssubunitsorprotomers.Monomericpro-teins.Top:Theenzymetriosephosphateisomerase.teinsconsistofasinglepolypeptidechain.DimericNotetheelegantandsymmetricalarrangementofal-proteinscontaintwopolypeptidechains.Homodimersternatingβsheetsandαhelices.(CourtesyofJRichard-containtwocopiesofthesamepolypeptidechain,whileinaheterodimerthepolypeptidesdiffer.Greekson.)Bottom:Two-domainstructureofthesubunitofaletters(α,β,γetc)areusedtodistinguishdifferentsub-homodimericenzyme,abacterialclassIIHMG-CoAre-unitsofaheterooligomericprotein,andsubscriptsindi-ductase.Asindicatedbythenumberedresidues,thecatethenumberofeachsubunittype.Forexample,α4singlepolypeptidebeginsinthelargedomain,entersdesignatesahomotetramericprotein,andα2β2γapro-thesmalldomain,andendsinthelargedomain.(Cour-teinwithfivesubunitsofthreedifferenttypes.tesyofCLawrence,VRodwell,andCStauffacher,PurdueSinceevensmallproteinscontainmanythousandsUniversity.)ofatoms,depictionsofproteinstructurethatindicatethepositionofeveryatomaregenerallytoocomplextobereadilyinterpreted.Simplifiedschematicdiagramsthusareusedtodepictkeyfeaturesofaprotein’ster-
43PROTEINS:HIGHERORDERSOFSTRUCTURE/35fromwater.Othersignificantcontributorsincludehy-drogenbondsandsaltbridgesbetweenthecarboxylatesofasparticandglutamicacidandtheoppositelychargedsidechainsofprotonatedlysyl,argininyl,andhistidylresidues.Whileindividuallyweakrelativetoatypicalcovalentbondof80–120kcal/mol,collectivelythesenumerousinteractionsconferahighdegreeofsta-bilitytothebiologicallyfunctionalconformationofaprotein,justasaVelcrofastenerharnessesthecumula-tivestrengthofmultipleplasticloopsandhooks.Someproteinscontaincovalentdisulfide(S⎯S)bondsthatlinkthesulfhydrylgroupsofcysteinylresidues.Formationofdisulfidebondsinvolvesoxida-tionofthecysteinylsulfhydrylgroupsandrequiresoxy-gen.Intrapolypeptidedisulfidebondsfurtherenhancethestabilityofthefoldedconformationofapeptide,whileinterpolypeptidedisulfidebondsstabilizethequaternarystructureofcertainoligomericproteins.THREE-DIMENSIONALSTRUCTUREISDETERMINEDBYX-RAYCRYSTALLOGRAPHYORBYNMRSPECTROSCOPYX-RayCrystallographySincethedeterminationofthethree-dimensionalstruc-tureofmyoglobinover40yearsago,thethree-dimen-sionalstructuresofthousandsofproteinshavebeende-Figure5–8.Domainstructure.Proteinkinasescon-terminedbyx-raycrystallography.Thekeytox-raytaintwodomains.Theupper,aminoterminaldomaincrystallographyistheprecipitationofaproteinunderbindsthephosphoryldonorATP(lightblue).Thelower,conditionsinwhichitformsorderedcrystalsthatdif-fractx-rays.Thisisgenerallyaccomplishedbyexposingcarboxylterminaldomainisshownbindingasyntheticsmalldropsoftheproteinsolutiontovariouscombina-peptidesubstrate(darkblue).tionsofpHandprecipitatingagentssuchassaltsandorganicsolutessuchaspolyethyleneglycol.Adetailedthree-dimensionalstructureofaproteincanbecon-tiaryandquaternarystructure.Ribbondiagrams(Fig-structedfromitsprimarystructureusingthepatternbyures5–6and5–8)tracetheconformationofthewhichitdiffractsabeamofmonochromaticx-rays.polypeptidebackbone,withcylindersandarrowsindi-Whilethedevelopmentofincreasinglycapablecom-catingregionsofαhelixandβsheet,respectively.Inanputer-basedtoolshasrenderedtheanalysisofcomplexevensimplerrepresentation,linesegmentsthatlinkthex-raydiffractionpatternsincreasinglyfacile,amajorαcarbonsindicatethepathofthepolypeptideback-stumblingblockremainstherequirementofinducingbone.Theseschematicdiagramsoftenincludethesidehighlypurifiedsamplesoftheproteinofinteresttochainsofselectedaminoacidsthatemphasizespecificcrystallize.Severallinesofevidence,includingtheabil-structure-functionrelationships.ityofsomecrystallizedenzymestocatalyzechemicalre-actions,indicatethatthevastmajorityofthestructuresMULTIPLEFACTORSSTABILIZEdeterminedbycrystallographyfaithfullyrepresenttheTERTIARY&QUATERNARYSTRUCTUREstructuresofproteinsinfreesolution.Higherordersofproteinstructurearestabilizedprimar-NuclearMagneticResonanceily—andoftenexclusively—bynoncovalentinterac-Spectroscopytions.Principalamongthesearehydrophobicinterac-tionsthatdrivemosthydrophobicaminoacidsideNuclearmagneticresonance(NMR)spectroscopy,achainsintotheinterioroftheprotein,shieldingthempowerfulcomplementtox-raycrystallography,mea-
4436/CHAPTER5surestheabsorbanceofradiofrequencyelectromagneticFoldingIsModularenergybycertainatomicnuclei.“NMR-active”isotopes11315Proteinfoldinggenerallyoccursviaastepwiseprocess.ofbiologicallyrelevantatomsincludeH,C,N,and31Inthefirststage,thenewlysynthesizedpolypeptideP.Thefrequency,orchemicalshift,atwhichapartic-emergesfromribosomes,andshortsegmentsfoldintoularnucleusabsorbsenergyisafunctionofboththesecondarystructuralunitsthatprovidelocalregionsoffunctionalgroupwithinwhichitresidesandtheprox-organizedstructure.Foldingisnowreducedtothese-imityofotherNMR-activenuclei.Two-dimensionallectionofanappropriatearrangementofthisrelativelyNMRspectroscopypermitsathree-dimensionalrepre-smallnumberofsecondarystructuralelements.Inthesentationofaproteintobeconstructedbydeterminingsecondstage,theforcesthatdrivehydrophobicregionstheproximityofthesenucleitooneanother.NMRintotheinterioroftheproteinawayfromsolventdrivespectroscopyanalyzesproteinsinaqueoussolution,ob-thepartiallyfoldedpolypeptideintoa“moltenglobule”viatingtheneedtoformcrystals.Itthusispossibletoinwhichthemodulesofsecondarystructurerearrangeobservechangesinconformationthataccompanylig-toarriveatthematureconformationoftheprotein.andbindingorcatalysisusingNMRspectroscopy.Thisprocessisorderlybutnotrigid.Considerableflexi-However,onlythespectraofrelativelysmallproteins,bilityexistsinthewaysandintheorderinwhichele-≤20kDainsize,canbeanalyzedwithcurrenttech-mentsofsecondarystructurecanberearranged.Ingen-nology.eral,eachelementofsecondaryorsupersecondarystructurefacilitatesproperfoldingbydirectingthefold-MolecularModelingingprocesstowardthenativeconformationandawayAnincreasinglyusefuladjuncttotheempiricaldetermi-fromunproductivealternatives.Foroligomericpro-nationofthethree-dimensionalstructureofproteinsisteins,individualprotomerstendtofoldbeforetheyas-theuseofcomputertechnologyformolecularmodel-sociatewithothersubunits.ing.Thetypesofmodelscreatedtaketwoforms.Inthefirst,theknownthree-dimensionalstructureofapro-teinisusedasatemplatetobuildamodeloftheproba-AuxiliaryProteinsAssistFoldingblestructureofahomologousprotein.Inthesecond,Underappropriateconditions,manyproteinswillcomputersoftwareisusedtomanipulatethestaticspontaneouslyrefoldafterbeingpreviouslydenaturedmodelprovidedbycrystallographytoexplorehowa(ie,unfolded)bytreatmentwithacidorbase,protein’sconformationmightchangewhenligandsarechaotropicagents,ordetergents.However,unliketheboundorwhentemperature,pH,orionicstrengthisfoldingprocessinvivo,refoldingunderlaboratorycon-altered.Scientistsalsoareexaminingthelibraryofditionsisafarslowerprocess.Moreover,someproteinsavailableproteinstructuresinanattempttodevisefailtospontaneouslyrefoldinvitro,oftenformingin-computerprogramsthatcanpredictthethree-dimen-solubleaggregates,disorderedcomplexesofunfoldedsionalconformationofaproteindirectlyfromitspri-orpartiallyfoldedpolypeptidesheldtogetherbyhy-marysequence.drophobicinteractions.Aggregatesrepresentunproduc-tivedeadendsinthefoldingprocess.Cellsemployaux-PROTEINFOLDINGiliaryproteinstospeedtheprocessoffoldingandtoguideittowardaproductiveconclusion.TheNativeConformationofaProteinIsThermodynamicallyFavoredChaperonesThenumberofdistinctcombinationsofphiandpsianglesspecifyingpotentialconformationsofevenarel-Chaperoneproteinsparticipateinthefoldingofoverativelysmall—15-kDa—polypeptideisunbelievablyhalfofmammalianproteins.Thehsp70(70-kDaheatvast.Proteinsareguidedthroughthisvastlabyrinthofshockprotein)familyofchaperonesbindsshortse-possibilitiesbythermodynamics.Sincethebiologicallyquencesofhydrophobicaminoacidsinnewlysyn-relevant—ornative—conformationofaproteingener-thesizedpolypeptides,shieldingthemfromsolvent.allyisthatwhichismostenergeticallyfavored,knowl-Chaperonespreventaggregation,thusprovidinganop-edgeofthenativeconformationisspecifiedinthepri-portunityfortheformationofappropriatesecondarymarysequence.However,ifoneweretowaitforastructuralelementsandtheirsubsequentcoalescencepolypeptidetofinditsnativeconformationbyrandomintoamoltenglobule.Thehsp60familyofchaperones,explorationofallpossibleconformations,theprocesssometimescalledchaperonins,differinsequenceandwouldrequirebillionsofyearstocomplete.Clearly,structurefromhsp70anditshomologs.Hsp60actsproteinfoldingincellstakesplaceinamoreorderlylaterinthefoldingprocess,oftentogetherwithanandguidedfashion.hsp70chaperone.Thecentralcavityofthedonut-
45PROTEINS:HIGHERORDERSOFSTRUCTURE/37shapedhsp60chaperoneprovidesashelteredenviron-sheep,andbovinespongiformencephalopathy(madmentinwhichapolypeptidecanfolduntilallhy-cowdisease)incattle.Priondiseasesmaymanifestdrophobicregionsareburiedinitsinterior,eliminatingthemselvesasinfectious,genetic,orsporadicdisorders.aggregation.Chaperoneproteinscanalso“rescue”pro-Becausenoviralorbacterialgeneencodingthepatho-teinsthathavebecomethermodynamicallytrappedinalogicprionproteincouldbeidentified,thesourceandmisfoldeddeadendbyunfoldinghydrophobicregionsmechanismoftransmissionofpriondiseaselongre-andprovidingasecondchancetofoldproductively.mainedelusive.Todayitisbelievedthatpriondiseasesareproteinconformationdiseasestransmittedbyalter-ProteinDisulfideIsomeraseingtheconformation,andhencethephysicalproper-ties,ofproteinsendogenoustothehost.Humanprion-Disulfidebondsbetweenandwithinpolypeptidesstabi-relatedprotein,PrP,aglycoproteinencodedonthelizetertiaryandquaternarystructure.However,disul-shortarmofchromosome20,normallyismonomericfidebondformationisnonspecific.Underoxidizingandrichinαhelix.Pathologicprionproteinsserveasconditions,agivencysteinecanformadisulfidebondthetemplatesfortheconformationaltransformationofwiththe⎯SHofanyaccessiblecysteinylresidue.BynormalPrP,knownasPrPc,intoPrPsc.PrPscisrichincatalyzingdisulfideexchange,theruptureofanS⎯Sβsheetwithmanyhydrophobicaminoacylsidechainsbondanditsreformationwithadifferentpartnercys-exposedtosolvent.PrPscmoleculesthereforeassociateteine,proteindisulfideisomerasefacilitatestheforma-stronglywithoneother,forminginsolubleprotease-re-tionofdisulfidebondsthatstabilizetheirnativeconfor-sistantaggregates.Sinceonepathologicprionorprion-mation.relatedproteincanserveastemplatefortheconforma-tionaltransformationofmanytimesitsnumberofPrPcProline-cis,trans-Isomerasemolecules,priondiseasescanbetransmittedbythepro-teinalonewithoutinvolvementofDNAorRNA.AllX-Propeptidebonds—whereXrepresentsanyresidue—aresynthesizedinthetransconfiguration.However,oftheX-Probondsofmatureproteins,ap-Alzheimer’sDiseaseproximately6%arecis.Thecisconfigurationispartic-Refoldingormisfoldingofanotherproteinendogenousularlycommoninβ-turns.Isomerizationfromtranstotohumanbraintissue,β-amyloid,isalsoaprominentcisiscatalyzedbytheenzymeproline-cis,trans-iso-featureofAlzheimer’sdisease.Whiletherootcauseofmerase(Figure5–9).Alzheimer’sdiseaseremainselusive,thecharacteristicsenileplaquesandneurofibrillarybundlescontainag-SEVERALNEUROLOGICDISEASESgregatesoftheproteinβ-amyloid,a4.3-kDapolypep-RESULTFROMALTEREDPROTEINtideproducedbyproteolyticcleavageofalargerproteinCONFORMATIONknownasamyloidprecursorprotein.InAlzheimer’sdiseasepatients,levelsofβ-amyloidbecomeelevated,Prionsandthisproteinundergoesaconformationaltransfor-mationfromasolubleαhelix–richstatetoastaterichThetransmissiblespongiformencephalopathies,orinβsheetandpronetoself-aggregation.Apolipopro-priondiseases,arefatalneurodegenerativediseasesteinEhasbeenimplicatedasapotentialmediatorofcharacterizedbyspongiformchanges,astrocyticgli-thisconformationaltransformation.omas,andneuronallossresultingfromthedepositionofinsolubleproteinaggregatesinneuralcells.Theyin-cludeCreutzfeldt-Jakobdiseaseinhumans,scrapieinCOLLAGENILLUSTRATESTHEROLEOFPOSTTRANSLATIONALPROCESSINGINPROTEINMATURATIONOOOProteinMaturationOftenInvolvesMakingHHα1′NN&BreakingCovalentBondsα1Nα1NThematurationofproteinsintotheirfinalstructuralR1α1′R1stateofteninvolvesthecleavageorformation(orboth)Oofcovalentbonds,aprocesstermedposttranslationalmodification.Manypolypeptidesareinitiallysynthe-Figure5–9.IsomerizationoftheN-α1prolylpeptidesizedaslargerprecursors,calledproproteins.Thebondfromacistoatransconfigurationrelativetothe“extra”polypeptidesegmentsintheseproproteinsbackboneofthepolypeptide.oftenserveasleadersequencesthattargetapolypeptide
4638/CHAPTER5toaparticularorganelleorfacilitateitspassagethroughriseperresiduenearlytwicethatofanαhelix.Theamembrane.Othersensurethatthepotentiallyharm-Rgroupsofeachpolypeptidestrandofthetriplehelixfulactivityofaproteinsuchastheproteasestrypsinpacksocloselythatinordertofit,onemustbeglycine.andchymotrypsinremainsinhibiteduntilthesepro-Thus,everythirdaminoacidresidueincollagenisateinsreachtheirfinaldestination.However,oncetheseglycineresidue.Staggeringofthethreestrandsprovidestransientrequirementsarefulfilled,thenowsuperflu-appropriatepositioningoftherequisiteglycinesouspeptideregionsareremovedbyselectiveproteoly-throughoutthehelix.Collagenisalsorichinprolinesis.Othercovalentmodificationsmaytakeplacethatandhydroxyproline,yieldingarepetitiveGly-X-Ypat-addnewchemicalfunctionalitiestoaprotein.Themat-tern(Figure5–10)inwhichYgenerallyisprolineorurationofcollagenillustratesbothoftheseprocesses.hydroxyproline.CollagentriplehelicesarestabilizedbyhydrogenCollagenIsaFibrousProteinbondsbetweenresiduesindifferentpolypeptidechains.Thehydroxylgroupsofhydroxyprolylresiduesalsopar-Collagenisthemostabundantofthefibrousproteinsticipateininterchainhydrogenbonding.Additionalthatconstitutemorethan25%oftheproteinmassinstabilityisprovidedbycovalentcross-linksformedbe-thehumanbody.Otherprominentfibrousproteinsin-tweenmodifiedlysylresiduesbothwithinandbetweencludekeratinandmyosin.Theseproteinsrepresentapolypeptidechains.primarysourceofstructuralstrengthforcells(ie,thecytoskeleton)andtissues.Skinderivesitsstrengthandflexibilityfromacrisscrossedmeshofcollagenandker-CollagenIsSynthesizedasaatinfibers,whilebonesandteetharebuttressedbyanLargerPrecursorunderlyingnetworkofcollagenfibersanalogoustotheCollagenisinitiallysynthesizedasalargerprecursorsteelstrandsinreinforcedconcrete.Collagenalsoispolypeptide,procollagen.Numerousprolylandlysylpresentinconnectivetissuessuchasligamentsandten-residuesofprocollagenarehydroxylatedbyprolylhy-dons.Thehighdegreeoftensilestrengthrequiredtodroxylaseandlysylhydroxylase,enzymesthatrequirefulfillthesestructuralrolesrequireselongatedproteinsascorbicacid(vitaminC).Hydroxyprolylandhydroxy-characterizedbyrepetitiveaminoacidsequencesandalysylresiduesprovideadditionalhydrogenbondingca-regularsecondarystructure.pabilitythatstabilizesthematureprotein.Inaddition,glucosylandgalactosyltransferasesattachglucosylorCollagenFormsaUniqueTripleHelixgalactosylresiduestothehydroxylgroupsofspecificTropocollagenconsistsofthreefibers,eachcontaininghydroxylysylresidues.about1000aminoacids,bundledtogetherinauniqueThecentralportionoftheprecursorpolypeptideconformation,thecollagentriplehelix(Figure5–10).Athenassociateswithothermoleculestoformthechar-maturecollagenfiberformsanelongatedrodwithanacteristictriplehelix.Thisprocessisaccompaniedbyaxialratioofabout200.Threeintertwinedpolypeptidetheremovaloftheglobularaminoterminalandcar-strands,whichtwisttotheleft,wraparoundonean-boxylterminalextensionsoftheprecursorpolypeptideotherinaright-handedfashiontoformthecollagenbyselectiveproteolysis.Certainlysylresiduesaremodi-triplehelix.Theopposinghandednessofthissuperhelixfiedbylysyloxidase,acopper-containingproteinthatanditscomponentpolypeptidesmakesthecollagenconvertsε-aminogroupstoaldehydes.Thealdehydestriplehelixhighlyresistanttounwinding—thesamecaneitherundergoanaldolcondensationtoformaprincipleusedinthesteelcablesofsuspensionbridges.C⎯⎯CdoublebondortoformaSchiffbase(eneimine)Acollagentriplehelixhas3.3residuesperturnandawiththeε-aminogroupofanunmodifiedlysylresidue,whichissubsequentlyreducedtoformaC⎯Nsinglebond.Thesecovalentbondscross-linktheindividualAminoacidpolypeptidesandimbuethefiberwithexceptionalsequence–Gly–X–Y–Gly–X–Y–Gly–X–Y–strengthandrigidity.2ºstructureNutritional&GeneticDisordersCanImpairCollagenMaturationThecomplexseriesofeventsincollagenmaturationTriplehelixprovideamodelthatillustratesthebiologicconse-quencesofincompletepolypeptidematuration.TheFigure5–10.Primary,secondary,andtertiarystruc-best-knowndefectincollagenbiosynthesisisscurvy,aturesofcollagen.resultofadietarydeficiencyofvitaminCrequiredby
47PROTEINS:HIGHERORDERSOFSTRUCTURE/39prolylandlysylhydroxylases.Theresultingdeficitinsizedpolypeptidefoldintosecondarystructuralthenumberofhydroxyprolineandhydroxylysineunits.Forcesthatburyhydrophobicregionsfromresiduesunderminestheconformationalstabilityofcol-solventthendrivethepartiallyfoldedpolypeptidelagenfibers,leadingtobleedinggums,swellingjoints,intoa“moltenglobule”inwhichthemodulesofsec-poorwoundhealing,andultimatelytodeath.Menkes’ondarystructurearerearrangedtogivethenativesyndrome,characterizedbykinkyhairandgrowthre-conformationoftheprotein.tardation,reflectsadietarydeficiencyofthecopperre-•Proteinsthatassistfoldingincludeproteindisulfidequiredbylysyloxidase,whichcatalyzesakeystepinisomerase,proline-cis,trans,-isomerase,andthechap-formationofthecovalentcross-linksthatstrengtheneronesthatparticipateinthefoldingofoverhalfofcollagenfibers.mammalianproteins.Chaperonesshieldnewlysyn-Geneticdisordersofcollagenbiosynthesisincludethesizedpolypeptidesfromsolventandprovideanseveralformsofosteogenesisimperfecta,characterizedenvironmentforelementsofsecondarystructuretobyfragilebones.InEhlers-Dahlossyndrome,agroupemergeandcoalesceintomoltenglobules.ofconnectivetissuedisordersthatinvolveimpairedin-•Techniquesforstudyofhigherordersofproteintegrityofsupportingstructures,defectsinthegenesstructureincludex-raycrystallography,NMRspec-thatencodeαcollagen-1,procollagenN-peptidase,ortroscopy,analyticalultracentrifugation,gelfiltration,lysylhydroxylaseresultinmobilejointsandskinabnor-andgelelectrophoresis.malities.•Silkfibroinandcollagenillustratethecloselinkageofproteinstructureandbiologicfunction.DiseasesofSUMMARYcollagenmaturationincludeEhlers-Danlossyndrome•Proteinsmaybeclassifiedonthebasisofthesolubil-andthevitaminCdeficiencydiseasescurvy.ity,shape,orfunctionorofthepresenceofapros-•Prions—proteinparticlesthatlacknucleicacid—theticgroupsuchasheme.Proteinsperformcomplexcausefataltransmissiblespongiformencephalopa-physicalandcatalyticfunctionsbypositioningspe-thiessuchasCreutzfeldt-Jakobdisease,scrapie,andcificchemicalgroupsinaprecisethree-dimensionalbovinespongiformencephalopathy.Priondiseasesarrangementthatisbothfunctionallyefficientandinvolveanalteredsecondary-tertiarystructureofaphysicallystrong.naturallyoccurringprotein,PrPc.WhenPrPcinter-•Thegene-encodedprimarystructureofapolypeptideactswithitspathologicisoformPrPSc,itsconforma-isthesequenceofitsaminoacids.Itssecondarytionistransformedfromapredominantlyα-helicalstructureresultsfromfoldingofpolypeptidesintostructuretotheβ-sheetstructurecharacteristicofhydrogen-bondedmotifssuchastheαhelix,thePrPSc.β-pleatedsheet,βbends,andloops.Combinationsofthesemotifscanformsupersecondarymotifs.•TertiarystructureconcernstherelationshipsbetweenREFERENCESsecondarystructuraldomains.QuaternarystructureofproteinswithtwoormorepolypeptidesBrandenC,ToozeJ:IntroductiontoProteinStructure.Garland,(oligomericproteins)isafeaturebasedonthespatial1991.relationshipsbetweenvarioustypesofpolypeptides.BurkhardP,StetefeldJ,StrelkovSV:Coiledcoils:Ahighlyversa-tileproteinfoldingmotif.TrendsCellBiol2001;11:82.•PrimarystructuresarestabilizedbycovalentpeptideCollingeJ:Priondiseasesofhumansandanimals:Theircausesandbonds.Higherordersofstructurearestabilizedbymolecularbasis.AnnuRevNeurosci2001;24:519.weakforces—multiplehydrogenbonds,salt(electro-FrydmanJ:Foldingofnewlytranslatedproteinsinvivo:Therolestatic)bonds,andassociationofhydrophobicRofmolecularchaperones.AnnuRevBiochem2001;70:603.groups.RadordS:Proteinfolding:Progressmadeandpromisesahead.•Thephi(Φ)angleofapolypeptideistheangleaboutTrendsBiochemSci2000;25:611.theCα⎯Nbond;thepsi(Ψ)angleisthatabouttheSchmidFX:Prolyisomerase:EnzymaticcatalysisofslowproteinCα-Cobond.Mostcombinationsofphi-psianglesfoldingreactions.AnnRevBiophysBiomolStruct1993;22:aredisallowedduetosterichindrance.Thephi-psi123.anglesthatformtheαhelixandtheβsheetfallSegrestMPetal:Theamphipathicalpha-helix:Amultifunctionalwithinthelowerandupperleft-handquadrantsofastructuralmotifinplasmalipoproteins.AdvProteinChem1995;45:1.Ramachandranplot,respectively.SotoC:Alzheimer’sandpriondiseaseasdisordersofproteincon-•Proteinfoldingisapoorlyunderstoodprocess.formation:ImplicationsforthedesignofnoveltherapeuticBroadlyspeaking,shortsegmentsofnewlysynthe-approaches.JMolMed1999;77:412.
48Proteins:Myoglobin&Hemoglobin6VictorW.Rodwell,PhD,&PeterJ.Kennelly,PhDBIOMEDICALIMPORTANCEMyoglobinIsRichinαHelixThehemeproteinsmyoglobinandhemoglobinmain-Oxygenstoredinredmusclemyoglobinisreleaseddur-tainasupplyofoxygenessentialforoxidativemetabo-ingO2deprivation(eg,severeexercise)foruseinmus-lism.Myoglobin,amonomericproteinofredmuscle,clemitochondriaforaerobicsynthesisofATP(seestoresoxygenasareserveagainstoxygendeprivation.Chapter12).A153-aminoacylresiduepolypeptideHemoglobin,atetramericproteinoferythrocytes,(MW17,000),myoglobinfoldsintoacompactshapetransportsO2tothetissuesandreturnsCO2andpro-thatmeasures4.5×3.5×2.5nm(Figure6–2).Unusu-tonstothelungs.Cyanideandcarbonmonoxidekillallyhighproportions,about75%,oftheresiduesarebecausetheydisruptthephysiologicfunctionofthepresentineightright-handed,7–20residueαhelices.hemeproteinscytochromeoxidaseandhemoglobin,re-Startingattheaminoterminal,thesearetermedhelicesspectively.Thesecondary-tertiarystructureofthesub-A–H.Typicalofglobularproteins,thesurfaceofmyo-unitsofhemoglobinresemblesmyoglobin.However,globinispolar,while—withonlytwoexceptions—thethetetramericstructureofhemoglobinpermitscooper-interiorcontainsonlynonpolarresiduessuchasLeu,ativeinteractionsthatarecentraltoitsfunction.Forex-Val,Phe,andMet.TheexceptionsareHisE7andHisample,2,3-bisphosphoglycerate(BPG)promotestheF8,theseventhandeighthresiduesinhelicesEandF,efficientreleaseofO2bystabilizingthequaternarywhichlieclosetothehemeironwheretheyfunctioninstructureofdeoxyhemoglobin.Hemoglobinandmyo-O2binding.globinillustratebothproteinstructure-functionrela-tionshipsandthemolecularbasisofgeneticdiseasessuchassicklecelldiseaseandthethalassemias.HistidinesF8&E7PerformUniqueRolesinOxygenBindingThehemeofmyoglobinliesinacrevicebetweenhelicesHEME&FERROUSIRONCONFERTHEEandForientedwithitspolarpropionategroupsfac-ABILITYTOSTORE&TOTRANSPORTingthesurfaceoftheglobin(Figure6–2).Theremain-OXYGENderresidesinthenonpolarinterior.Thefifthcoordina-tionpositionoftheironislinkedtoaringnitrogenofMyoglobinandhemoglobincontainheme,acyclictheproximalhistidine,HisF8.Thedistalhistidine,tetrapyrroleconsistingoffourmoleculesofpyrroleHisE7,liesonthesideofthehemeringoppositetolinkedbyα-methylenebridges.ThisplanarnetworkofHisF8.conjugateddoublebondsabsorbsvisiblelightandcol-orshemedeepred.Thesubstituentsattheβ-positionsofhemearemethyl(M),vinyl(V),andpropionate(Pr)TheIronMovesTowardthePlaneofthegroupsarrangedintheorderM,V,M,V,M,Pr,Pr,M+HemeWhenOxygenIsBound(Figure6–1).Oneatomofferrousiron(Fe2)residesatthecenteroftheplanartetrapyrrole.OtherproteinsTheironofunoxygenatedmyoglobinlies0.03nmwithmetal-containingtetrapyrroleprostheticgroups(0.3Å)outsidetheplaneofthehemering,towardHisincludethecytochromes(FeandCu)andchlorophyllF8.Thehemetherefore“puckers”slightly.WhenO2(Mg)(seeChapter12).Oxidationandreductionoftheoccupiesthesixthcoordinationposition,theironFeandCuatomsofcytochromesisessentialtotheirbi-movestowithin0.01nm(0.1Å)oftheplaneoftheologicfunctionascarriersofelectrons.Bycontrast,oxi-hemering.Oxygenationofmyoglobinthusisaccompa-dationoftheFe2+ofmyoglobinorhemoglobintoFe3+niedbymotionoftheiron,ofHisF8,andofresiduesdestroystheirbiologicactivity.linkedtoHisF8.40
49PROTEINS:MYOGLOBIN&HEMOGLOBIN/41ApomyoglobinProvidesaHinderedEnvironmentforHemeIronWhenO2bindstomyoglobin,thebondbetweenthefirstNoxygenatomandtheFe+isperpendiculartotheplaneof2thehemering.ThebondlinkingthefirstandsecondNFe2+Noxygenatomsliesatanangleof121degreestotheplaneoftheheme,orientingthesecondoxygenawayfromtheNdistalhistidine(Figure6–3,left).Isolatedhemebinds–Ocarbonmonoxide(CO)25,000timesmorestronglythanOoxygen.SinceCOispresentinsmallquantitiesintheat-mosphereandarisesincellsfromthecatabolismofheme,whyisitthatCOdoesnotcompletelydisplaceO2fromhemeiron?Theacceptedexplanationisthattheapopro-O–teinsofmyoglobinandhemoglobincreateahinderedOenvironment.WhileCOcanbindtoisolatedhemeinitsFigure6–1.Heme.Thepyrroleringsandmethylenepreferredorientation,ie,withallthreeatoms(Fe,C,andbridgecarbonsarecoplanar,andtheironatom(Fe2+)O)perpendiculartotheplaneoftheheme,inmyoglobinresidesinalmostthesameplane.Thefifthandsixthco-andhemoglobinthedistalhistidinestericallyprecludesordinationpositionsofFe2+aredirectedperpendicularthisorientation.Bindingatalessfavoredanglereducesto—anddirectlyaboveandbelow—theplaneofthethestrengthoftheheme-CObondtoabout200timeshemering.Observethenatureofthesubstituentthatoftheheme-O2bond(Figure6–3,right)atwhichgroupsontheβcarbonsofthepyrrolerings,thecen-levelthegreatexcessofO2overCOnormallypresentdominates.Nevertheless,about1%ofmyoglobintypi-tralironatom,andthelocationofthepolarsideofthecallyispresentcombinedwithcarbonmonoxide.hemering(atabout7o’clock)thatfacesthesurfaceofthemyoglobinmolecule.THEOXYGENDISSOCIATIONCURVESFORMYOGLOBIN&HEMOGLOBINSUITTHEIRPHYSIOLOGICROLESOO–FG2CD2WhyismyoglobinunsuitableasanO2transportpro-CF9H24teinbutwellsuitedforO2storage?TherelationshipHC5F6betweentheconcentration,orpartialpressure,ofOC3C7CD12G1F8C5(PO2)andthequantityofO2boundisexpressedasanCD7O2saturationisotherm(Figure6–4).Theoxygen-E7C1E1F1G5B14D1B16D7H16E5NNE7E7NNEF3EF1G15B5OOB1A16NA1E20OC+G19H3NH5AB1FeFeA1NNH1GH4F8F8NNFigure6–2.Amodelofmyoglobinatlowresolution.Onlytheα-carbonatomsareshown.Theα-helicalre-Figure6–3.Anglesforbondingofoxygenandcar-gionsarenamedAthroughH.(BasedonDickersonREin:bonmonoxidetothehemeironofmyoglobin.Thedis-TheProteins,2nded.Vol2.NeurathH[editor].AcademictalE7histidinehindersbondingofCOatthepreferredPress,1964.Reproducedwithpermission.)(180degree)angletotheplaneofthehemering.
5042/CHAPTER6100HemoglobinIsTetramericMyoglobinHemoglobinsaretetramerscomprisedofpairsoftwo80Oxygenatedblooddifferentpolypeptidesubunits.Greeklettersareusedtoleavingthelungsdesignateeachsubunittype.Thesubunitcomposition60oftheprincipalhemoglobinsareα2β2(HbA;normaladulthemoglobin),α2γ2(HbF;fetalhemoglobin),α2S2Reducedblood40returningfromtissues(HbS;sicklecellhemoglobin),andα2δ2(HbA2;aminoradulthemoglobin).TheprimarystructuresofPercentsaturation20theβ,γ,andδchainsofhumanhemoglobinarehighlyHemoglobinconserved.020406080100120140Myoglobin&theSubunitsGaseouspressureofoxygen(mmHg)ofHemoglobinShareAlmostIdenticalSecondaryandTertiaryStructuresFigure6–4.Oxygen-bindingcurvesofbothhemo-Despitedifferencesinthekindandnumberofaminoglobinandmyoglobin.Arterialoxygentensionisaboutacidspresent,myoglobinandtheβpolypeptideofhe-100mmHg;mixedvenousoxygentensionisabout40moglobinAhavealmostidenticalsecondaryandter-mmHg;capillary(activemuscle)oxygentensionistiarystructures.Similaritiesincludethelocationoftheabout20mmHg;andtheminimumoxygentensionre-hemeandtheeighthelicalregionsandthepresenceofquiredforcytochromeoxidaseisabout5mmHg.Asso-aminoacidswithsimilarpropertiesatcomparableloca-ciationofchainsintoatetramericstructure(hemoglo-tions.Althoughitpossessessevenratherthaneightheli-bin)resultsinmuchgreateroxygendeliverythancalregions,theαpolypeptideofhemoglobinalsowouldbepossiblewithsinglechains.(Modified,withcloselyresemblesmyoglobin.permission,fromScriverCRetal[editors]:TheMolecularandMetabolicBasesofInheritedDisease,7thed.OxygenationofHemoglobinMcGraw-Hill,1995.)TriggersConformationalChangesintheApoproteinHemoglobinsbindfourmoleculesofO2pertetramer,bindingcurveformyoglobinishyperbolic.Myoglobinoneperheme.AmoleculeofO2bindstoahemoglobinthereforeloadsO2readilyatthePO2ofthelungcapil-tetramermorereadilyifotherOmoleculesarealreadylarybed(100mmHg).However,sincemyoglobinre-2bound(Figure6–4).Termedcooperativebinding,leasesonlyasmallfractionofitsboundO2atthePO2thisphenomenonpermitshemoglobintomaximizevaluestypicallyencounteredinactivemuscle(20mmboththequantityofO2loadedatthePO2ofthelungsHg)orothertissues(40mmHg),itrepresentsaninef-andthequantityofO2releasedatthePO2ofthepe-fectivevehiclefordeliveryofO2.However,whenripheraltissues.Cooperativeinteractions,anexclusivestrenuousexerciselowersthePO2ofmuscletissuetopropertyofmultimericproteins,arecriticallyimpor-about5mmHg,myoglobinreleasesO2formitochon-tanttoaerobiclife.drialsynthesisofATP,permittingcontinuedmuscularactivity.P50ExpressestheRelativeAffinitiesofDifferentHemoglobinsforOxygenTHEALLOSTERICPROPERTIESOFHEMOGLOBINSRESULTFROMTHEIRThequantityP50,ameasureofO2concentration,istheQUATERNARYSTRUCTURESpartialpressureofO2thathalf-saturatesagivenhemo-globin.Dependingontheorganism,P50canvaryThepropertiesofindividualhemoglobinsareconse-widely,butinallinstancesitwillexceedthePO2ofthequencesoftheirquaternaryaswellasoftheirsecondaryperipheraltissues.Forexample,valuesofP50forHbAandtertiarystructures.Thequaternarystructureofhe-andfetalHbFare26and20mmHg,respectively.Inmoglobinconfersstrikingadditionalproperties,absenttheplacenta,thisdifferenceenablesHbFtoextractoxy-frommonomericmyoglobin,whichadaptsittoitsgenfromtheHbAinthemother’sblood.However,uniquebiologicroles.Theallosteric(Gkallos“other,”HbFissuboptimalpostpartumsinceitshighaffinitysteros“space”)propertiesofhemoglobinprovide,inad-forO2dictatesthatitcandeliverlessO2tothetissues.dition,amodelforunderstandingotherallostericpro-Thesubunitcompositionofhemoglobintetramersteins(seeChapter11).undergoescomplexchangesduringdevelopment.The
51PROTEINS:MYOGLOBIN&HEMOGLOBIN/43humanfetusinitiallysynthesizesaζ2ε2tetramer.BytheHistidineF8endofthefirsttrimester,ζandγsubunitshavebeenre-FhelixNplacedbyαandεsubunits,formingHbF(α2γ2),theChemoglobinoflatefetallife.Whilesynthesisofβsub-CHHCunitsbeginsinthethirdtrimester,βsubunitsdonotNcompletelyreplaceγsubunitstoyieldadultHbA(α2β2)Stericuntilsomeweekspostpartum(Figure6–5).repulsionFePorphyrinplaneOxygenationofHemoglobinIsAccompaniedbyLargeConformationalChanges+O2ThebindingofthefirstO2moleculetodeoxyHbshiftsFhelixthehemeirontowardstheplaneofthehemeringfromCNapositionabout0.6nmbeyondit(Figure6–6).Thismotionistransmittedtotheproximal(F8)histidineHCCHandtotheresiduesattachedthereto,whichinturnNcausestheruptureofsaltbridgesbetweenthecarboxylterminalresiduesofallfoursubunits.Asaconsequence,Feonepairofα/βsubunitsrotates15degreeswithrespecttotheother,compactingthetetramer(Figure6–7).OProfoundchangesinsecondary,tertiary,andquater-Onarystructureaccompanythehigh-affinityO2-inducedtransitionofhemoglobinfromthelow-affinityT(taut)Figure6–6.TheironatommovesintotheplaneofstatetotheR(relaxed)state.Thesechangessignifi-thehemeonoxygenation.HistidineF8anditsassoci-cantlyincreasetheaffinityoftheremainingunoxy-atedresiduesarepulledalongwiththeironatom.genatedhemesforO2,assubsequentbindingeventsre-(Slightlymodifiedandreproduced,withpermission,quiretheruptureoffewersaltbridges(Figure6–8).fromStryerL:Biochemistry,4thed.Freeman,1995.)ThetermsTandRalsoareusedtorefertothelow-affinityandhigh-affinityconformationsofallostericen-zymes,respectively.50αchainγchainα1β2α1β2(fetal)40Axisβchain(adult)30α220and∋ζchainsα2β1β1(embryonic)1015°δchainTformRformGlobinchainsynthesis(%oftotal)0363Birth6Figure6–7.DuringtransitionoftheTformtotheRGestation(months)Age(months)formofhemoglobin,onepairofsubunits(α2/β2)ro-tatesthrough15degreesrelativetotheotherpairFigure6–5.Developmentalpatternofthequater-(α1/β1).Theaxisofrotationiseccentric,andtheα2/β2narystructureoffetalandnewbornhemoglobins.(Re-pairalsoshiftstowardtheaxissomewhat.Inthedia-produced,withpermission,fromGanongWF:Reviewofgram,theunshadedα1/β1pairisshownfixedwhiletheMedicalPhysiology,20thed.McGraw-Hill,2001.)coloredα2/β2pairbothshiftsandrotates.
5244/CHAPTER6Tstructureα1α2O2O2O2O2O2β1β2O2O2O2O2O2O2O2O2O2O2O2RstructureFigure6–8.TransitionfromtheTstructuretotheRstructure.Inthismodel,saltbridges(thinlines)linkingthesubunitsintheTstructurebreakprogressivelyasoxy-genisadded,andeventhosesaltbridgesthathavenotyetrupturedareprogressivelyweakened(wavylines).ThetransitionfromTtoRdoesnottakeplaceafterafixednumberofoxygenmoleculeshavebeenboundbutbecomesmoreprobableaseachsuccessiveoxygenbinds.Thetransitionbetweenthetwostructuresisinfluencedbyprotons,carbondioxide,chloride,andBPG;thehighertheirconcentration,themoreoxygenmustbeboundtotriggerthetransition.FullyoxygenatedmoleculesintheTstructureandfullydeoxygenatedmoleculesintheRstructurearenotshownbecausetheyareunstable.(Modifiedandredrawn,withpermission,fromPerutzMF:Hemoglobinstructureandrespiratorytransport.SciAm[Dec]1978;239:92.)AfterReleasingO2attheTissues,HemoglobinTransportsCO2&ProtonstotheLungsInadditiontotransportingO2fromthelungstope-ripheraltissues,hemoglobintransportsCO2,theby-productofrespiration,andprotonsfromperipheraltis-Deoxyhemoglobinbindsoneprotonforeverytwosuestothelungs.HemoglobincarriesCO2asO2moleculesreleased,contributingsignificantlytothecarbamatesformedwiththeaminoterminalnitrogensbufferingcapacityofblood.ThesomewhatlowerpHofofthepolypeptidechains.peripheraltissues,aidedbycarbamation,stabilizestheTstateandthusenhancesthedeliveryofO2.Inthelungs,theprocessreverses.AsO2bindstodeoxyhemo-Oglobin,protonsarereleasedandcombinewithbicar-++H||−bonatetoformcarbonicacid.DehydrationofH2CO3,CO2++NHHb⎯=32HHb⎯N⎯C⎯Ocatalyzedbycarbonicanhydrase,formsCO2,whichisexhaled.BindingofoxygenthusdrivestheexhalationofCO2(Figure6–9).ThisreciprocalcouplingofprotonCarbamateschangethechargeonaminoterminalsandO2bindingistermedtheBohreffect.TheBohrfrompositivetonegative,favoringsaltbondformationeffectisdependentuponcooperativeinteractionsbe-betweentheαandβchains.tweenthehemesofthehemoglobintetramer.Myo-Hemoglobincarbamatesaccountforabout15%ofglobin,amonomer,exhibitsnoBohreffect.theCO2invenousblood.MuchoftheremainingCO2iscarriedasbicarbonate,whichisformedinerythro-ProtonsAriseFromRuptureofSaltBondscytesbythehydrationofCO2tocarbonicacidWhenOBinds(H2CO3),aprocesscatalyzedbycarbonicanhydrase.At2thepHofvenousblood,H2CO3dissociatesintobicar-ProtonsresponsiblefortheBohreffectarisefromrup-bonateandaproton.tureofsaltbridgesduringthebindingofO2toTstate
53PROTEINS:MYOGLOBIN&HEMOGLOBIN/45Exhaled2CO2+2H2OCARBONICANHYDRASE2H2CO3PERIPHERALTISSUES2HCO–+2H+Hb•4O32Thehemoglobintetramerbindsonemoleculeof4O2BPGinthecentralcavityformedbyitsfoursubunits.+–However,thespacebetweentheHhelicesoftheβ2H+2HCO3chainsliningthecavityissufficientlywidetoaccom-4OHb•2H+modateBPGonlywhenhemoglobinisintheTstate.2(buffer)BPGformssaltbridgeswiththeterminalaminogroups2H2CO3ofbothβchainsviaValNA1andwithLysEF6andLUNGSCARBONICANHYDRASEHisH21(Figure6–10).BPGthereforestabilizesde-oxygenated(Tstate)hemoglobinbyformingadditional2CO2+2H2OsaltbridgesthatmustbebrokenpriortoconversiontotheRstate.GeneratedbytheKrebscycleResidueH21oftheγsubunitoffetalhemoglobin(HbF)isSerratherthanHis.SinceSercannotformaFigure6–9.TheBohreffect.Carbondioxidegener-saltbridge,BPGbindsmoreweaklytoHbFthantoatedinperipheraltissuescombineswithwatertoformHbA.ThelowerstabilizationaffordedtotheTstatebycarbonicacid,whichdissociatesintoprotonsandbicar-BPGaccountsforHbFhavingahigheraffinityforO2bonateions.DeoxyhemoglobinactsasabufferbythanHbA.bindingprotonsanddeliveringthemtothelungs.Inthelungs,theuptakeofoxygenbyhemoglobinre-leasesprotonsthatcombinewithbicarbonateion,formingcarbonicacid,whichwhendehydratedbycar-bonicanhydrasebecomescarbondioxide,whichthenisexhaled.HisH21LysEF6hemoglobin.ConversiontotheoxygenatedRstatebreakssaltbridgesinvolvingβ-chainresidueHis146.BPGValNA1ThesubsequentdissociationofprotonsfromHis146+ValNA1α-NH3drivestheconversionofbicarbonatetocarbonicacid(Figure6–9).UponthereleaseofO2,theTstructureanditssaltbridgesre-form.ThisconformationalLysEF6changeincreasesthepKaoftheβ-chainHis146residues,whichbindprotons.Byfacilitatingthere-for-mationofsaltbridges,anincreaseinprotonconcentra-tionenhancesthereleaseofO2fromoxygenated(RHisH21state)hemoglobin.Conversely,anincreaseinPO2pro-motesprotonrelease.Figure6–10.Modeofbindingof2,3-bisphospho-2,3-Bisphosphoglycerate(BPG)Stabilizesglyceratetohumandeoxyhemoglobin.BPGinteractstheTStructureofHemoglobinwiththreepositivelychargedgroupsoneachβchain.AlowPO2inperipheraltissuespromotesthesynthesis(BasedonArnoneA:X-raydiffractionstudyofbindingofinerythrocytesof2,3-bisphosphoglycerate(BPG)from2,3-diphosphoglyceratetohumandeoxyhemoglobin.Na-theglycolyticintermediate1,3-bisphosphoglycerate.ture1972;237:146.Reproducedwithpermission.)
5446/CHAPTER6AdaptationtoHighAltitudeInhemoglobinM,histidineF8(HisF8)hasbeenreplacedbytyrosine.TheironofHbMformsatightPhysiologicchangesthataccompanyprolongedexpo-ioniccomplexwiththephenolateanionoftyrosinethatsuretohighaltitudeincludeanincreaseinthenumber+stabilizestheFe3form.Inα-chainhemoglobinMvari-oferythrocytesandintheirconcentrationsofhemoglo-ants,theR-TequilibriumfavorstheTstate.OxygenbinandofBPG.ElevatedBPGlowerstheaffinityofaffinityisreduced,andtheBohreffectisabsent.HbAforO2(decreasesP50),whichenhancesreleaseofβ-ChainhemoglobinMvariantsexhibitR-Tswitching,O2atthetissues.andtheBohreffectisthereforepresent.Mutations(eg,hemoglobinChesapeake)thatfavorNUMEROUSMUTANTHUMANtheRstateincreaseO2affinity.ThesehemoglobinsHEMOGLOBINSHAVEBEENIDENTIFIEDthereforefailtodeliveradequateO2toperipheraltis-sues.Theresultingtissuehypoxialeadstopoly-Mutationsinthegenesthatencodetheαorβsubunitscythemia,anincreasedconcentrationoferythrocytes.ofhemoglobinpotentiallycanaffectitsbiologicfunc-tion.However,almostalloftheover800knownmu-tanthumanhemoglobinsarebothextremelyrareandHemoglobinSbenign,presentingnoclinicalabnormalities.Whenamutationdoescompromisebiologicfunction,thecon-InHbS,thenonpolaraminoacidvalinehasreplacedditionistermedahemoglobinopathy.TheURLthepolarsurfaceresidueGlu6oftheβsubunit,gener-http://globin.cse.psu.edu/(GlobinGeneServer)pro-atingahydrophobic“stickypatch”onthesurfaceofvidesinformationabout—andlinksfor—normalandtheβsubunitofbothoxyHbSanddeoxyHbS.Bothmutanthemoglobins.HbAandHbScontainacomplementarystickypatchontheirsurfacesthatisexposedonlyinthedeoxy-genated,Rstate.Thus,atlowPO2,deoxyHbScanpoly-Methemoglobin&HemoglobinMmerizetoformlong,insolublefibers.Bindingofdeoxy-Inmethemoglobinemia,thehemeironisferricratherHbAterminatesfiberpolymerization,sinceHbAlacksthanferrous.MethemoglobinthuscanneitherbindnorthesecondstickypatchnecessarytobindanotherHbtransportO2.Normally,theenzymemethemoglobinmolecule(Figure6–11).ThesetwistedhelicalfibersreductasereducestheFe3+ofmethemoglobintoFe2+.distorttheerythrocyteintoacharacteristicsickleshape,MethemoglobincanarisebyoxidationofFe2+toFe3+renderingitvulnerabletolysisintheintersticesoftheasasideeffectofagentssuchassulfonamides,fromsplenicsinusoids.TheyalsocausemultiplesecondaryhereditaryhemoglobinM,orconsequenttoreducedclinicaleffects.AlowPO2suchasthatathighaltitudesactivityoftheenzymemethemoglobinreductase.exacerbatesthetendencytopolymerize.OxyADeoxyAOxySDeoxySβααβDeoxyADeoxySFigure6–11.Representationofthestickypatch()onhemoglobinSandits“receptor”()ondeoxyhemoglobinAanddeoxyhemoglobinS.Thecomplementarysurfacesallowdeoxyhe-moglobinStopolymerizeintoafibrousstructure,butthepresenceofdeoxyhemoglobinAwillterminatethepolymerizationbyfailingtoprovidestickypatches.(Modifiedandreproduced,withpermission,fromStryerL:Biochemistry,4thed.Freeman,1995.)
55PROTEINS:MYOGLOBIN&HEMOGLOBIN/47BIOMEDICALIMPLICATIONSdifferentprimarystructures,myoglobinandthesub-unitsofhemoglobinhavenearlyidenticalsecondaryMyoglobinuriaandtertiarystructures.Followingmassivecrushinjury,myoglobinreleased•Heme,anessentiallyplanar,slightlypuckered,cyclicfromdamagedmusclefiberscolorstheurinedarkred.tetrapyrrole,hasacentralFe2+linkedtoallfourni-Myoglobincanbedetectedinplasmafollowingamy-trogenatomsoftheheme,tohistidineF8,and,inocardialinfarction,butassayofserumenzymes(seeoxyMbandoxyHb,alsotoO2.Chapter7)providesamoresensitiveindexofmyocar-•TheO2-bindingcurveformyoglobinishyperbolic,dialinjury.butforhemoglobinitissigmoidal,aconsequenceofcooperativeinteractionsinthetetramer.Cooperativ-AnemiasitymaximizestheabilityofhemoglobinbothtoloadO2atthePO2ofthelungsandtodeliverO2attheAnemias,reductionsinthenumberofredbloodcellsorPO2ofthetissues.ofhemoglobinintheblood,canreflectimpairedsyn-•Relativeaffinitiesofdifferenthemoglobinsforoxy-thesisofhemoglobin(eg,inirondeficiency;ChaptergenareexpressedasP50,thePO2thathalf-saturates51)orimpairedproductionoferythrocytes(eg,infolicthemwithO2.HemoglobinssaturateatthepartialacidorvitaminB12deficiency;Chapter45).Diagnosispressuresoftheirrespectiverespiratoryorgan,eg,theofanemiasbeginswithspectroscopicmeasurementoflungorplacenta.bloodhemoglobinlevels.•Onoxygenationofhemoglobin,theiron,histidineF8,andlinkedresiduesmovetowardthehemering.ThalassemiasConformationalchangesthataccompanyoxygena-tionincluderuptureofsaltbondsandlooseningofThegeneticdefectsknownasthalassemiasresultfromquaternarystructure,facilitatingbindingofaddi-thepartialortotalabsenceofoneormoreαorβchainstionalO2.ofhemoglobin.Over750differentmutationshave•2,3-Bisphosphoglycerate(BPG)inthecentralcavitybeenidentified,butonlythreearecommon.EithertheofdeoxyHbformssaltbondswiththeβsubunitsαchain(alphathalassemias)orβchain(betathal-thatstabilizedeoxyHb.Onoxygenation,thecentralassemias)canbeaffected.Asuperscriptindicates00cavitycontracts,BPGisextruded,andthequaternarywhetherasubunitiscompletelyabsent(αorβ)or++structureloosens.whetheritssynthesisisreduced(αorβ).Apartfrommarrowtransplantation,treatmentissymptomatic.•HemoglobinalsofunctionsinCO2andprotonCertainmutanthemoglobinsarecommoninmanytransportfromtissuestolungs.ReleaseofO2frompopulations,andapatientmayinheritmorethanoneoxyHbatthetissuesisaccompaniedbyuptakeoftype.HemoglobindisordersthuspresentacomplexprotonsduetoloweringofthepKaofhistidinepatternofclinicalphenotypes.TheuseofDNAprobesresidues.fortheirdiagnosisisconsideredinChapter40.•Insicklecellhemoglobin(HbS),Valreplacestheβ6GluofHbA,creatinga“stickypatch”thathasacomplementondeoxyHb(butnotonoxyHb).De-GlycosylatedHemoglobin(HbA1c)oxyHbSpolymerizesatlowO2concentrations,Whenbloodglucoseenterstheerythrocytesitglycosy-formingfibersthatdistorterythrocytesintosicklelatestheε-aminogroupoflysineresiduesandtheshapes.aminoterminalsofhemoglobin.Thefractionofhemo-•Alphaandbetathalassemiasareanemiasthatresultglobinglycosylated,normallyabout5%,isproportion-fromreducedproductionofαandβsubunitsofatetobloodglucoseconcentration.Sincethehalf-lifeofHbA,respectively.anerythrocyteistypically60days,thelevelofglycosy-latedhemoglobin(HbA1c)reflectsthemeanbloodglu-REFERENCEScoseconcentrationoverthepreceding6–8weeks.MeasurementofHbA1cthereforeprovidesvaluablein-BettatiSetal:Allostericmechanismofhaemoglobin:Ruptureofformationformanagementofdiabetesmellitus.salt-bridgesraisestheoxygenaffinityoftheT-structure.JMolBiol1998;281:581.BunnHF:Pathogenesisandtreatmentofsicklecelldisease.NEnglSUMMARYJMed1997;337:762.FaustinoPetal:Dominantlytransmittedbeta-thalassemiaarising•Myoglobinismonomeric;hemoglobinisatetramerfromtheproductionofseveralaberrantmRNAspeciesandoftwosubunittypes(α2β2inHbA).Despitehavingoneabnormalpeptide.Blood1998;91:685.
5648/CHAPTER6ManningJMetal:Normalandabnormalproteinsubunitinterac-UnzaiSetal:RateconstantsforO2andCObindingtothealphationsinhemoglobins.JBiolChem1998;273:19359.andbetasubunitswithintheRandTstatesofhumanhemo-MarioN,BaudinB,GiboudeauJ:Qualitativeandquantitativeglobin.JBiolChem1998;273:23150.analysisofhemoglobinvariantsbycapillaryisoelectricfocus-WeatherallDJetal:Thehemoglobinopathies.Chapter181inTheing.JChromatogrBBiomedSciAppl1998;706:123.MetabolicandMolecularBasesofInheritedDisease,8thed.ReedW,VichinskyEP:NewconsiderationsinthetreatmentofScriverCRetal(editors).McGraw-Hill,2000.sicklecelldisease.AnnuRevMed1998;49:461.
57Enzymes:MechanismofAction7VictorW.Rodwell,PhD,&PeterJ.Kennelly,PhDBIOMEDICALIMPORTANCEwiththeabilitytosimultaneouslyconductandinde-pendentlycontrolabroadspectrumofchemicalEnzymesarebiologicpolymersthatcatalyzethechemi-processes.calreactionswhichmakelifeasweknowitpossible.Thepresenceandmaintenanceofacompleteandbal-ancedsetofenzymesisessentialforthebreakdownofENZYMESARECLASSIFIEDBYREACTIONnutrientstosupplyenergyandchemicalbuildingTYPE&MECHANISMblocks;theassemblyofthosebuildingblocksintopro-Asystemofenzymenomenclaturethatiscomprehen-teins,DNA,membranes,cells,andtissues;andthehar-sive,consistent,andatthesametimeeasytousehasnessingofenergytopowercellmotilityandmuscleprovedelusive.Thecommonnamesformostenzymescontraction.WiththeexceptionofafewcatalyticRNAderivefromtheirmostdistinctivecharacteristic:theirmolecules,orribozymes,thevastmajorityofenzymesabilitytocatalyzeaspecificchemicalreaction.Ingen-areproteins.Deficienciesinthequantityorcatalyticac-eral,anenzyme’snameconsistsofatermthatidentifiestivityofkeyenzymescanresultfromgeneticdefects,thetypeofreactioncatalyzedfollowedbythesuffixnutritionaldeficits,ortoxins.Defectiveenzymescanre--ase.Forexample,dehydrogenasesremovehydrogensultfromgeneticmutationsorinfectionbyviralorbac-atoms,proteaseshydrolyzeproteins,andisomerasescat-terialpathogens(eg,Vibriocholerae).Medicalscientistsalyzerearrangementsinconfiguration.Oneormoreaddressimbalancesinenzymeactivitybyusingpharma-modifiersusuallyprecedethisname.Unfortunately,cologicagentstoinhibitspecificenzymesandareinves-whilemanymodifiersnamethespecificsubstratein-tigatinggenetherapyasameanstoremedydeficitsinvolved(xanthineoxidase),othersidentifythesourceofenzymelevelorfunction.theenzyme(pancreaticribonuclease),specifyitsmodeofregulation(hormone-sensitivelipase),ornameadis-ENZYMESAREEFFECTIVE&HIGHLYtinguishingcharacteristicofitsmechanism(acysteineSPECIFICCATALYSTSprotease).Whenitwasdiscoveredthatmultipleformsofsomeenzymesexisted,alphanumericdesignatorsTheenzymesthatcatalyzetheconversionofoneorwereaddedtodistinguishbetweenthem(eg,RNAmorecompounds(substrates)intooneormorediffer-polymeraseIII;proteinkinaseCβ).Toaddresstheam-entcompounds(products)enhancetheratesofthebiguityandconfusionarisingfromtheseinconsistenciescorrespondingnoncatalyzedreactionbyfactorsofat6innomenclatureandthecontinuingdiscoveryofnewleast10.Likeallcatalysts,enzymesareneithercon-enzymes,theInternationalUnionofBiochemists(IUB)sumednorpermanentlyalteredasaconsequenceofdevelopedacomplexbutunambiguoussystemofen-theirparticipationinareaction.zymenomenclature.IntheIUBsystem,eachenzymeInadditiontobeinghighlyefficient,enzymesarehasauniquenameandcodenumberthatreflectthealsoextremelyselectivecatalysts.Unlikemostcatalyststypeofreactioncatalyzedandthesubstratesinvolved.usedinsyntheticchemistry,enzymesarespecificbothEnzymesaregroupedintosixclasses,eachwithseveralforthetypeofreactioncatalyzedandforasinglesub-subclasses.Forexample,theenzymecommonlycalledstrateorasmallsetofcloselyrelatedsubstrates.En-“hexokinase”isdesignated“ATP:D-hexose-6-phospho-zymesarealsostereospecificcatalystsandtypicallycat-transferaseE.C.2.7.1.1.”Thisidentifieshexokinaseasaalyzereactionsonlyofspecificstereoisomersofagivenmemberofclass2(transferases),subclass7(transferofacompound—forexample,D-butnotL-sugars,L-butphosphorylgroup),sub-subclass1(alcoholisthephos-notD-aminoacids.Sincetheybindsubstratesthroughphorylacceptor).Finally,theterm“hexose-6”indicatesatleast“threepointsofattachment,”enzymescaneventhatthealcoholphosphorylatedisthatofcarbonsixofconvertnonchiralsubstratestochiralproducts.Figureahexose.ListedbelowarethesixIUBclassesofen-7–1illustrateswhytheenzyme-catalyzedreductionofzymesandthereactionstheycatalyze.thenonchiralsubstratepyruvateproducesL-lactateratheraracemicmixtureofD-andL-lactate.Theex-1.Oxidoreductasescatalyzeoxidationsandreduc-quisitespecificityofenzymecatalystsimbueslivingcellstions.49
5850/CHAPTER74ProstheticGroupsAreTightlyIntegratedIntoanEnzyme’sStructureProstheticgroupsaredistinguishedbytheirtight,stable13incorporationintoaprotein’sstructurebycovalentor1noncovalentforces.Examplesincludepyridoxalphos-3phate,flavinmononucleotide(FMN),flavindinu-cleotide(FAD),thiaminpyrophosphate,biotin,and22themetalionsofCo,Cu,Mg,Mn,Se,andZn.Metalsarethemostcommonprostheticgroups.TheroughlyEnzymesiteSubstrateone-thirdofallenzymesthatcontaintightlyboundmetalionsaretermedmetalloenzymes.MetalionsthatFigure7–1.Planarrepresentationofthe“three-participateinredoxreactionsgenerallyarecomplexedpointattachment”ofasubstratetotheactivesiteofantoprostheticgroupssuchasheme(Chapter6)oriron-enzyme.Althoughatoms1and4areidentical,oncesulfurclusters(Chapter12).Metalsalsomayfacilitateatoms2and3areboundtotheircomplementarysitesthebindingandorientationofsubstrates,theformationontheenzyme,onlyatom1canbind.Onceboundtoofcovalentbondswithreactionintermediates(Co2+inanenzyme,apparentlyidenticalatomsthusmaybedis-coenzymeB12),orinteractionwithsubstratestorendertinguishable,permittingastereospecificchemicalthemmoreelectrophilic(electron-poor)ornucleo-change.philic(electron-rich).CofactorsAssociateReversiblyWith2.TransferasescatalyzetransferofgroupssuchasEnzymesorSubstratesmethylorglycosylgroupsfromadonormoleculetoanacceptormolecule.Cofactorsservefunctionssimilartothoseofprostheticgroupsbutbindinatransient,dissociablemannerei-3.HydrolasescatalyzethehydrolyticcleavageofthertotheenzymeortoasubstratesuchasATP.Un-C⎯C,C⎯O,C⎯N,P⎯O,andcertainotherlikethestablyassociatedprostheticgroups,cofactorsbonds,includingacidanhydridebonds.thereforemustbepresentinthemediumsurrounding4.LyasescatalyzecleavageofC⎯C,C⎯O,C⎯N,theenzymeforcatalysistooccur.Themostcommonandotherbondsbyelimination,leavingdoublecofactorsalsoaremetalions.Enzymesthatrequireabonds,andalsoaddgroupstodoublebonds.metalioncofactoraretermedmetal-activatedenzymes5.Isomerasescatalyzegeometricorstructuraltodistinguishthemfromthemetalloenzymesforchangeswithinasinglemolecule.whichmetalionsserveasprostheticgroups.6.Ligasescatalyzethejoiningtogetheroftwomole-cules,coupledtothehydrolysisofapyrophospho-CoenzymesServeasSubstrateShuttlesrylgroupinATPorasimilarnucleosidetriphos-phate.Coenzymesserveasrecyclableshuttles—orgrouptransferreagents—thattransportmanysubstratesfromDespitethemanyadvantagesoftheIUBsystem,theirpointofgenerationtotheirpointofutilization.textstendtorefertomostenzymesbytheirolderandAssociationwiththecoenzymealsostabilizessubstratesshorter,albeitsometimesambiguousnames.suchashydrogenatomsorhydrideionsthatareunsta-bleintheaqueousenvironmentofthecell.OtherchemicalmoietiestransportedbycoenzymesincludePROSTHETICGROUPS,COFACTORS,methylgroups(folates),acylgroups(coenzymeA),and&COENZYMESPLAYIMPORTANToligosaccharides(dolichol).ROLESINCATALYSISManyCoenzymes,Cofactors,&ProstheticManyenzymescontainsmallnonproteinmoleculesandGroupsAreDerivativesofBVitaminsmetalionsthatparticipatedirectlyinsubstratebindingorcatalysis.Termedprostheticgroups,cofactors,andThewater-solubleBvitaminssupplyimportantcompo-coenzymes,theseextendtherepertoireofcatalyticca-nentsofnumerouscoenzymes.Manycoenzymescon-pabilitiesbeyondthoseaffordedbythelimitednumbertain,inaddition,theadenine,ribose,andphosphoryloffunctionalgroupspresentontheaminoacylsidemoietiesofAMPorADP(Figure7–2).Nicotinamidechainsofpeptides.andriboflavinarecomponentsoftheredoxcoenzymes
59ENZYMES:MECHANISMOFACTION/51OArg145NHNH2+OHCNNH2NH2OCH2OHHCOOONHCCNHTyr248HHHHOOHHis196NCOPO–OCH22+ZnNH2NH2OOHis69CNNNGlu72NONNHOPOCH2Figure7–3.Two-dimensionalrepresentationofaO–Odipeptidesubstrate,glycyl-tyrosine,boundwithintheactivesiteofcarboxypeptidaseA.HHHOORFigure7–2.StructureofNAD+andNADP+.Fortributetotheextensivesizeandthree-dimensionalchar-NAD+,R=H.ForNADP+,R=PO2−.acteroftheactivesite.3ENZYMESEMPLOYMULTIPLENADandNADPandFMNandFAD,respectively.MECHANISMSTOFACILITATEPantothenicacidisacomponentoftheacylgroupcar-CATALYSISriercoenzymeA.Asitspyrophosphate,thiaminpartici-patesindecarboxylationofα-ketoacidsandfolicacidFourgeneralmechanismsaccountfortheabilityofen-andcobamidecoenzymesfunctioninone-carbonme-zymestoachievedramaticcatalyticenhancementofthetabolism.ratesofchemicalreactions.CatalysisbyProximityCATALYSISOCCURSATTHEACTIVESITEFormoleculestoreact,theymustcomewithinbond-Theextremesubstratespecificityandhighcatalyticeffi-formingdistanceofoneanother.Thehighertheircon-ciencyofenzymesreflecttheexistenceofanenviron-centration,themorefrequentlytheywillencounteronementthatisexquisitelytailoredtoasinglereaction.anotherandthegreaterwillbetherateoftheirreaction.Termedtheactivesite,thisenvironmentgenerallyWhenanenzymebindssubstratemoleculesinitsactivetakestheformofacleftorpocket.Theactivesitesofsite,itcreatesaregionofhighlocalsubstrateconcentra-multimericenzymesoftenarelocatedattheinterfacetion.Thisenvironmentalsoorientsthesubstratemole-betweensubunitsandrecruitresiduesfrommorethanculesspatiallyinapositionidealforthemtointeract,re-onemonomer.Thethree-dimensionalactivesitebothsultinginrateenhancementsofatleastathousandfold.shieldssubstratesfromsolventandfacilitatescatalysis.Substratesbindtotheactivesiteataregioncomple-Acid-BaseCatalysismentarytoaportionofthesubstratethatwillnotun-dergochemicalchangeduringthecourseofthereac-Theionizablefunctionalgroupsofaminoacylsidetion.Thissimultaneouslyalignsportionsofthechainsand(wherepresent)ofprostheticgroupscon-substratethatwillundergochangewiththechemicaltributetocatalysisbyactingasacidsorbases.Acid-basefunctionalgroupsofpeptidylaminoacylresidues.Thecatalysiscanbeeitherspecificorgeneral.By“specific”activesitealsobindsandorientscofactorsorprostheticwemeanonlyprotons(HO+)orOH–ions.Inspecific3groups.Manyaminoacylresiduesdrawnfromdiverseacidorspecificbasecatalysis,therateofreactionisportionsofthepolypeptidechain(Figure7–3)con-sensitivetochangesintheconcentrationofprotonsbut
6052/CHAPTER7independentoftheconcentrationsofotheracids(pro-enzyme’sactivesitefailedtoaccountforthedynamictondonors)orbases(protonacceptors)presentinsolu-changesthataccompanycatalysis.Thisdrawbackwastionorattheactivesite.Reactionswhoseratesarere-addressedbyDanielKoshland’sinducedfitmodel,sponsivetoalltheacidsorbasespresentaresaidtobewhichstatesthatwhensubstratesapproachandbindtosubjecttogeneralacidorgeneralbasecatalysis.anenzymetheyinduceaconformationalchange,achangeanalogoustoplacingahand(substrate)intoaCatalysisbyStrainglove(enzyme)(Figure7–5).Acorollaryisthattheen-zymeinducesreciprocalchangesinitssubstrates,har-Enzymesthatcatalyzelyticreactionswhichinvolvenessingtheenergyofbindingtofacilitatethetransfor-breakingacovalentbondtypicallybindtheirsubstratesmationofsubstratesintoproducts.Theinducedfitinaconformationslightlyunfavorableforthebondmodelhasbeenamplyconfirmedbybiophysicalstudiesthatwillundergocleavage.Theresultingstrainofenzymemotionduringsubstratebinding.stretchesordistortsthetargetedbond,weakeningitandmakingitmorevulnerabletocleavage.HIVPROTEASEILLUSTRATESACID-BASECATALYSISCovalentCatalysisEnzymesoftheasparticproteasefamily,whichin-Theprocessofcovalentcatalysisinvolvestheformationcludesthedigestiveenzymepepsin,thelysosomalofacovalentbondbetweentheenzymeandoneormorecathepsins,andtheproteaseproducedbythehumanim-substrates.Themodifiedenzymethenbecomesareac-munodeficiencyvirus(HIV),shareacommoncatalytictant.Covalentcatalysisintroducesanewreactionpath-mechanism.Catalysisinvolvestwoconservedaspartylwaythatisenergeticallymorefavorable—andthereforeresidueswhichactasacid-basecatalysts.Inthefirststagefaster—thanthereactionpathwayinhomogeneousso-ofthereaction,anaspartatefunctioningasageneralbaselution.Thechemicalmodificationoftheenzymeis,(AspX,Figure7–6)extractsaprotonfromawatermole-however,transient.Oncompletionofthereaction,thecule,makingitmorenucleophilic.Thisresultingnucle-enzymereturnstoitsoriginalunmodifiedstate.Itsroleophilethenattackstheelectrophiliccarbonylcarbonofthusremainscatalytic.Covalentcatalysisisparticularlythepeptidebondtargetedforhydrolysis,formingacommonamongenzymesthatcatalyzegrouptransfertetrahedraltransitionstateintermediate.Asecondas-reactions.Residuesontheenzymethatparticipateinco-partate(AspY,Figure7–6)thenfacilitatesthedecompo-valentcatalysisgenerallyarecysteineorserineandocca-sitionofthistetrahedralintermediatebydonatingapro-sionallyhistidine.Covalentcatalysisoftenfollowsatontotheaminogroupproducedbyruptureofthe“ping-pong”mechanism—oneinwhichthefirstsub-peptidebond.Twodifferentactivesiteaspartatesthusstrateisboundanditsproductreleasedpriortothecanactsimultaneouslyasageneralbaseorasageneralbindingofthesecondsubstrate(Figure7–4).acid.Thisispossiblebecausetheirimmediateenviron-mentfavorsionizationofonebutnottheother.SUBSTRATESINDUCECONFORMATIONALCHANGESCHYMOTRYPSIN&FRUCTOSE-2,6-INENZYMESBISPHOSPHATASEILLUSTRATEEarlyinthelastcentury,EmilFischercomparedtheCOVALENTCATALYSIShighlyspecificfitbetweenenzymesandtheirsubstratesChymotrypsintothatofalockanditskey.Whilethe“lockandkeymodel”accountedfortheexquisitespecificityofen-Whilecatalysisbyasparticproteasesinvolvesthedirectzyme-substrateinteractions,theimpliedrigidityofthehydrolyticattackofwateronapeptidebond,catalysisPyrGluAlaCHOCH2NH2KGCH2NH2CHOECHOEEECH2NH2EEECHOAlaPyrKGGluFigure7–4.Ping-pongmechanismfortransamination.E⎯CHOandE⎯CH2NH2representtheenzyme-pyridoxalphosphateandenzyme-pyridoxaminecomplexes,respectively.(Ala,alanine;Pyr,pyruvate;KG,α-ketoglutarate;Glu,glutamate).
61ENZYMES:MECHANISMOFACTION/53OR′N..CRABHH..O..1HOOHOOCCCHCH22ABAspXAspYOR′N..CRH2OHABHHOOOOCCCH2CH2AspYAspXFigure7–5.Two-dimensionalrepresentationofKoshland’sinducedfitmodeloftheactivesiteofaOR′lyase.BindingofthesubstrateA⎯Binducesconforma-NH+CRtionalchangesintheenzymethatalignscatalyticHHOresidueswhichparticipateincatalysisandstrainsthebondbetweenAandB,facilitatingitscleavage.3HOOOOCCbytheserineproteasechymotrypsininvolvespriorfor-mationofacovalentacylenzymeintermediate.ACH2CH2highlyreactiveserylresidue,serine195,participatesinAspYAspXacharge-relaynetworkwithhistidine57andaspartate102.Farapartinprimarystructure,intheactivesiteFigure7–6.Mechanismforcatalysisbyanaspartictheseresiduesarewithinbond-formingdistanceofoneproteasesuchasHIVprotease.Curvedarrowsindicateanother.AlignedintheorderAsp102-His57-Ser195,directionsofelectronmovement.1AspartateXactstheyconstitutea“charge-relaynetwork”thatfunctionsasabasetoactivateawatermoleculebyabstractingaasa“protonshuttle.”proton.2TheactivatedwatermoleculeattackstheBindingofsubstrateinitiatesprotonshiftsthatinef-peptidebond,formingatransienttetrahedralinterme-fecttransferthehydroxylprotonofSer195toAsp102diate.3AspartateYactsasanacidtofacilitatebreak-(Figure7–7).Theenhancednucleophilicityoftheseryldownofthetetrahedralintermediateandreleaseoftheoxygenfacilitatesitsattackonthecarbonylcarbonofsplitproductsbydonatingaprotontothenewlythepeptidebondofthesubstrate,formingacovalentformedaminogroup.Subsequentshuttlingofthepro-acyl-enzymeintermediate.ThehydrogenonAsp102tononAspXtoAspYrestorestheproteasetoitsinitialthenshuttlesthroughHis57totheaminogroupliber-atedwhenthepeptidebondiscleaved.Theportionofstate.theoriginalpeptidewithafreeaminogroupthenleavestheactivesiteandisreplacedbyawatermolecule.Thecharge-relaynetworknowactivatesthewatermoleculebywithdrawingaprotonthroughHis57toAsp102.Theresultinghydroxideionattackstheacyl-enzymein-
6254/CHAPTER7HOtermediateandareverseprotonshuttlereturnsaprotontoSer195,restoringitsoriginalstate.WhilemodifiedR1NCR2duringtheprocessofcatalysis,chymotrypsinemergesunchangedoncompletionofthereaction.Trypsinand1OOHNHNOelastaseemployasimilarcatalyticmechanism,buttheCSer195numbersoftheresiduesintheirSer-His-AspprotonAsp102His57shuttlesdiffer.HOFructose-2,6-BisphosphataseR1NCR2Fructose-2,6-bisphosphatase,aregulatoryenzymeofgluconeogenesis(Chapter19),catalyzesthehydrolytic2OOHNHNOreleaseofthephosphateoncarbon2offructose2,6-Ser195bisphosphate.Figure7–8illustratestherolesofsevenAsp102His57activesiteresidues.Catalysisinvolvesa“catalytictriad”OofoneGluandtwoHisresiduesandacovalentphos-R1NH2CR2phohistidylintermediate.3OOHNNOCATALYTICRESIDUESARESer195HIGHLYCONSERVEDAsp102His57MembersofanenzymefamilysuchastheasparticorOserineproteasesemployasimilarmechanismtocatalyzeHCR2acommonreactiontypebutactondifferentsubstrates.OOEnzymefamiliesappeartoarisethroughgeneduplica-4OOHNNHSer195tioneventsthatcreateasecondcopyofthegenewhichencodesaparticularenzyme.TheproteinsencodedbyAsp102His57thetwogenescanthenevolveindependentlytorecog-nizedifferentsubstrates—resulting,forexample,inOchymotrypsin,whichcleavespeptidebondsonthecar-HOCRboxylterminalsideoflargehydrophobicaminoacids;2andtrypsin,whichcleavespeptidebondsonthecar-5OOHNHNOboxylterminalsideofbasicaminoacids.ThecommonSer195ancestryofenzymescanbeinferredfromthepresenceAsp102His57ofspecificaminoacidsinthesamepositionineachHOOCRfamilymember.Theseresiduesaresaidtobeconserved2residues.Proteinsthatsharealargenumberofcon-servedresiduesaresaidtobehomologoustoonean-6OOHNHNOother.Table7–1illustratestheprimarystructuralcon-Ser195servationoftwocomponentsofthecharge-relayAsp102His57networkforseveralserineproteases.AmongthemostFigure7–7.Catalysisbychymotrypsin.1Thehighlyconservedresiduesarethosethatparticipatedi-charge-relaysystemremovesaprotonfromSer195,rectlyincatalysis.makingitastrongernucleophile.2ActivatedSer195attacksthepeptidebond,formingatransienttetrahedralISOZYMESAREDISTINCTENZYMEintermediate.3ReleaseoftheaminoterminalpeptideFORMSTHATCATALYZETHEisfacilitatedbydonationofaprotontothenewlyformedaminogroupbyHis57ofthecharge-relaysys-SAMEREACTIONtem,yieldinganacyl-Ser195intermediate.4His57andHigherorganismsoftenelaborateseveralphysicallydis-Asp102collaboratetoactivateawatermolecule,whichtinctversionsofagivenenzyme,eachofwhichcat-attackstheacyl-Ser195,formingasecondtetrahedralin-alyzesthesamereaction.Likethemembersofothertermediate.5Thecharge-relaysystemdonatesapro-proteinfamilies,theseproteincatalystsorisozymestontoSer195,facilitatingbreakdownoftetrahedralin-arisethroughgeneduplication.Isozymesmayexhibittermediatetoreleasethecarboxylterminalpeptide6.subtledifferencesinpropertiessuchassensitivityto
63ENZYMES:MECHANISMOFACTION/55particularregulatoryfactors(Chapter9)orsubstrateLys356Lys356affinity(eg,hexokinaseandglucokinase)thatadaptArgArg+352+352themtospecifictissuesorcircumstances.Someiso-6–P+6–P+zymesmayalsoenhancesurvivalbyprovidinga“back-up”copyofanessentialenzyme.2–2–Arg307Arg307–O–OHGlu+Glu+327+HP327P++THECATALYTICACTIVITYOFENZYMESHisHis392392FACILITATESTHEIRDETECTIONArg257His2581Arg257His2582Theminutequantitiesofenzymespresentincellscom-E•Fru-2,6-P2E-P•Fru-6-Pplicatedeterminationoftheirpresenceandconcentra-tion.However,theabilitytorapidlytransformthou-sandsofmoleculesofaspecificsubstrateintoproductsLys356Lys356imbueseachenzymewiththeabilitytorevealitspres-ArgArg+352+352ence.Assaysofthecatalyticactivityofenzymesarefre-H++quentlyusedinresearchandclinicallaboratories.HOUnderappropriateconditions(seeChapter8),therateArg307Arg307ofthecatalyticreactionbeingmonitoredisproportion-––GluP+GluPi+atetotheamountofenzymepresent,whichallowsits+H+327327++concentrationtobeinferred.HisHis392392Arg257His2583Arg257His2584Enzyme-LinkedImmunoassaysE-P•H2OE•PiThesensitivityofenzymeassayscanalsobeexploitedtoFigure7–8.Catalysisbyfructose-2,6-bisphos-detectproteinsthatlackcatalyticactivity.Enzyme-phatase.(1)Lys356andArg257,307,and352stabilizelinkedimmunoassays(ELISAs)useantibodiescova-thequadruplenegativechargeofthesubstratebylentlylinkedtoa“reporterenzyme”suchasalkalinecharge-chargeinteractions.Glu327stabilizestheposi-phosphataseorhorseradishperoxidase,enzymeswhosetivechargeonHis392.(2)ThenucleophileHis392at-productsarereadilydetected.WhenserumorothertackstheC-2phosphorylgroupandtransfersittoHissamplestobetestedareplacedinaplasticmicrotiterplate,theproteinsadheretotheplasticsurfaceandare258,formingaphosphoryl-enzymeintermediate.Fruc-immobilized.Anyremainingabsorbingareasofthewelltose6-phosphateleavestheenzyme.(3)Nucleophilicarethen“blocked”byaddinganonantigenicproteinattackbyawatermolecule,possiblyassistedbyGlu327suchasbovineserumalbumin.Asolutionofantibodyactingasabase,formsinorganicphosphate.(4)Inor-covalentlylinkedtoareporterenzymeisthenadded.ganicorthophosphateisreleasedfromArg257andArgTheantibodiesadheretotheimmobilizedantigenand307.(Reproduced,withpermission,fromPilkisSJetal:6-thesearethemselvesimmobilized.ExcessfreeantibodyPhosphofructo-2-kinase/fructose-2,6-bisphosphatase:Amoleculesarethenremovedbywashing.Thepresencemetabolicsignalingenzyme.AnnuRevBiochemandquantityofboundantibodyarethendetermined1995;64:799.)byaddingthesubstrateforthereporterenzyme.Table7–1.Aminoacidsequencesintheneighborhoodofthecatalyticsitesofseveralbovineproteases.Regionsshownarethoseoneithersideofthecatalyticsiteseryl(S)andhistidyl(H)residues.EnzymeSequenceAroundSerineSSequenceAroundHistidineHTrypsinDSCQDGSGGPVVCSGKVVSAAHCYKSGChymotrypsinASSCMGDSGGPLVCKKNVVTAAHGGVTTChymotrypsinBSSCMGDSGGPLVCQKNVVTAAHCGVTTThrombinDACEGDSGGPFVMKSPVLTAAHCLLYP
6456/CHAPTER7NAD(P)+-DependentDehydrogenasesAretatedbytheuseofradioactivesubstrates.AnalternativestrategyistodeviseasyntheticsubstratewhoseproductAssayedSpectrophotometricallyabsorbslight.Forexample,p-nitrophenylphosphateisThephysicochemicalpropertiesofthereactantsinananartificialsubstrateforcertainphosphatasesandforenzyme-catalyzedreactiondictatetheoptionsforthechymotrypsinthatdoesnotabsorbvisiblelight.How-assayofenzymeactivity.Spectrophotometricassaysex-ever,followinghydrolysis,theresultingp-nitrophen-ploittheabilityofasubstrateorproducttoabsorbylateanionabsorbslightat419nm.light.ThereducedcoenzymesNADHandNADPH,Anotherquitegeneralapproachistoemploya“cou-writtenasNAD(P)H,absorblightatawavelengthofpled”assay(Figure7–10).Typically,adehydrogenase340nm,whereastheiroxidizedformsNAD(P)+donotwhosesubstrateistheproductoftheenzymeofinterest(Figure7–9).WhenNAD(P)+isreduced,theab-isaddedincatalyticexcess.Therateofappearanceorsorbanceat340nmthereforeincreasesinproportiondisappearanceofNAD(P)Hthendependsontherateto—andataratedeterminedby—thequantityofoftheenzymereactiontowhichthedehydrogenasehasNAD(P)Hproduced.Conversely,foradehydrogenasebeencoupled.thatcatalyzestheoxidationofNAD(P)H,adecreaseinabsorbanceat340nmwillbeobserved.Ineachcase,therateofchangeinopticaldensityat340nmwillbeTHEANALYSISOFCERTAINENZYMESproportionatetothequantityofenzymepresent.AIDSDIAGNOSISManyEnzymesAreAssayedbyCouplingOfthethousandsofdifferentenzymespresentinthehumanbody,thosethatfulfillfunctionsindispensabletoaDehydrogenasetocellvitalityarepresentthroughoutthebodytissues.Theassayofenzymeswhosereactionsarenotaccompa-Otherenzymesorisozymesareexpressedonlyinspe-niedbyachangeinabsorbanceorfluorescenceisgener-cificcelltypes,duringcertainperiodsofdevelopment,allymoredifficult.Insomeinstances,theproductorre-orinresponsetospecificphysiologicorpathophysio-mainingsubstratecanbetransformedintoamorelogicchanges.Analysisofthepresenceanddistributionreadilydetectedcompound.Inotherinstances,there-ofenzymesandisozymes—whoseexpressionisnor-actionproductmayhavetobeseparatedfromunre-mallytissue-,time-,orcircumstance-specific—oftenactedsubstratepriortomeasurement—aprocessfacili-aidsdiagnosis.1.0GlucoseATP,Mg2+0.8HEXOKINASEADP,Mg2+0.6Glucose6-phosphateNADP+0.4GLUCOSE-6-PHOSPHATEOpticaldensityNADHDEHYDROGENASENADPH+H+0.26-Phosphogluconolactone+Figure7–10.CoupledenzymeassayforhexokinaseNAD0activity.Theproductionofglucose6-phosphateby200250300350400hexokinaseiscoupledtotheoxidationofthisproductbyglucose-6-phosphatedehydrogenaseinthepres-Wavelength(nm)enceofaddedenzymeandNADP+.WhenanexcessofFigure7–9.AbsorptionspectraofNAD+andNADH.glucose-6-phosphatedehydrogenaseispresent,theDensitiesarefora44mg/Lsolutioninacellwitha1cmrateofformationofNADPH,whichcanbemeasuredatlightpath.NADP+andNADPHhavespectrumsanalo-340nm,isgovernedbytherateofformationofglucosegoustoNAD+andNADH,respectively.6-phosphatebyhexokinase.
65ENZYMES:MECHANISMOFACTION/57NonfunctionalPlasmaEnzymesAidheart)andM(formuscle).ThesubunitscancombineDiagnosis&PrognosisasshownbelowtoyieldcatalyticallyactiveisozymesofL-lactatedehydrogenase:Certainenzymes,proenzymes,andtheirsubstratesarepresentatalltimesinthecirculationofnormalindivid-ualsandperformaphysiologicfunctionintheblood.LactateExamplesofthesefunctionalplasmaenzymesincludeDehydrogenaselipoproteinlipase,pseudocholinesterase,andtheproen-IsozymeSubunitszymesofbloodcoagulationandbloodclotdissolutionI1HHHH(Chapters9and51).ThemajorityoftheseenzymesareI2HHHMsynthesizedinandsecretedbytheliver.I3HHMMPlasmaalsocontainsnumerousotherenzymesthatIHMMM4performnoknownphysiologicfunctioninblood.IMMMM5Theseapparentlynonfunctionalplasmaenzymesarisefromtheroutinenormaldestructionoferythrocytes,leukocytes,andothercells.TissuedamageornecrosisDistinctgeneswhoseexpressionisdifferentiallyregu-resultingfrominjuryordiseaseisgenerallyaccompa-latedinvarioustissuesencodetheHandMsubunits.niedbyincreasesinthelevelsofseveralnonfunctionalSinceheartexpressestheHsubunitalmostexclusively,plasmaenzymes.Table7–2listsseveralenzymesusedisozymeI1predominatesinthistissue.Bycontrast,indiagnosticenzymology.isozymeI5predominatesinliver.Smallquantitiesoflactatedehydrogenasearenormallypresentinplasma.IsozymesofLactateDehydrogenaseAreFollowingamyocardialinfarctionorinliverdisease,UsedtoDetectMyocardialInfarctionsthedamagedtissuesreleasecharacteristiclactatedehy-L-Lactatedehydrogenaseisatetramericenzymewhosedrogenaseisoformsintotheblood.Theresultingeleva-foursubunitsoccurintwoisoforms,designatedH(fortioninthelevelsoftheI1orI5isozymesisdetectedbyseparatingthedifferentoligomersoflactatedehydroge-nasebyelectrophoresisandassayingtheircatalyticac-tivity(Figure7–11).Table7–2.Principalserumenzymesusedinclinicaldiagnosis.ManyoftheenzymesarenotENZYMESFACILITATEDIAGNOSISspecificforthediseaselisted.OFGENETICDISEASESSerumEnzymeMajorDiagnosticUseWhilemanydiseaseshavelongbeenknowntoresultfromalterationsinanindividual’sDNA,toolsfortheAminotransferasesdetectionofgeneticmutationshaveonlyrecentlybe-Aspartateaminotransfer-Myocardialinfarctioncomewidelyavailable.Thesetechniquesrelyuponthease(AST,orSGOT)catalyticefficiencyandspecificityofenzymecatalysts.AlanineaminotransferaseViralhepatitisForexample,thepolymerasechainreaction(PCR)re-(ALT,orSGPT)liesupontheabilityofenzymestoserveascatalyticam-AmylaseAcutepancreatitisplifierstoanalyzetheDNApresentinbiologicandCeruloplasminHepatolenticulardegenerationforensicsamples.InthePCRtechnique,athermostable(Wilson’sdisease)DNApolymerase,directedbyappropriateoligonu-cleotideprimers,producesthousandsofcopiesofaCreatinekinaseMuscledisordersandmyocar-sampleofDNAthatwaspresentinitiallyatlevelstoodialinfarctionlowfordirectdetection.γ-GlutamyltranspeptidaseVariousliverdiseasesThedetectionofrestrictionfragmentlengthpoly-morphisms(RFLPs)facilitatesprenataldetectionofLactatedehydrogenaseMyocardialinfarctionhereditarydisorderssuchassicklecelltrait,beta-(isozymes)thalassemia,infantphenylketonuria,andHuntington’sLipaseAcutepancreatitisdisease.DetectionofRFLPsinvolvescleavageofdou-Phosphatase,acidMetastaticcarcinomaoftheble-strandedDNAbyrestrictionendonucleases,whichprostatecandetectsubtlealterationsinDNAthataffecttheirrecognizedsites.Chapter40providesfurtherdetailsPhosphatase,alkalineVariousbonedisorders,ob-concerningtheuseofPCRandrestrictionenzymesfor(isozymes)structiveliverdiseasesdiagnosis.
6658/CHAPTER7+–(Lactate)SH2LACTATES(Pyruvate)DEHYDROGENASEHeartANAD+NADH+H+NormalBReducedPMSOxidizedPMSLiverCOxidizedNBTReducedNBT(colorless)(blueformazan)54321Figure7–11.Normalandpathologicpatternsoflactatedehydrogenase(LDH)isozymesinhumanserum.LDHisozymesofserumwereseparatedbyelectrophoresisandvisualizedusingthecoupledreac-tionschemeshownontheleft.(NBT,nitrobluetetrazolium;PMS,phenazinemethylsulfate).Atrightisshownthestainedelectropherogram.PatternAisserumfromapatientwithamyocardialinfarct;Bisnor-malserum;andCisserumfromapatientwithliverdisease.ArabicnumeralsdenotespecificLDHisozymes.RECOMBINANTDNAPROVIDESANresultingmodifiedprotein,termedafusionprotein,IMPORTANTTOOLFORSTUDYINGcontainsadomaintailoredtointeractwithaspecificENZYMESaffinitysupport.OnepopularapproachistoattachanoligonucleotidethatencodessixconsecutivehistidineRecombinantDNAtechnologyhasemergedasanim-residues.Theexpressed“Histag”proteinbindstochro-portantassetinthestudyofenzymes.Highlypurifiedmatographicsupportsthatcontainanimmobilizeddiva-2+samplesofenzymesarenecessaryforthestudyoftheirlentmetalionsuchasNi.Alternatively,thesubstrate-structureandfunction.TheisolationofanindividualbindingdomainofglutathioneS-transferase(GST)canenzyme,particularlyonepresentinlowconcentration,serveasa“GSTtag.”Figure7–12illustratesthepurifi-fromamongthethousandsofproteinspresentinacellcationofaGST-fusionproteinusinganaffinitysupportcanbeextremelydifficult.Ifthegenefortheenzymeofcontainingboundglutathione.Fusionproteinsalsointeresthasbeencloned,itgenerallyispossibletopro-oftenencodeacleavagesiteforahighlyspecificproteaseducelargequantitiesofitsencodedproteininEsch-suchasthrombinintheregionthatlinksthetwopor-erichiacolioryeast.However,notallanimalproteinstionsoftheprotein.Thispermitsremovaloftheaddedcanbeexpressedinactiveforminmicrobialcells,norfusiondomainfollowingaffinitypurification.domicrobesperformcertainposttranslationalprocess-ingtasks.Forthesereasons,agenemaybeexpressedinSite-DirectedMutagenesisProvidesculturedanimalcellsystemsemployingthebaculovirusMechanisticInsightsexpressionvectortotransformculturedinsectcells.FormoredetailsconcerningrecombinantDNAtechniques,OncetheabilitytoexpressaproteinfromitsclonedseeChapter40.genehasbeenestablished,itispossibletoemploysite-directedmutagenesistochangespecificaminoacylRecombinantFusionProteinsArePurifiedresiduesbyalteringtheircodons.Usedincombinationwithkineticanalysesandx-raycrystallography,thisap-byAffinityChromatographyproachfacilitatesidentificationofthespecificrolesofRecombinantDNAtechnologycanalsobeusedtocre-givenaminoacylresiduesinsubstratebindingandcatal-atemodifiedproteinsthatarereadilypurifiedbyaffinityysis.Forexample,theinferencethataparticularchromatography.Thegeneofinterestislinkedtoanaminoacylresiduefunctionsasageneralacidcanbeoligonucleotidesequencethatencodesacarboxylortestedbyreplacingitwithanaminoacylresidueinca-aminoterminalextensiontotheencodedprotein.Thepableofdonatingaproton.
67ENZYMES:MECHANISMOFACTION/59GSTTEnzyme•Catalyticmechanismsemployedbyenzymesincludetheintroductionofstrain,approximationofreac-tants,acid-basecatalysis,andcovalentcatalysis.PlasmidencodingGSTClonedDNAwiththrombinsite(T)encodingenzyme•Aminoacylresiduesthatparticipateincatalysisarehighlyconservedamongallclassesofagivenenzymeactivity.•Substratesandenzymesinducemutualconforma-Ligatetogethertionalchangesinoneanotherthatfacilitatesubstraterecognitionandcatalysis.•Thecatalyticactivityofenzymesrevealstheirpres-GSTTEnzymeence,facilitatestheirdetection,andprovidesthebasisforenzyme-linkedimmunoassays.Transfectcells,add•Manyenzymescanbeassayedspectrophotometri-inducingagent,thenbreakcellscallybycouplingthemtoanNAD(P)+-dependentdehydrogenase.Applytoglutathione(GSH)•Assayofplasmaenzymesaidsdiagnosisandprogno-affinitycolumnsis.Forexample,amyocardialinfarctionelevatesserumlevelsoflactatedehydrogenaseisozymeI1.SepharoseGSHGSTTEnzyme•Restrictionendonucleasesfacilitatediagnosisofge-beadneticdiseasesbyrevealingrestrictionfragmentlengthElutewithGSH,polymorphisms.treatwiththrombin•Site-directedmutagenesis,usedtochangeresiduessuspectedofbeingimportantincatalysisorsubstrateGSHGSTTEnzymebinding,providesinsightsintothemechanismsofenzymeaction.Figure7–12.UseofglutathioneS-transferase(GST)•RecombinantfusionproteinssuchasHis-taggedorGSTfusionenzymesarereadilypurifiedbyaffinityfusionproteinstopurifyrecombinantproteins.(GSH,chromatography.glutathione.)REFERENCESSUMMARYConyersGBetal:Metalrequirementsofadiadenosinepyrophos-phatasefromBartonellabacilliformis.Magneticresonanceand•Enzymesarehighlyeffectiveandextremelyspecific2+kineticstudiesoftheroleofMn.Biochemistry2000;catalysts.39:2347.•Organicandinorganicprostheticgroups,cofactors,FershtA:StructureandMechanisminProteinScience:AGuidetoandcoenzymesplayimportantrolesincatalysis.EnzymeCatalysisandProteinFolding.Freeman,1999.Coenzymes,manyofwhicharederivativesofBvita-SucklingCJ:EnzymeChemistry.Chapman&Hall,1990.mins,serveas“shuttles.”WalshCT:EnzymaticReactionMechanisms.Freeman,1979.
68Enzymes:Kinetics8VictorW.Rodwell,PhD,&PeterJ.Kennelly,PhDBIOMEDICALIMPORTANCEAB+→P+Q(2)EnzymekineticsisthefieldofbiochemistryconcernedUnidirectionalarrowsarealsousedtodescribereac-withthequantitativemeasurementoftheratesofen-tionsinlivingcellswheretheproductsofreaction(2)zyme-catalyzedreactionsandthesystematicstudyoffac-areimmediatelyconsumedbyasubsequentenzyme-torsthataffecttheserates.Kineticanalysespermitscien-catalyzedreaction.TherapidremovalofproductPortiststoreconstructthenumberandorderoftheQthereforeprecludesoccurrenceofthereversereac-individualstepsbywhichenzymestransformsubstratestion,renderingequation(2)functionallyirreversibleintoproducts.Thestudyofenzymekineticsalsorepre-underphysiologicconditions.sentstheprincipalwaytoidentifypotentialtherapeuticagentsthatselectivelyenhanceorinhibittheratesofspe-cificenzyme-catalyzedprocesses.Togetherwithsite-CHANGESINFREEENERGYDETERMINEdirectedmutagenesisandothertechniquesthatprobeTHEDIRECTION&EQUILIBRIUMSTATEproteinstructure,kineticanalysiscanalsorevealdetailsOFCHEMICALREACTIONSofthecatalyticmechanism.Acomplete,balancedsetofenzymeactivitiesisoffundamentalimportanceformain-TheGibbsfreeenergychangeΔG(alsocalledeitherthetaininghomeostasis.Anunderstandingofenzymekinet-freeenergyorGibbsenergy)describesboththedirec-icsthusisimportantforunderstandinghowphysiologictioninwhichachemicalreactionwilltendtoproceedstressessuchasanoxia,metabolicacidosisoralkalosis,andtheconcentrationsofreactantsandproductsthattoxins,andpharmacologicagentsaffectthatbalance.willbepresentatequilibrium.ΔGforachemicalreac-tionequalsthesumofthefreeenergiesofformationofCHEMICALREACTIONSAREDESCRIBEDthereactionproductsΔGPminusthesumofthefree0energiesofformationofthesubstratesΔGS.ΔGde-USINGBALANCEDEQUATIONSnotesthechangeinfreeenergythataccompaniestransi-Abalancedchemicalequationliststheinitialchemicaltionfromthestandardstate,one-molarconcentrationsspecies(substrates)presentandthenewchemicalofsubstratesandproducts,toequilibrium.Amoreuse-0′0species(products)formedforaparticularchemicalre-fulbiochemicaltermisΔG,whichdefinesΔGata−7action,allintheircorrectproportionsorstoichiome-standardstateof10Mprotons,pH7.0(Chapter10).try.Forexample,balancedequation(1)belowdescribesIfthefreeenergyoftheproductsislowerthanthatof00′thereactionofonemoleculeeachofsubstratesAandBthesubstrates,thesignsofΔGandΔGwillbenega-toformonemoleculeeachofproductsPandQ.tive,indicatingthatthereactionaswrittenisfavoredinthedirectionlefttoright.SuchreactionsarereferredtoAB+←→PQ+(1)asspontaneous.ThesignandthemagnitudeofthefreeenergychangedeterminehowfarthereactionwillThedoublearrowsindicatereversibility,anintrinsicproceed.Equation(3)—propertyofallchemicalreactions.Thus,forreaction(1),ifAandBcanformPandQ,thenPandQcanΔGR0=−TlnKeq(3)alsoformAandB.Designationofaparticularreactantasa“substrate”or“product”isthereforesomewhatar-—illustratestherelationshipbetweentheequilibriumbitrarysincetheproductsforareactionwritteninone0constantKeqandΔG,whereRisthegasconstant(1.98directionarethesubstratesforthereversereaction.Thecal/mol/°Kor8.31J/mol/°K)andTistheabsoluteterm“products”is,however,oftenusedtodesignatetemperatureindegreesKelvin.Keqisequaltotheprod-thereactantswhoseformationisthermodynamicallyfa-uctoftheconcentrationsofthereactionproducts,eachvored.Reactionsforwhichthermodynamicfactorsraisedtothepoweroftheirstoichiometry,dividedbystronglyfavorformationoftheproductstowhichthetheproductofthesubstrates,eachraisedtothepowerarrowpointsoftenarerepresentedwithasinglearrowoftheirstoichiometry.asiftheywere“irreversible”:60
69ENZYMES:KINETICS/61ForthereactionA+B→P+Q—characteristicchangesinfreeenergy,ΔG,andΔGareFDassociatedwitheachpartialreaction.[][]PQKeq=(4)[][]ABERL+−→←ERLLLGΔF(8)andforreaction(5)ERLLL→←E−ΔRL+GD(9)→(5)AA+←PERL+−−←→ERLGG+=+ΔΔΔFDG(8-10)[]PKeq=(6)2Fortheoverallreaction(10),ΔGisthesumofΔGand[]AFΔG.Asforanyequationoftwoterms,itisnotpossi-0D—ΔGmaybecalculatedfromequation(3)ifthecon-bletoinferfromΔGeitherthesignorthemagnitudecentrationsofsubstratesandproductspresentatequi-ofΔGorΔG.0FDlibriumareknown.IfΔGisanegativenumber,KeqManyreactionsinvolvemultipletransitionstates,willbegreaterthanunityandtheconcentrationofeachwithanassociatedchangeinfreeenergy.Fortheseproductsatequilibriumwillexceedthatofsubstrates.Ifreactions,theoverallΔGrepresentsthesumofallof0ΔGispositive,Keqwillbelessthanunityandthefor-thefreeenergychangesassociatedwiththeformationmationofsubstrateswillbefavored.anddecayofallofthetransitionstates.Therefore,itis0Noticethat,sinceΔGisafunctionexclusivelyofnotpossibletoinferfromtheoverallGthenum-theinitialandfinalstatesofthereactingspecies,itcanberortypeoftransitionstatesthroughwhichthere-provideinformationonlyaboutthedirectionandequi-actionproceeds.Statedanotherway:overallthermo-0libriumstateofthereaction.ΔGisindependentofthedynamicstellsusnothingaboutkinetics.mechanismofthereactionandthereforeprovidesnoinformationconcerningratesofreactions.Conse-ΔGFDefinestheActivationEnergyquently—andasexplainedbelow—althoughareaction00′RegardlessofthesignormagnitudeofΔG,ΔGforthemayhavealargenegativeΔGorΔG,itmaynever-Fthelesstakeplaceatanegligiblerate.overwhelmingmajorityofchemicalreactionshasapos-itivesign.Theformationoftransitionstateintermedi-atesthereforerequiressurmountingofenergybarriers.THERATESOFREACTIONSForthisreason,ΔGisoftentermedtheactivationen-FAREDETERMINEDBYTHEIRergy,Eact,theenergyrequiredtosurmountagivenen-ACTIVATIONENERGYergybarrier.Theease—andhencethefrequency—withwhichthisbarrierisovercomeisinverselyrelatedtoReactionsProceedviaTransitionStatesEact.ThethermodynamicparametersthatdeterminehowfastareactionproceedsthusaretheΔGvaluesforTheconceptofthetransitionstateisfundamentaltoFformationofthetransitionstatesthroughwhichthere-understandingthechemicalandthermodynamicbasisactionproceeds.Forasimplereaction,wheremeansofcatalysis.Equation(7)depictsadisplacementreac-“proportionateto,”tioninwhichanenteringgroupEdisplacesaleavinggroupL,attachedinitiallytoR.−Eact(11)Ratee∝RTERL+−−→←ERL+(7)Midwaythroughthedisplacement,thebondbetweenTheactivationenergyforthereactionproceedingintheRandLhasweakenedbuthasnotyetbeencompletelyoppositedirectiontothatdrawnisequalto−ΔG.Dsevered,andthenewbondbetweenEandRisasyetincompletelyformed.Thistransientintermediate—inNUMEROUSFACTORSAFFECTwhichneitherfreesubstratenorproductexists—istermedthetransitionstate,ERL.DottedlinesTHEREACTIONRATErepresentthe“partial”bondsthatareundergoingfor-Thekinetictheory—alsocalledthecollisiontheory—mationandrupture.ofchemicalkineticsstatesthatfortwomoleculestoReaction(7)canbethoughtofasconsistingoftworeacttheymust(1)approachwithinbond-formingdis-“partialreactions,”thefirstcorrespondingtotheforma-tanceofoneanother,or“collide”;and(2)mustpossesstion(F)andthesecondtothesubsequentdecay(D)ofsufficientkineticenergytoovercometheenergybarrierthetransitionstateintermediate.Asforallreactions,forreachingthetransitionstate.Itthereforefollows
7062/CHAPTER8thatanythingwhichincreasesthefrequencyorenergyofwhichcanalsobewrittenascollisionbetweensubstrateswillincreasetherateofthereactioninwhichtheyparticipate.ABBP++→(15)thecorrespondingrateexpressionisTemperatureRaisingthetemperatureincreasesthekineticenergyofRate∝[][][]ABB(16)molecules.AsillustratedinFigure8–1,thetotalnum-orberofmoleculeswhosekineticenergyexceedstheen-ergybarrierEact(verticalbar)forformationofproductsRate∝[][]AB2(17)increasesfromlow(A),throughintermediate(B),tohigh(C)temperatures.IncreasingthekineticenergyofForthegeneralcasewhennmoleculesofAreactwithmoleculesalsoincreasestheirmotionandthereforethemmoleculesofB,frequencywithwhichtheycollide.Thiscombinationofmorefrequentandmorehighlyenergeticandproduc-nAmB+→P(18)tivecollisionsincreasesthereactionrate.therateexpressionisReactantConcentrationnmRate∝[][]AB(19)Thefrequencywithwhichmoleculescollideisdirectlyproportionatetotheirconcentrations.FortwodifferentReplacingtheproportionalityconstantwithanequalmoleculesAandB,thefrequencywithwhichtheycol-signbyintroducingaproportionalityorrateconstantlidewilldoubleiftheconcentrationofeitherAorBiskcharacteristicofthereactionunderstudygivesequa-doubled.IftheconcentrationsofbothAandBaredou-tions(20)and(21),inwhichthesubscripts1and−1bled,theprobabilityofcollisionwillincreasefourfold.refertotherateconstantsfortheforwardandreverseForachemicalreactionproceedingatconstanttem-reactions,respectively.peraturethatinvolvesonemoleculeeachofAandB,Rate=kAB[][]nm11(20)ABP+→(12)thenumberofmoleculesthatpossesskineticenergyRate−−11=kP[](21)sufficienttoovercometheactivationenergybarrierwillbeaconstant.ThenumberofcollisionswithsufficientenergytoproduceproductPthereforewillbedirectlyKeqIsaRatioofRateConstantsproportionatetothenumberofcollisionsbetweenAWhileallchemicalreactionsaretosomeextentre-andBandthustotheirmolarconcentrations,denotedversible,atequilibriumtheoverallconcentrationsofre-bysquarebrackets.actantsandproductsremainconstant.Atequilibrium,therateofconversionofsubstratestoproductsthere-Rate∝[][]AB(13)foreequalstherateatwhichproductsareconvertedtosubstrates.Similarly,forthereactionrepresentedbyABP+→2(14)Rate11=Rate−(22)Therefore,kAB[][]nm=kP[](23)11−Energybarrier∞andABCk1[]P(24)=k[][]ABnm−1NumberofmoleculesTheratioofk1tok−1istermedtheequilibriumcon-0stant,Keq.Thefollowingimportantpropertiesofasys-∞Kineticenergytematequilibriummustbekeptinmind:Figure8–1.Theenergybarrierforchemical(1)Theequilibriumconstantisaratioofthereactionrateconstants(notthereactionrates).reactions.
71ENZYMES:KINETICS/63(2)Atequilibrium,thereactionrates(nottherateoΔGR=−TlnKeq(25)constants)oftheforwardandbackreactionsareequal.Ifweincludethepresenceoftheenzyme(E)inthecal-(3)Equilibriumisadynamicstate.Althoughthereisculationoftheequilibriumconstantforareaction,nonetchangeintheconcentrationofsubstratesorproducts,individualsubstrateandproductABEnz++→←P+Q+Enz(26)moleculesarecontinuallybeinginterconverted.theexpressionfortheequilibriumconstant,(4)ThenumericvalueoftheequilibriumconstantKeqcanbecalculatedeitherfromtheconcentra-[][][]PQEnzKeq=(27)tionsofsubstratesandproductsatequilibriumor[][][]ABEnzfromtheratiok1/k−1.reducestooneidenticaltothatforthereactionintheabsenceoftheenzyme:THEKINETICSOFENZYMATICCATALYSIS[][]PQ(28)Keq=[][]ABEnzymesLowertheActivationEnergyBarrierforaReactionEnzymesthereforehavenoeffectonKeq.Allenzymesacceleratereactionratesbyprovidingtran-sitionstateswithaloweredΔGforformationoftheFtransitionstates.However,theymaydifferinthewayMULTIPLEFACTORSAFFECTTHERATESthisisachieved.WherethemechanismorthesequenceOFENZYME-CATALYZEDREACTIONSofchemicalstepsattheactivesiteisessentiallythesameTemperatureasthoseforthesamereactionproceedingintheabsenceofacatalyst,theenvironmentoftheactivesitelowersRaisingthetemperatureincreasestherateofbothuncat-GFbystabilizingthetransitionstateintermediates.Asalyzedandenzyme-catalyzedreactionsbyincreasingthediscussedinChapter7,stabilizationcaninvolve(1)kineticenergyandthecollisionfrequencyofthereact-acid-basegroupssuitablypositionedtotransferprotonsingmolecules.However,heatenergycanalsoincreasetoorfromthedevelopingtransitionstateintermediate,thekineticenergyoftheenzymetoapointthatexceeds(2)suitablypositionedchargedgroupsormetalionstheenergybarrierfordisruptingthenoncovalentinter-thatstabilizedevelopingcharges,or(3)theimpositionactionsthatmaintaintheenzyme’sthree-dimensionalofstericstrainonsubstratessothattheirgeometryap-structure.Thepolypeptidechainthenbeginstounfold,proachesthatofthetransitionstate.HIVprotease(Fig-ordenature,withanaccompanyingrapidlossofcat-ure7–6)illustratescatalysisbyanenzymethatlowersalyticactivity.Thetemperaturerangeoverwhichantheactivationbarrierbystabilizingatransitionstatein-enzymemaintainsastable,catalyticallycompetentcon-termediate.formationdependsupon—andtypicallymoderatelyCatalysisbyenzymesthatproceedsviaauniquere-exceeds—thenormaltemperatureofthecellsinwhichactionmechanismtypicallyoccurswhenthetransitionitresides.Enzymesfromhumansgenerallyexhibitsta-stateintermediateformsacovalentbondwiththeen-bilityattemperaturesupto45–55°C.Bycontrast,zyme(covalentcatalysis).Thecatalyticmechanismofenzymesfromthethermophilicmicroorganismsthatre-theserineproteasechymotrypsin(Figure7–7)illus-sideinvolcanichotspringsorunderseahydrothermaltrateshowanenzymeutilizescovalentcatalysistopro-ventsmaybestableuptoorabove100°C.videauniquereactionpathway.TheQ10,ortemperaturecoefficient,isthefactorbywhichtherateofabiologicprocessincreasesforaENZYMESDONOTAFFECTK10°Cincreaseintemperature.Forthetemperatureseqoverwhichenzymesarestable,theratesofmostbio-Enzymesacceleratereactionratesbyloweringtheacti-logicprocessestypicallydoublefora10°Criseintem-vationbarrierΔGF.Whiletheymayundergotransientperature(Q10=2).Changesintheratesofenzyme-modificationduringtheprocessofcatalysis,enzymescatalyzedreactionsthataccompanyariseorfallinbodyemergeunchangedatthecompletionofthereaction.temperatureconstituteaprominentsurvivalfeatureforThepresenceofanenzymethereforehasnoeffecton“cold-blooded”lifeformssuchaslizardsorfish,whose0ΔGfortheoverallreaction,whichisafunctionsolelybodytemperaturesaredictatedbytheexternalenviron-oftheinitialandfinalstatesofthereactants.Equationment.However,formammalsandotherhomeothermic(25)showstherelationshipbetweentheequilibriumorganisms,changesinenzymereactionrateswithtem-constantforareactionandthestandardfreeenergyperatureassumephysiologicimportanceonlyincir-changeforthatreaction:cumstancessuchasfeverorhypothermia.
7264/CHAPTER8HydrogenIonConcentrationtherateoftheforwardreaction.Assaysofenzymeactiv-ityalmostalwaysemployalarge(103–107)molarexcessTherateofalmostallenzyme-catalyzedreactionsex-ofsubstrateoverenzyme.Undertheseconditions,viishibitsasignificantdependenceonhydrogenioncon-proportionatetotheconcentrationofenzyme.Measur-centration.MostintracellularenzymesexhibitoptimalingtheinitialvelocitythereforepermitsonetoestimateactivityatpHvaluesbetween5and9.Therelationshipthequantityofenzymepresentinabiologicsample.ofactivitytohydrogenionconcentration(Figure8–2)reflectsthebalancebetweenenzymedenaturationathighorlowpHandeffectsonthechargedstateoftheSUBSTRATECONCENTRATIONAFFECTSenzyme,thesubstrates,orboth.ForenzymeswhoseREACTIONRATEmechanisminvolvesacid-basecatalysis,theresiduesin-Inwhatfollows,enzymereactionsaretreatedasiftheyvolvedmustbeintheappropriatestateofprotonationhadonlyasinglesubstrateandasingleproduct.Whileforthereactiontoproceed.Thebindingandrecogni-mostenzymeshavemorethanonesubstrate,theprinci-tionofsubstratemoleculeswithdissociablegroupsalsoplesdiscussedbelowapplywithequalvaliditytoen-typicallyinvolvestheformationofsaltbridgeswiththezymeswithmultiplesubstrates.enzyme.ThemostcommonchargedgroupsaretheForatypicalenzyme,assubstrateconcentrationisnegativecarboxylategroupsandthepositivelychargedincreased,viincreasesuntilitreachesamaximumvaluegroupsofprotonatedamines.GainorlossofcriticalVmax(Figure8–3).Whenfurtherincreasesinsubstratechargedgroupsthuswilladverselyaffectsubstratebind-concentrationdonotfurtherincreasevi,theenzymeisingandthuswillretardorabolishcatalysis.saidtobe“saturated”withsubstrate.Notethattheshapeofthecurvethatrelatesactivitytosubstratecon-ASSAYSOFENZYME-CATALYZEDcentration(Figure8–3)ishyperbolic.Atanygivenin-REACTIONSTYPICALLYMEASUREstant,onlysubstratemoleculesthatarecombinedwithTHEINITIALVELOCITYtheenzymeasanEScomplexcanbetransformedintoproduct.Second,theequilibriumconstantforthefor-Mostmeasurementsoftheratesofenzyme-catalyzedre-mationoftheenzyme-substratecomplexisnotinfi-actionsemployrelativelyshorttimeperiods,conditionsnitelylarge.Therefore,evenwhenthesubstrateispre-thatapproximateinitialrateconditions.Underthesesentinexcess(pointsAandBofFigure8–4),onlyaconditions,onlytracesofproductaccumulate,hencefractionoftheenzymemaybepresentasanEScom-therateofthereversereactionisnegligible.Theinitialplex.AtpointsAorB,increasingordecreasing[S]velocity(vi)ofthereactionthusisessentiallythatofthereforewillincreaseordecreasethenumberofEScomplexeswithacorrespondingchangeinvi.AtpointC(Figure8–4),essentiallyalltheenzymeispresentasXtheEScomplex.Sincenofreeenzymeremainsavailable100forformingES,furtherincreasesin[S]cannotincreasetherateofthereaction.Underthesesaturatingcondi-tions,vidependssolelyon—andthusislimitedby—SH+E–therapiditywithwhichfreeenzymeisreleasedtocom-binewithmoresubstrate.%Vmax0LowHighCVmax/2pHviBFigure8–2.EffectofpHonenzymeactivity.Con-−V/2sider,forexample,anegativelychargedenzyme(EH)Amaxthatbindsapositivelychargedsubstrate(SH+).Shownistheproportion(%)ofSH+[\\\]andofEH−[///]asaKm[S]functionofpH.Onlyinthecross-hatchedareadoboththeenzymeandthesubstratebearanappropriateFigure8–3.Effectofsubstrateconcentrationonthecharge.initialvelocityofanenzyme-catalyzedreaction.
73ENZYMES:KINETICS/65=S=EABCFigure8–4.Representationofanenzymeatlow(A),athigh(C),andatasubstrateconcentrationequaltoKm(B).PointsA,B,andCcorrespondtothosepointsinFigure8–3.THEMICHAELIS-MENTEN&HILLequalto[S].ReplacingKm+[S]with[S]reducesequa-EQUATIONSMODELTHEEFFECTStion(29)toOFSUBSTRATECONCENTRATIONv=Vmax[]Sv≈≈Vmax[]SV(31)TheMichaelis-MentenEquationiimaxKm+S[][S]TheMichaelis-Mentenequation(29)illustratesinmathematicaltermstherelationshipbetweeninitialre-Thus,when[S]greatlyexceedsKm,thereactionvelocityactionvelocityviandsubstrateconcentration[S],ismaximal(Vmax)andunaffectedbyfurtherincreasesinshowngraphicallyinFigure8–3.substrateconcentration.(3)When[S]=Km(pointBinFigures8–3andVmax[]S8–4).vi=(29)Km+S[]Vmax[]SVVmax[]Smax(32)vi===TheMichaelisconstantKmisthesubstrateconcen-Km+S[]22[]Strationatwhichviishalfthemaximalvelocity(Vmax/2)attainableataparticularconcentrationofEquation(32)statesthatwhen[S]equalsKm,theinitialenzyme.Kmthushasthedimensionsofsubstratecon-velocityishalf-maximal.Equation(32)alsorevealsthatcentration.ThedependenceofinitialreactionvelocityKmis—andmaybedeterminedexperimentallyfrom—on[S]andKmmaybeillustratedbyevaluatingthethesubstrateconcentrationatwhichtheinitialvelocityMichaelis-Mentenequationunderthreeconditions.ishalf-maximal.(1)When[S]ismuchlessthanKm(pointAinFig-ures8–3and8–4),thetermKm+[S]isessentiallyequalALinearFormoftheMichaelis-MententoKm.ReplacingKm+[S]withKmreducesequation(29)toEquationIsUsedtoDetermineKm&VmaxThedirectmeasurementofthenumericvalueofVmaxVmax[]SVmax[]S⎛Vmax⎞(30)andthereforethecalculationofKmoftenrequiresim-v11=v≈≈⎜⎟S[]practicallyhighconcentrationsofsubstratetoachieveKmm+[]SK⎝Km⎠saturatingconditions.AlinearformoftheMichaelis-where≈means“approximatelyequalto.”SinceVmaxMentenequationcircumventsthisdifficultyandper-andKmarebothconstants,theirratioisaconstant.InmitsVmaxandKmtobeextrapolatedfrominitialveloc-otherwords,when[S]isconsiderablybelowKm,vi∝itydataobtainedatlessthansaturatingconcentrationsk[S].Theinitialreactionvelocitythereforeisdirectlyofsubstrate.Startingwithequation(29),proportionateto[S].(2)When[S]ismuchgreaterthanKm(pointCinv=Vmax[]S(29)iFigures8–3and8–4),thetermKm+[S]isessentiallyKm+S[]
7466/CHAPTER8invertStatedanotherway,thesmallerthetendencyoftheen-zymeanditssubstratetodissociate,thegreatertheaffin-1=Km+[]S(33)ityoftheenzymeforitssubstrate.WhiletheMichaelisv1Vmax[]SconstantKmoftenapproximatesthedissociationcon-stantKd,thisisbynomeansalwaysthecase.Foratypi-factorcalenzyme-catalyzedreaction,1Km[]S(34)=+k1k2vSiVmax[]Vmax[]S(39)ES+→←ES→EP+andsimplifyk−111=⎛Km⎞1(35)thevalueof[S]thatgivesvi=Vmax/2is⎜⎟+vSi⎝Vmax⎠[]Vmax[]S=kk−12+=K(40)Equation(35)istheequationforastraightline,y=axmk1+b,wherey=1/viandx=1/[S].Aplotof1/viasyasafunctionof1/[S]asxthereforegivesastraightlineWhenk−1»k2,thenwhoseyinterceptis1/VmaxandwhoseslopeisKm/Vmax.SuchaplotiscalledadoublereciprocalorLineweaver-Burkplot(Figure8–5).Settingtheytermkkk−−121+≈(41)ofequation(36)equaltozeroandsolvingforxrevealsandthatthexinterceptis−1/Km.−−b1(36)[]S≈≈k1K(42)0=+axb;therefore,x==daKmk−1Kmisthusmosteasilycalculatedfromthexintercept.Hence,1/Kmonlyapproximates1/KdunderconditionswheretheassociationanddissociationoftheEScom-KmMayApproximateaBindingConstantplexisrapidrelativetotherate-limitingstepincataly-sis.Forthemanyenzyme-catalyzedreactionsforwhichTheaffinityofanenzymeforitssubstrateistheinversek−1+k2isnotapproximatelyequaltok−1,1/KmwillofthedissociationconstantKdfordissociationoftheunderestimate1/Kd.enzymesubstratecomplexES.k1TheHillEquationDescribestheBehaviorES+→←ES(37)ofEnzymesThatExhibitCooperativek−1BindingofSubstrateKk−1(38)Whilemostenzymesdisplaythesimplesaturationki-d=neticsdepictedinFigure8–3andareadequatelyde-k1scribedbytheMichaelis-Mentenexpression,someen-zymesbindtheirsubstratesinacooperativefashionanalogoustothebindingofoxygenbyhemoglobin(Chapter6).CooperativebehaviormaybeencounteredKmformultimericenzymesthatbindsubstrateatmultiple1Slope=vVmaxsites.Forenzymesthatdisplaypositivecooperativityinibindingsubstrate,theshapeofthecurvethatrelateschangesinvitochangesin[S]issigmoidal(Figure18–6).NeithertheMichaelis-Mentenexpressionnorits–K1deriveddouble-reciprocalplotscanbeusedtoevaluatemVmaxcooperativesaturationkinetics.Enzymologiststherefore0employagraphicrepresentationoftheHillequation1[S]originallyderivedtodescribethecooperativebindingofO2byhemoglobin.Equation(43)representstheHillFigure8–5.DoublereciprocalorLineweaver-Burkequationarrangedinaformthatpredictsastraightline,plotof1/viversus1/[S]usedtoevaluateKmandVmax.wherek′isacomplexconstant.
75ENZYMES:KINETICS/67∞firstsubstratemoleculethenenhancestheaffinityoftheenzymeforbindingadditionalsubstrate.Thegreaterthevalueforn,thehigherthedegreeofcooperativityandthemoresigmoidalwillbetheplotofviversus[S].Aperpendiculardroppedfromthepointwheretheytermlogvi/(Vmax−vi)iszerointersectsthexaxisatasubstrateconcentrationtermedS50,thesubstratecon-vicentrationthatresultsinhalf-maximalvelocity.Sthus50isanalogoustotheP50foroxygenbindingtohemoglo-bin(Chapter6).KINETICANALYSISDISTINGUISHESCOMPETITIVEFROM0∞NONCOMPETITIVEINHIBITION[S]InhibitorsofthecatalyticactivitiesofenzymesprovideFigure8–6.Representationofsigmoidsubstratebothpharmacologicagentsandresearchtoolsforstudysaturationkinetics.ofthemechanismofenzymeaction.Inhibitorscanbeclassifiedbasedupontheirsiteofactionontheenzyme,onwhetherornottheychemicallymodifytheenzyme,oronthekineticparameterstheyinfluence.Kinetically,logv1(43)=log[S]n−′logkwedistinguishtwoclassesofinhibitorsbaseduponVmax−v1whetherraisingthesubstrateconcentrationdoesorEquation(43)statesthatwhen[S]islowrelativetok′,doesnotovercometheinhibition.theinitialreactionvelocityincreasesasthenthpowerof[S].CompetitiveInhibitorsTypicallyAgraphoflogvi/(Vmax−vi)versuslog[S]givesaResembleSubstratesstraightline(Figure8–7),wheretheslopeofthelinenTheeffectsofcompetitiveinhibitorscanbeovercomeistheHillcoefficient,anempiricalparameterwhosebyraisingtheconcentrationofthesubstrate.Mostfre-valueisafunctionofthenumber,kind,andstrengthofquently,incompetitiveinhibitiontheinhibitor,I,theinteractionsofthemultiplesubstrate-bindingsitesbindstothesubstrate-bindingportionoftheactivesiteontheenzyme.Whenn=1,allbindingsitesbehavein-andblocksaccessbythesubstrate.Thestructuresofdependently,andsimpleMichaelis-Mentenkineticbe-mostclassiccompetitiveinhibitorsthereforetendtore-haviorisobserved.Ifnisgreaterthan1,theenzymeissemblethestructuresofasubstrateandthusaretermedsaidtoexhibitpositivecooperativity.Bindingofthesubstrateanalogs.Inhibitionoftheenzymesuccinatedehydrogenasebymalonateillustratescompetitiveinhi-bitionbyasubstrateanalog.Succinatedehydrogenase1catalyzestheremovalofonehydrogenatomfromeachiofthetwomethylenecarbonsofsuccinate(Figure8–8).i–vBothsuccinateanditsstructuralanalogmalonatev(−OOC⎯CH⎯COO−)canbindtotheactivesiteof0Slope=n2maxVsuccinatedehydrogenase,forminganESoranEIcom-plex,respectively.However,sincemalonatecontainsLog–1H–4S50–3HCCOO––2HHCCOO–Log[S]––OOCCHOOCCHSUCCINATEFigure8–7.AgraphicrepresentationofalinearHDEHYDROGENASEformoftheHillequationisusedtoevaluateS50,theSuccinateFumaratesubstrateconcentrationthatproduceshalf-maximalvelocity,andthedegreeofcooperativityn.Figure8–8.Thesuccinatedehydrogenasereaction.
7668/CHAPTER8onlyonemethylenecarbon,itcannotundergodehy-drogenation.TheformationanddissociationoftheEIcomplexisadynamicprocessdescribedby1k1viEnzI→←EnzI+(44)k−1+Inhibitor–1NoinhibitorK′mforwhichtheequilibriumconstantKis–11iKmVmax01[]EnzI[]k1(45)[S]K1==[]EnzIk−1Figure8–9.Lineweaver-Burkplotofcompetitivein-hibition.NotethecompletereliefofinhibitionathighIneffect,acompetitiveinhibitoractsbydecreasing[S](ie,low1/[S]).thenumberoffreeenzymemoleculesavailabletobindsubstrate,ie,toformES,andthuseventuallytoformproduct,asdescribedbelow:Forsimplecompetitiveinhibition,theintercepton±IE-IthexaxisisE±SE-S−1⎛[]I⎞x=+⎜1⎟(47)E+P(46)Kmi⎝K⎠AcompetitiveinhibitorandsubstrateexertreciprocalOnceKmhasbeendeterminedintheabsenceofin-effectsontheconcentrationoftheEIandEScom-hibitor,Kicanbecalculatedfromequation(47).Kival-plexes.Sincebindingsubstrateremovesfreeenzymeuesareusedtocomparedifferentinhibitorsofthesameavailabletocombinewithinhibitor,increasingthe[S]enzyme.ThelowerthevalueforKi,themoreeffectivedecreasestheconcentrationoftheEIcomplexandtheinhibitor.Forexample,thestatindrugsthatactasraisesthereactionvelocity.Theextenttowhich[S]competitiveinhibitorsofHMG-CoAreductase(Chap-mustbeincreasedtocompletelyovercometheinhibi-ter26)haveKivaluesseveralordersofmagnitudelowertiondependsupontheconcentrationofinhibitorpre-thantheKmforthesubstrateHMG-CoA.sent,itsaffinityfortheenzymeKi,andtheKmoftheenzymeforitssubstrate.SimpleNoncompetitiveInhibitorsLowerDoubleReciprocalPlotsFacilitatetheVmaxbutDoNotAffectKmEvaluationofInhibitorsInnoncompetitiveinhibition,bindingoftheinhibitorDoublereciprocalplotsdistinguishbetweencompeti-doesnotaffectbindingofsubstrate.Formationofbothtiveandnoncompetitiveinhibitorsandsimplifyevalua-EIandEIScomplexesisthereforepossible.However,tionofinhibitionconstantsKi.viisdeterminedatsev-whiletheenzyme-inhibitorcomplexcanstillbindsub-eralsubstrateconcentrationsbothinthepresenceandstrate,itsefficiencyattransformingsubstratetoprod-intheabsenceofinhibitor.Forclassiccompetitiveinhi-uct,reflectedbyVmax,isdecreased.Noncompetitivebition,thelinesthatconnecttheexperimentaldatainhibitorsbindenzymesatsitesdistinctfromthesub-pointsmeetattheyaxis(Figure8–9).Sincetheyinter-strate-bindingsiteandgenerallybearlittleornostruc-ceptisequalto1/Vmax,thispatternindicatesthatwhenturalresemblancetothesubstrate.1/[S]approaches0,viisindependentofthepresenceForsimplenoncompetitiveinhibition,EandEIofinhibitor.Note,however,thattheinterceptonthepossessidenticalaffinityforsubstrate,andtheEIScom-xaxisdoesvarywithinhibitorconcentration—andthatplexgeneratesproductatanegligiblerate(Figure8–10).since−1/Km′issmallerthan1/Km,Km′(the“apparentMorecomplexnoncompetitiveinhibitionoccurswhenKm”)becomeslargerinthepresenceofincreasingcon-bindingoftheinhibitordoesaffecttheapparentaffinitycentrationsofinhibitor.Thus,acompetitiveinhibitoroftheenzymeforsubstrate,causingthelinestointer-hasnoeffectonVmaxbutraisesK′m,theapparentceptineitherthethirdorfourthquadrantsofadoubleKmforthesubstrate.reciprocalplot(notshown).
77ENZYMES:KINETICS/69ABPQ1EEAEAB-EPQEQEviABPQ1–+InhibitorV′max–1KNoinhibitor1EAEQmVmaxEEAB-EPQE01[S]EBEPFigure8–10.Lineweaver-Burkplotforsimplenon-BAQPcompetitiveinhibition.APBQEEA-FPFFB-EQEIrreversibleInhibitors“Poison”EnzymesFigure8–11.RepresentationsofthreeclassesofBi-Intheaboveexamples,theinhibitorsformadissocia-Bireactionmechanisms.Horizontallinesrepresenttheble,dynamiccomplexwiththeenzyme.Fullyactiveen-enzyme.Arrowsindicatetheadditionofsubstratesandzymecanthereforeberecoveredsimplybyremovingtheinhibitorfromthesurroundingmedium.However,departureofproducts.Top:AnorderedBi-Bireaction,avarietyofotherinhibitorsactirreversiblybychemi-characteristicofmanyNAD(P)H-dependentoxidore-callymodifyingtheenzyme.Thesemodificationsgen-ductases.Center:ArandomBi-Bireaction,characteris-erallyinvolvemakingorbreakingcovalentbondswithticofmanykinasesandsomedehydrogenases.Bot-aminoacylresiduesessentialforsubstratebinding,catal-tom:Aping-pongreaction,characteristicofysis,ormaintenanceoftheenzyme’sfunctionalconfor-aminotransferasesandserineproteases.mation.Sincethesecovalentchangesarerelativelysta-ble,anenzymethathasbeen“poisoned”byanirreversibleinhibitorremainsinhibitedevenafterre-movaloftheremaininginhibitorfromthesurroundingreactionsbecausethegroupundergoingtransferisusu-medium.allypasseddirectly,inasinglestep,fromonesubstratetotheother.SequentialBi-BireactionscanbefurtherdistinguishedbasedonwhetherthetwosubstratesaddMOSTENZYME-CATALYZEDREACTIONSinarandomorinacompulsoryorder.Forrandom-INVOLVETWOORMORESUBSTRATESorderreactions,eithersubstrateAorsubstrateBmayWhilemanyenzymeshaveasinglesubstrate,manyoth-combinefirstwiththeenzymetoformanEAoranEBershavetwo—andsometimesmorethantwo—sub-complex(Figure8–11,center).Forcompulsory-orderstratesandproducts.Thefundamentalprinciplesdis-reactions,AmustfirstcombinewithEbeforeBcancussedabove,whileillustratedforsingle-substratecombinewiththeEAcomplex.Oneexplanationforaenzymes,applyalsotomultisubstrateenzymes.Thecompulsory-ordermechanismisthattheadditionofAmathematicalexpressionsusedtoevaluatemultisub-inducesaconformationalchangeintheenzymethatstratereactionsare,however,complex.WhiledetailedalignsresidueswhichrecognizeandbindB.kineticanalysisofmultisubstratereactionsexceedsthescopeofthischapter,two-substrate,two-productreac-Ping-PongReactionstions(termed“Bi-Bi”reactions)areconsideredbelow.Theterm“ping-pong”appliestomechanismsinSequentialorSinglewhichoneormoreproductsarereleasedfromtheen-zymebeforeallthesubstrateshavebeenadded.Ping-DisplacementReactionspongreactionsinvolvecovalentcatalysisandatran-Insequentialreactions,bothsubstratesmustcombinesient,modifiedformoftheenzyme(Figure7–4).withtheenzymetoformaternarycomplexbeforePing-pongBi-Bireactionsaredoubledisplacementre-catalysiscanproceed(Figure8–11,top).Sequentialre-actions.Thegroupundergoingtransferisfirstdis-actionsaresometimesreferredtoassingledisplacementplacedfromsubstrateAbytheenzymetoformproduct
7870/CHAPTER8Increasing[S2]1viFigure8–12.Lineweaver-Burkplotforatwo-sub-strateping-pongreaction.Anincreaseinconcentra-tionofonesubstrate(S1)whilethatoftheothersub-1strate(S2)ismaintainedconstantchangesboththexS1andyintercepts,butnottheslope.Pandamodifiedformoftheenzyme(F).Thesubse-othercombinationsofproductinhibitorandvariablequentgrouptransferfromFtothesecondsubstrateB,substratewillproduceformsofcomplexnoncompeti-formingproductQandregeneratingE,constitutesthetiveinhibition.seconddisplacement(Figure8–11,bottom).MostBi-BiReactionsConformtoSUMMARYMichaelis-MentenKinetics•Thestudyofenzymekinetics—thefactorsthataffectMostBi-Bireactionsconformtoasomewhatmoretheratesofenzyme-catalyzedreactions—revealsthecomplexformofMichaelis-Mentenkineticsinwhichindividualstepsbywhichenzymestransformsub-Vmaxreferstothereactionrateattainedwhenbothsub-stratesintoproducts.stratesarepresentatsaturatinglevels.Eachsubstrate•ΔG,theoverallchangeinfreeenergyforareaction,hasitsowncharacteristicKmvaluewhichcorrespondsisindependentofreactionmechanismandprovidestotheconcentrationthatyieldshalf-maximalvelocitynoinformationconcerningratesofreactions.whenthesecondsubstrateispresentatsaturatinglevels.•EnzymesdonotaffectKeq.Keq,aratioofreactionAsforsingle-substratereactions,double-reciprocalplotsrateconstants,maybecalculatedfromtheconcentra-canbeusedtodetermineVmaxandKm.viismeasuredastionsofsubstratesandproductsatequilibriumorafunctionoftheconcentrationofonesubstrate(thefromtheratiok1/k−1.variablesubstrate)whiletheconcentrationoftheother•ReactionsproceedviatransitionstatesinwhichΔGsubstrate(thefixedsubstrate)ismaintainedconstant.IfFistheactivationenergy.Temperature,hydrogenionthelinesobtainedforseveralfixed-substrateconcentra-concentration,enzymeconcentration,substratecon-tionsareplottedonthesamegraph,itispossibletodis-centration,andinhibitorsallaffecttheratesofen-tinguishbetweenaping-pongenzyme,whichyieldszyme-catalyzedreactions.parallellines,andasequentialmechanism,whichyields•Ameasurementoftherateofanenzyme-catalyzedapatternofintersectinglines(Figure8–12).reactiongenerallyemploysinitialrateconditions,forProductinhibitionstudiesareusedtocomplementwhichtheessentialabsenceofproductprecludesthekineticanalysesandtodistinguishbetweenorderedandreversereaction.randomBi-Bireactions.Forexample,inarandom-orderBi-Bireaction,eachproductwillbeacompetitive•AlinearformoftheMichaelis-Mentenequationsim-inhibitorregardlessofwhichsubstrateisdesignatedtheplifiesdeterminationofKmandVmax.variablesubstrate.However,forasequentialmecha-•AlinearformoftheHillequationisusedtoevaluatenism(Figure8–11,bottom),onlyproductQwillgivethecooperativesubstrate-bindingkineticsexhibitedthepatternindicativeofcompetitiveinhibitionwhenAbysomemultimericenzymes.Theslopen,theHillisthevariablesubstrate,whileonlyproductPwillpro-coefficient,reflectsthenumber,nature,andstrengthducethispatternwithBasthevariablesubstrate.Theoftheinteractionsofthesubstrate-bindingsites.A
79ENZYMES:KINETICS/71valueofngreaterthan1indicatespositivecoopera-REFERENCEStivity.FershtA:StructureandMechanisminProteinScience:AGuideto•Theeffectsofcompetitiveinhibitors,whichtypicallyEnzymeCatalysisandProteinFolding.Freeman,1999.resemblesubstrates,areovercomebyraisingthecon-SchultzAR:EnzymeKinetics:FromDiastasetoMulti-enzymeSys-centrationofthesubstrate.Noncompetitivein-tems.CambridgeUnivPress,1994.hibitorslowerVmaxbutdonotaffectKm.SegelIH:EnzymeKinetics.WileyInterscience,1975.•Substratesmayaddinarandomorder(eithersub-stratemaycombinefirstwiththeenzyme)orinacompulsoryorder(substrateAmustbindbeforesub-strateB).•Inping-pongreactions,oneormoreproductsarere-leasedfromtheenzymebeforeallthesubstrateshaveadded.
80Enzymes:RegulationofActivities9VictorW.Rodwell,PhD,&PeterJ.Kennelly,PhDBIOMEDICALIMPORTANCEconcentrationgeneratecorrespondingchangesinme-taboliteflux(Figure9–1).Responsestochangesinsub-The19th-centuryphysiologistClaudeBernardenunci-stratelevelrepresentanimportantbutpassivemeansforatedtheconceptualbasisformetabolicregulation.Hecoordinatingmetaboliteflowandmaintaininghomeo-observedthatlivingorganismsrespondinwaysthatarestasisinquiescentcells.However,theyofferlimitedbothquantitativelyandtemporallyappropriatetoper-scopeforrespondingtochangesinenvironmentalvari-mitthemtosurvivethemultiplechallengesposedbyables.Themechanismsthatregulateenzymeactivityinchangesintheirexternalandinternalenvironments.anactivemannerinresponsetointernalandexternalWalterCannonsubsequentlycoinedtheterm“homeo-signalsarediscussedbelow.stasis”todescribetheabilityofanimalstomaintainaconstantintracellularenvironmentdespitechangesintheirexternalenvironment.Wenowknowthatorgan-MetaboliteFlowTendsismsrespondtochangesintheirexternalandinternaltoBeUnidirectionalenvironmentbybalanced,coordinatedchangesintheDespitetheexistenceofshort-termoscillationsinratesofspecificmetabolicreactions.Manyhumandis-metaboliteconcentrationsandenzymelevels,livingeases,includingcancer,diabetes,cysticfibrosis,andcellsexistinadynamicsteadystateinwhichthemeanAlzheimer’sdisease,arecharacterizedbyregulatorydys-concentrationsofmetabolicintermediatesremainrela-functionstriggeredbypathogenicagentsorgeneticmu-tivelyconstantovertime(Figure9–2).Whileallchemi-tations.Forexample,manyoncogenicviruseselaboratecalreactionsaretosomeextentreversible,inlivingcellsprotein-tyrosinekinasesthatmodifytheregulatorythereactionproductsserveassubstratesfor—andareeventswhichcontrolpatternsofgeneexpression,con-removedby—otherenzyme-catalyzedreactions.Manytributingtotheinitiationandprogressionofcancer.Thenominallyreversiblereactionsthusoccurunidirection-toxinfromVibriocholerae,thecausativeagentofcholera,ally.Thissuccessionofcoupledmetabolicreactionsisdisablessensor-responsepathwaysinintestinalepithelialaccompaniedbyanoverallchangeinfreeenergythatcellsbyADP-ribosylatingtheGTP-bindingproteinsfavorsunidirectionalmetaboliteflow(Chapter10).The(G-proteins)thatlinkcellsurfacereceptorstoadenylylunidirectionalflowofmetabolitesthroughapathwaycyclase.Theconsequentactivationofthecyclasetriggerswithalargeoverallnegativechangeinfreeenergyistheflowofwaterintotheintestines,resultinginmassiveanalogoustotheflowofwaterthroughapipeinwhichdiarrheaanddehydration.Yersiniapestis,thecausativeoneendislowerthantheother.Bendsorkinksintheagentofplague,elaboratesaprotein-tyrosinephos-pipesimulateindividualenzyme-catalyzedstepswithaphatasethathydrolyzesphosphorylgroupsonkeycy-smallnegativeorpositivechangeinfreeenergy.Flowoftoskeletalproteins.Knowledgeoffactorsthatcontrolthewaterthroughthepipeneverthelessremainsunidirec-ratesofenzyme-catalyzedreactionsthusisessentialtoantionalduetotheoverallchangeinheight,whichcorre-understandingofthemolecularbasisofdisease.Thisspondstotheoverallchangeinfreeenergyinapathwaychapteroutlinesthepatternsbywhichmetabolic(Figure9–3).processesarecontrolledandprovidesillustrativeexam-ples.Subsequentchaptersprovideadditionalexamples.COMPARTMENTATIONENSURESREGULATIONOFMETABOLITEFLOWMETABOLICEFFICIENCYCANBEACTIVEORPASSIVE&SIMPLIFIESREGULATIONEnzymesthatoperateattheirmaximalratecannotre-Ineukaryotes,anabolicandcatabolicpathwaysthatin-spondtoanincreaseinsubstrateconcentration,andterconvertcommonproductsmaytakeplaceinspecificcanrespondonlytoaprecipitousdecreaseinsubstratesubcellularcompartments.Forexample,manyoftheconcentration.Formostenzymes,therefore,theaver-enzymesthatdegradeproteinsandpolysaccharidesre-ageintracellularconcentrationoftheirsubstratetendssideinsideorganellescalledlysosomes.Similarly,fattytobeclosetotheKmvalue,sothatchangesinsubstrateacidbiosynthesisoccursinthecytosol,whereasfatty72
81ENZYMES:REGULATIONOFACTIVITIES/73ΔVBVΔVAAKmΔSΔSB[S]Figure9–1.Differentialresponseoftherateofanenzyme-catalyzedreaction,ΔV,tothesameincremen-Figure9–3.Hydrostaticanalogyforapathwaywithtalchangeinsubstrateconcentrationatasubstratearate-limitingstep(A)andastepwithaΔGvaluenearconcentrationofKm(ΔVA)orfaraboveKm(ΔVB).zero(B).acidoxidationtakesplacewithinmitochondria(Chap-generationfromthoseofNADPHthatparticipateinters21and22).Segregationofcertainmetabolicpath-thereductivestepsinmanybiosyntheticpathways.wayswithinspecializedcelltypescanprovidefurtherphysicalcompartmentation.Alternatively,possessionofControllinganEnzymeThatCatalyzesoneormoreuniqueintermediatescanpermitapparentlyaRate-LimitingReactionRegulatesopposingpathwaystocoexistevenintheabsenceofanEntireMetabolicPathwayphysicalbarriers.Forexample,despitemanysharedin-termediatesandenzymes,bothglycolysisandgluconeo-Whilethefluxofmetabolitesthroughmetabolicpath-genesisarefavoredenergetically.Thiscannotbetrueifwaysinvolvescatalysisbynumerousenzymes,activeallthereactionswerethesame.Ifonepathwaywasfa-controlofhomeostasisisachievedbyregulationofonlyvoredenergetically,theotherwouldbeaccompaniedbyasmallnumberofenzymes.Theidealenzymeforregu-achangeinfreeenergyGequalinmagnitudebutop-latoryinterventionisonewhosequantityorcatalyticef-positeinsign.Simultaneousspontaneityofbothpath-ficiencydictatesthatthereactionitcatalyzesisslowrel-waysresultsfromsubstitutionofoneormorereactionsativetoallothersinthepathway.Decreasingthebydifferentreactionsfavoredthermodynamicallyinthecatalyticefficiencyorthequantityofthecatalystfortheoppositedirection.Theglycolyticenzymephospho-“bottleneck”orrate-limitingreactionimmediatelyre-fructokinase(Chapter17)isreplacedbythegluco-ducesmetabolitefluxthroughtheentirepathway.Con-neogenicenzymefructose-1,6-bisphosphatase(Chapterversely,anincreaseineitheritsquantityorcatalyticef-19).Theabilityofenzymestodiscriminatebetweentheficiencyenhancesfluxthroughthepathwayasawhole.++structurallysimilarcoenzymesNADandNADPalsoForexample,acetyl-CoAcarboxylasecatalyzesthesyn-resultsinaformofcompartmentation,sinceitsegre-thesisofmalonyl-CoA,thefirstcommittedreactionofgatestheelectronsofNADHthataredestinedforATPfattyacidbiosynthesis(Chapter21).Whensynthesisofmalonyl-CoAisinhibited,subsequentreactionsoffattyacidsynthesisceaseduetolackofsubstrates.Enzymesthatcatalyzerate-limitingstepsserveasnatural“gover-Largenors”ofmetabolicflux.Thus,theyconstituteefficientmoleculestargetsforregulatoryinterventionbydrugs.Forexam-ple,inhibitionby“statin”drugsofHMG-CoAreduc-tase,whichcatalyzestherate-limitingreactionofcho-SmallSmallNutrients~P~PWasteslesterogenesis,curtailssynthesisofcholesterol.moleculesmoleculesSmallREGULATIONOFENZYMEQUANTITYmoleculesThecatalyticcapacityoftherate-limitingreactioninametabolicpathwayistheproductoftheconcentrationFigure9–2.Anidealizedcellinsteadystate.Noteofenzymemoleculesandtheirintrinsiccatalyticeffi-thatmetaboliteflowisunidirectional.ciency.Itthereforefollowsthatcatalyticcapacitycanbe
8274/CHAPTER9influencedbothbychangingthequantityofenzymeEnzymelevelsinmammaliantissuesrespondtoapresentandbyalteringitsintrinsiccatalyticefficiency.widerangeofphysiologic,hormonal,ordietaryfactors.Forexample,glucocorticoidsincreasetheconcentrationoftyrosineaminotransferasebystimulatingks,andControlofEnzymeSynthesisglucagon—despiteitsantagonisticphysiologiceffects—increasesksfourfoldtofivefold.RegulationofliverEnzymeswhoseconcentrationsremainessentiallycon-arginasecaninvolvechangeseitherinksorinkdeg.Afterstantovertimearetermedconstitutiveenzymes.Byaprotein-richmeal,liverarginaselevelsriseandargi-contrast,theconcentrationsofmanyotherenzymesde-ninesynthesisdecreases(Chapter29).Arginaselevelspenduponthepresenceofinducers,typicallysub-alsoriseinstarvation,butherearginasedegradationde-stratesorstructurallyrelatedcompounds,thatinitiatecreases,whereasksremainsunchanged.Similarly,injec-theirsynthesis.Escherichiacoligrownonglucosewill,tionofglucocorticoidsandingestionoftryptophanforexample,onlycatabolizelactoseafteradditionofabothelevatelevelsoftryptophanoxygenase.Whiletheβ-galactoside,aninducerthatinitiatessynthesisofahormoneraisesksforoxygenasesynthesis,tryptophanβ-galactosidaseandagalactosidepermease(Figure39–3).specificallylowerskdegbystabilizingtheoxygenaseInducibleenzymesofhumansincludetryptophanpyr-againstproteolyticdigestion.rolase,threoninedehydrase,tyrosine-α-ketoglutarateaminotransferase,enzymesoftheureacycle,HMG-CoAreductase,andcytochromeP450.Conversely,anexcessMULTIPLEOPTIONSAREAVAILABLEFORofametabolitemaycurtailsynthesisofitscognateenzymeviarepression.BothinductionandrepressionREGULATINGCATALYTICACTIVITYinvolveciselements,specificDNAsequenceslocatedup-Inhumans,theinductionofproteinsynthesisisacom-streamofregulatedgenes,andtrans-actingregulatoryplexmultistepprocessthattypicallyrequireshourstoproteins.Themolecularmechanismsofinductionandproducesignificantchangesinoverallenzymelevel.ByrepressionarediscussedinChapter39.contrast,changesinintrinsiccatalyticefficiencyef-fectedbybindingofdissociableligands(allostericreg-ulation)orbycovalentmodificationachieveregula-ControlofEnzymeDegradationtionofenzymicactivitywithinseconds.Changesinproteinlevelservelong-termadaptiverequirements,Theabsolutequantityofanenzymereflectsthenetbal-whereaschangesincatalyticefficiencyarebestsuitedancebetweenenzymesynthesisandenzymedegrada-forrapidandtransientalterationsinmetaboliteflux.tion,whereksandkdegrepresenttherateconstantsfortheoverallprocessesofsynthesisanddegradation,re-spectively.Changesinboththeksandkdegofspecificenzymesoccurinhumansubjects.ALLOSTERICEFFECTORSREGULATECERTAINENZYMESEnzymeFeedbackinhibitionreferstoinhibitionofanenzymekkinabiosyntheticpathwaybyanendproductofthatsdegpathway.Forexample,forthebiosynthesisofDfromAAminoacidscatalyzedbyenzymesEnz1throughEnz3,Proteinturnoverrepresentsthenetresultofen-Enz1EnzEnz23zymesynthesisanddegradation.Bymeasuringtheratesofincorporationof15N-labeledaminoacidsintopro-ABCD→→→15teinandtheratesoflossofNfromprotein,Schoen-heimerdeducedthatbodyproteinsareinastateof“dy-highconcentrationsofDinhibitconversionofAtoB.namicequilibrium”inwhichtheyarecontinuouslyInhibitionresultsnotfromthe“backingup”ofinter-synthesizedanddegraded.Mammalianproteinsarede-mediatesbutfromtheabilityofDtobindtoandin-gradedbothbyATPandubiquitin-dependentpath-hibitEnz1.Typically,Dbindsatanallostericsitespa-waysandbyATP-independentpathways(Chapter29).tiallydistinctfromthecatalyticsiteofthetargetSusceptibilitytoproteolyticdegradationcanbeinflu-enzyme.Feedbackinhibitorsthusareallostericeffectorsencedbythepresenceofligandssuchassubstrates,andtypicallybearlittleornostructuralsimilaritytothecoenzymes,ormetalionsthatalterproteinconforma-substratesoftheenzymestheyinhibit.Inthisexample,tion.IntracellularligandsthuscaninfluencetheratesatthefeedbackinhibitorDactsasanegativeallostericwhichspecificenzymesaredegraded.effectorofEnz1.
83ENZYMES:REGULATIONOFACTIVITIES/75Inabranchedbiosyntheticpathway,theinitialreac-tionsparticipateinthesynthesisofseveralproducts.ABFigure9–4showsahypotheticalbranchedbiosyntheticpathwayinwhichcurvedarrowsleadfromfeedbackin-hibitorstotheenzymeswhoseactivitytheyinhibit.TheS1S2S3S4sequencesS3→A,S4→B,S4→C,andS3→→DCeachrepresentlinearreactionsequencesthatarefeed-back-inhibitedbytheirendproducts.ThepathwaysofS5Dnucleotidebiosynthesis(Chapter34)providespecificexamples.Thekineticsoffeedbackinhibitionmaybecompeti-Figure9–5.Multiplefeedbackinhibitioninative,noncompetitive,partiallycompetitive,ormixed.branchedbiosyntheticpathway.Superimposedonsim-Feedbackinhibitors,whichfrequentlyarethesmallplefeedbackloops(dashed,curvedarrows)aremulti-moleculebuildingblocksofmacromolecules(eg,aminoplefeedbackloops(solid,curvedarrows)thatregulateacidsforproteins,nucleotidesfornucleicacids),typi-enzymescommontobiosynthesisofseveralendprod-callyinhibitthefirstcommittedstepinaparticularucts.biosyntheticsequence.Amuch-studiedexampleisinhi-bitionofbacterialaspartatetranscarbamoylasebyCTP(seebelowandChapter34).phosphate(CTP).Followingtreatmentwithmercuri-Multiplefeedbackloopscanprovideadditionalfineals,ATCaselosesitssensitivitytoinhibitionbyCTPcontrol.Forexample,asshowninFigure9–5,thepres-butretainsitsfullactivityforsynthesisofcarbamoylas-enceofexcessproductBdecreasestherequirementforpartate.ThissuggeststhatCTPisboundatadifferentsubstrateS2.However,S2isalsorequiredforsynthesis(allosteric)sitefromeithersubstrate.ATCaseconsistsofA,C,andD.ExcessBshouldthereforealsocurtailofmultiplecatalyticandregulatorysubunits.Eachcat-synthesisofallfourendproducts.Tocircumventthisalyticsubunitcontainsfouraspartate(substrate)sitespotentialdifficulty,eachendproducttypicallyonlyandeachregulatorysubunitatleasttwoCTP(regula-partiallyinhibitscatalyticactivity.Theeffectofanex-tory)sites(Chapter34).cessoftwoormoreendproductsmaybestrictlyaddi-tiveor,alternatively,maybegreaterthantheirindivid-ualeffect(cooperativefeedbackinhibition).Allosteric&CatalyticSitesAreSpatiallyDistinctThelackofstructuralsimilaritybetweenafeedbackin-AspartateTranscarbamoylaseIsaModelhibitorandthesubstratefortheenzymewhoseactivityAllostericEnzymeitregulatessuggeststhattheseeffectorsarenotisostericAspartatetranscarbamoylase(ATCase),thecatalystforwithasubstratebutallosteric(“occupyanotherthefirstreactionuniquetopyrimidinebiosynthesisspace”).JacquesMonodthereforeproposedtheexis-(Figure34–7),isfeedback-inhibitedbycytidinetri-tenceofallostericsitesthatarephysicallydistinctfromthecatalyticsite.Allostericenzymesthusarethosewhoseactivityattheactivesitemaybemodulatedbythepresenceofeffectorsatanallostericsite.Thishy-ABpothesishasbeenconfirmedbymanylinesofevidence,includingx-raycrystallographyandsite-directedmuta-genesis,demonstratingtheexistenceofspatiallydistinctS1S2S3S4activeandallostericsitesonavarietyofenzymes.CS5DAllostericEffectsMayBeonKmoronVmaxTorefertothekineticsofallostericinhibitionas“com-Figure9–4.Sitesoffeedbackinhibitioninapetitive”or“noncompetitive”withsubstratecarriesbranchedbiosyntheticpathway.S1–S5areintermedi-misleadingmechanisticimplications.WereferinsteadatesinthebiosynthesisofendproductsA–D.Straighttotwoclassesofregulatedenzymes:K-seriesandV-se-arrowsrepresentenzymescatalyzingtheindicatedcon-riesenzymes.ForK-seriesallostericenzymes,thesub-versions.CurvedarrowsrepresentfeedbackloopsandstratesaturationkineticsarecompetitiveinthesenseindicatesitesoffeedbackinhibitionbyspecificendthatKmisraisedwithoutaneffectonVmax.ForV-seriesproducts.allostericenzymes,theallostericinhibitorlowersVmax
8476/CHAPTER9withoutaffectingtheKm.AlterationsinKmorVmaxREGULATORYCOVALENTprobablyresultfromconformationalchangesatthecat-MODIFICATIONSCANBEalyticsiteinducedbybindingoftheallostericeffectorREVERSIBLEORIRREVERSIBLEattheallostericsite.ForaK-seriesallostericenzyme,thisconformationalchangemayweakenthebondsbe-Inmammaliancells,thetwomostcommonformsoftweensubstrateandsubstrate-bindingresidues.ForacovalentmodificationarepartialproteolysisandV-seriesallostericenzyme,theprimaryeffectmaybetophosphorylation.Becausecellslacktheabilitytore-altertheorientationorchargeofcatalyticresidues,low-unitethetwoportionsofaproteinproducedbyhydrol-eringVmax.IntermediateeffectsonKmandVmax,how-ysisofapeptidebond,proteolysisconstitutesanirre-ever,maybeobservedconsequenttotheseconforma-versiblemodification.Bycontrast,phosphorylationisationalchanges.reversiblemodificationprocess.Thephosphorylationofproteinsonseryl,threonyl,ortyrosylresidues,catalyzedFEEDBACKREGULATIONbyproteinkinases,isthermodynamicallyspontaneous.ISNOTSYNONYMOUSWITHEquallyspontaneousisthehydrolyticremovalofthesephosphorylgroupsbyenzymescalledproteinphos-FEEDBACKINHIBITIONphatases.Inbothmammalianandbacterialcells,endproducts“feedback”andcontroltheirownsynthesis,inmanyPROTEASESMAYBESECRETEDASinstancesbyfeedbackinhibitionofanearlybiosyn-CATALYTICALLYINACTIVEPROENZYMEStheticenzyme.Wemust,however,distinguishbetweenfeedbackregulation,aphenomenologictermdevoidCertainproteinsaresynthesizedandsecretedasinactiveofmechanisticimplications,andfeedbackinhibition,precursorproteinsknownasproproteins.Thepropro-amechanismforregulationofenzymeactivity.Forex-teinsofenzymesaretermedproenzymesorzymogens.ample,whiledietarycholesteroldecreaseshepaticsyn-Selectiveproteolysisconvertsaproproteinbyoneorthesisofcholesterol,thisfeedbackregulationdoesnotmoresuccessiveproteolytic“clips”toaformthatex-involvefeedbackinhibition.HMG-CoAreductase,thehibitsthecharacteristicactivityofthematureprotein,rate-limitingenzymeofcholesterologenesis,isaffected,eg,itsenzymaticactivity.Proteinssynthesizedaspro-butcholesteroldoesnotfeedback-inhibititsactivity.proteinsincludethehormoneinsulin(proprotein=Regulationinresponsetodietarycholesterolinvolvesproinsulin),thedigestiveenzymespepsin,trypsin,andcurtailmentbycholesteroloracholesterolmetaboliteofchymotrypsin(proproteins=pepsinogen,trypsinogen,theexpressionofthegenethatencodesHMG-CoAre-andchymotrypsinogen,respectively),severalfactorsofductase(enzymerepression)(Chapter26).thebloodclottingandbloodclotdissolutioncascades(seeChapter51),andtheconnectivetissueproteincol-MANYHORMONESACTTHROUGHlagen(proprotein=procollagen).ALLOSTERICSECONDMESSENGERSProenzymesFacilitateRapidNerveimpulses—andbindingofhormonestocellsur-MobilizationofanActivityinResponsefacereceptors—elicitchangesintherateofenzyme-toPhysiologicDemandcatalyzedreactionswithintargetcellsbyinducingthere-leaseorsynthesisofspecializedallostericeffectorscalledThesynthesisandsecretionofproteasesascatalyticallysecondmessengers.Theprimaryor“first”messengerisinactiveproenzymesprotectsthetissueoforigin(eg,thehormonemoleculeornerveimpulse.Secondmes-thepancreas)fromautodigestion,suchascanoccurinsengersinclude3′,5′-cAMP,synthesizedfromATPbypancreatitis.Certainphysiologicprocessessuchasdi-theenzymeadenylylcyclaseinresponsetothehormonegestionareintermittentbutfairlyregularandpre-2+epinephrine,andCa,whichisstoredinsidetheendo-dictable.Otherssuchasbloodclotformation,clotdis-plasmicreticulumofmostcells.Membranedepolariza-solution,andtissuerepairarebrought“online”onlyintionresultingfromanerveimpulseopensamembraneresponsetopressingphysiologicorpathophysiologicchannelthatreleasescalciumionintothecytoplasm,need.Theprocessesofbloodclotformationanddis-whereitbindstoandactivatesenzymesinvolvedinthesolutionclearlymustbetemporallycoordinatedtoregulationofcontractionandthemobilizationofstoredachievehomeostasis.Enzymesneededintermittentlyglucosefromglycogen.Glucosethensuppliesthein-butrapidlyoftenaresecretedinaninitiallyinactivecreasedenergydemandsofmusclecontraction.Otherformsincethesecretionprocessornewsynthesisofthesecondmessengersinclude3′,5′-cGMPandpolyphos-requiredproteinsmightbeinsufficientlyrapidforre-phoinositols,producedbythehydrolysisofinositolsponsetoapressingpathophysiologicdemandsuchasphospholipidsbyhormone-regulatedphospholipases.thelossofblood.
85ENZYMES:REGULATIONOFACTIVITIES/77113141516146149245Pro-CT113141516146149245π-CT14-15147-14811316146149245α-CTSSSSFigure9–6.Selectiveproteolysisandassociatedconformationalchangesformtheactivesiteofchymotrypsin,whichincludestheAsp102-His57-Ser195catalytictriad.Successiveproteolysisformsprochymotrypsin(pro-CT),π-chymotrypsin(π-CT),andul-timatelyα-chymotrypsin(α-CT),anactiveproteasewhosethreepeptidesremainasso-ciatedbycovalentinter-chaindisulfidebonds.ActivationofProchymotrypsincatalyzingtransferoftheterminalphosphorylgroupofRequiresSelectiveProteolysisATPtothehydroxylgroupsofseryl,threonyl,ortyro-sylresidues,formingO-phosphoseryl,O-phosphothre-Selectiveproteolysisinvolvesoneormorehighlyspe-onyl,orO-phosphotyrosylresidues,respectively(Figurecificproteolyticclipsthatmayormaynotbeaccompa-9–7).Someproteinkinasestargetthesidechainsofhis-niedbyseparationoftheresultingpeptides.Mostim-tidyl,lysyl,arginyl,andaspartylresidues.Theunmodi-portantly,selectiveproteolysisoftenresultsinfiedformoftheproteincanberegeneratedbyhy-conformationalchangesthat“create”thecatalyticsitedrolyticremovalofphosphorylgroups,catalyzedbyofanenzyme.NotethatwhileHis57andAsp102re-proteinphosphatases.sideontheBpeptideofα-chymotrypsin,Ser195re-Atypicalmammaliancellpossessesover1000phos-sidesontheCpeptide(Figure9–6).Theconforma-phorylatedproteinsandseveralhundredproteinkinasestionalchangesthataccompanyselectiveproteolysisofandproteinphosphatasesthatcatalyzetheirintercon-prochymotrypsin(chymotrypsinogen)alignthethreeversion.Theeaseofinterconversionofenzymesbe-residuesofthecharge-relaynetwork,creatingthecat-tweentheirphospho-anddephospho-formsinpartalyticsite.Notealsothatcontactandcatalyticresiduescanbelocatedondifferentpeptidechainsbutstillbewithinbond-formingdistanceofboundsubstrate.ATPADP2+REVERSIBLECOVALENTMODIFICATIONMgREGULATESKEYMAMMALIANENZYMESKINASEMammalianproteinsarethetargetsofawiderangeofEnzSerOHEnzSerOPO2–3covalentmodificationprocesses.ModificationssuchasPHOSPHATASEglycosylation,hydroxylation,andfattyacidacylationintroducenewstructuralfeaturesintonewlysynthe-Mg2+sizedproteinsthattendtopersistforthelifetimeofthePiH2Oprotein.Amongthecovalentmodificationsthatregu-lateproteinfunction(eg,methylation,adenylylation),Figure9–7.Covalentmodificationofaregulateden-themostcommonbyfarisphosphorylation-dephos-zymebyphosphorylation-dephosphorylationofaserylphorylation.Proteinkinasesphosphorylateproteinsbyresidue.
8678/CHAPTER9accountsforthefrequencyofphosphorylation-dephos-Table9–1.Examplesofmammalianenzymesphorylationasamechanismforregulatorycontrol.whosecatalyticactivityisalteredbycovalentPhosphorylation-dephosphorylationpermitsthefunc-phosphorylation-dephosphorylation.tionalpropertiesoftheaffectedenzymetobealteredonlyforaslongasitservesaspecificneed.Oncethe1ActivityStateneedhaspassed,theenzymecanbeconvertedbacktoitsoriginalform,poisedtorespondtothenextstimula-EnzymeLowHightoryevent.AsecondfactorunderlyingthewidespreadAcetyl-CoAcarboxylaseEPEuseofproteinphosphorylation-dephosphorylationliesGlycogensynthaseEPEinthechemicalpropertiesofthephosphorylgroupit-PyruvatedehydrogenaseEPEself.Inordertoalteranenzyme’sfunctionalproperties,HMG-CoAreductaseEPEanymodificationofitschemicalstructuremustinflu-GlycogenphosphorylaseEEPencetheprotein’sthree-dimensionalconfiguration.CitratelyaseEEPThehighchargedensityofprotein-boundphosphorylPhosphorylasebkinaseEEPgroups—generally−2atphysiologicpH—andtheirHMG-CoAreductasekinaseEEPpropensitytoformsaltbridgeswitharginylresidues1E,dephosphoenzyme;EP,phosphoenzyme.makethempotentagentsformodifyingproteinstruc-tureandfunction.Phosphorylationgenerallytargetsaminoacidsdistantfromthecatalyticsiteitself.Conse-quentconformationalchangestheninfluenceanen-phosphorylationatdifferentsites,orbetweenphosphory-zyme’sintrinsiccatalyticefficiencyorotherproperties.lationsitesandallostericsitesprovidesthebasisforInthissense,thesitesofphosphorylationandotherco-regulatorynetworksthatintegratemultipleenviron-valentmodificationscanbeconsideredanotherformofmentalinputsignalstoevokeanappropriatecoordi-allostericsite.However,inthiscasethe“allostericli-natedcellularresponse.Inthesesophisticatedregula-gand”bindscovalentlytotheprotein.torynetworks,individualenzymesrespondtodifferentenvironmentalsignals.Forexample,ifanenzymecanPROTEINPHOSPHORYLATIONbephosphorylatedatasinglesitebymorethanoneproteinkinase,itcanbeconvertedfromacatalyticallyISEXTREMELYVERSATILEefficienttoaninefficient(inactive)form,orviceversa,Proteinphosphorylation-dephosphorylationisahighlyinresponsetoanyoneofseveralsignals.Iftheproteinversatileandselectiveprocess.Notallproteinsaresub-kinaseisactivatedinresponsetoasignaldifferentfromjecttophosphorylation,andofthemanyhydroxylthesignalthatactivatestheproteinphosphatase,thegroupsonaprotein’ssurface,onlyoneorasmallsubsetphosphoproteinbecomesadecisionnode.Thefunc-aretargeted.Whilethemostcommonenzymefunctiontionaloutput,generallycatalyticactivity,reflectstheaffectedistheprotein’scatalyticefficiency,phosphory-phosphorylationstate.Thisstateordegreeofphos-lationcanalsoaltertheaffinityforsubstrates,locationphorylationisdeterminedbytherelativeactivitiesofwithinthecell,orresponsivenesstoregulationbyal-theproteinkinaseandproteinphosphatase,areflectionlostericligands.Phosphorylationcanincreaseanen-ofthepresenceandrelativestrengthoftheenviron-zyme’scatalyticefficiency,convertingittoitsactivementalsignalsthatactthrougheach.Theabilityofforminoneprotein,whilephosphorylationofanothermanyproteinkinasesandproteinphosphatasestotar-convertsitintoanintrinsicallyinefficient,orinactive,getmorethanoneproteinprovidesameansforanen-form(Table9–1).vironmentalsignaltocoordinatelyregulatemultipleManyproteinscanbephosphorylatedatmultiplemetabolicprocesses.Forexample,theenzymes3-hy-sitesoraresubjecttoregulationbothbyphosphoryla-droxy-3-methylglutaryl-CoAreductaseandacetyl-CoAtion-dephosphorylationandbythebindingofallostericcarboxylase—therate-controllingenzymesforcholes-ligands.Phosphorylation-dephosphorylationatanyoneterolandfattyacidbiosynthesis,respectively—aresitecanbecatalyzedbymultipleproteinkinasesorpro-phosphorylatedandinactivatedbytheAMP-activatedteinphosphatases.Manyproteinkinasesandmostpro-proteinkinase.Whenthisproteinkinaseisactivatedei-teinphosphatasesactonmorethanoneproteinandaretherthroughphosphorylationbyyetanotherproteinthemselvesinterconvertedbetweenactiveandinactivekinaseorinresponsetothebindingofitsallostericacti-formsbythebindingofsecondmessengersorbycova-vator5′-AMP,thetwomajorpathwaysresponsibleforlentmodificationbyphosphorylation-dephosphoryla-thesynthesisoflipidsfromacetyl-CoAbothareinhib-tion.ited.Interconvertibleenzymesandtheenzymesrespon-Theinterplaybetweenproteinkinasesandproteinsiblefortheirinterconversiondonotactasmereonphosphatases,betweenthefunctionalconsequencesofandoffswitchesworkingindependentlyofoneanother.
87ENZYMES:REGULATIONOFACTIVITIES/79Theyformthebuildingblocksofbiomolecularcom-activesite.Secretionasaninactiveproenzymefacili-putersthatmaintainhomeostasisincellsthatcarryouttatesrapidmobilizationofactivityinresponsetoin-acomplexarrayofmetabolicprocessesthatmustbejuryorphysiologicneedandmayprotectthetissueregulatedinresponsetoabroadspectrumofenviron-oforigin(eg,autodigestionbyproteases).mentalfactors.•Bindingofmetabolitesandsecondmessengerstositesdistinctfromthecatalyticsiteofenzymestrig-CovalentModificationRegulatesgersconformationalchangesthatalterVmaxortheMetaboliteFlowKm.•Phosphorylationbyproteinkinasesofspecificseryl,Regulationofenzymeactivitybyphosphorylation-threonyl,ortyrosylresidues—andsubsequentde-dephosphorylationhasanalogiestoregulationbyfeed-phosphorylationbyproteinphosphatases—regulatesbackinhibition.Bothprovideforshort-term,readilytheactivityofmanyhumanenzymes.Theproteinki-reversibleregulationofmetaboliteflowinresponsetonasesandphosphatasesthatparticipateinregulatoryspecificphysiologicsignals.Bothactwithoutalteringcascadeswhichrespondtohormonalorsecondmes-geneexpression.Bothactonearlyenzymesofapro-sengersignalsconstitutea“bio-organiccomputer”tracted,oftenbiosyntheticmetabolicsequence,andthatcanprocessandintegratecomplexenvironmen-bothactatallostericratherthancatalyticsites.Feed-talinformationtoproduceanappropriateandcom-backinhibition,however,involvesasingleproteinandprehensivecellularresponse.lackshormonalandneuralfeatures.Bycontrast,regula-tionofmammalianenzymesbyphosphorylation-dephosphorylationinvolvesseveralproteinsandATPREFERENCESandisunderdirectneuralandhormonalcontrol.BrayD:Proteinmoleculesascomputationalelementsinlivingcells.Nature1995;376:307.SUMMARYGravesDJ,MartinBL,WangJH:Co-andPost-translationalModi-ficationofProteins:ChemicalPrinciplesandBiologicalEffects.•Homeostasisinvolvesmaintainingarelativelycon-OxfordUnivPress,1994.stantintracellularandintra-organenvironmentde-JohnsonLN,BarfordD:Theeffectofphosphorylationonthespitewidefluctuationsintheexternalenvironmentstructureandfunctionofproteins.AnnuRevBiophysBio-viaappropriatechangesintheratesofbiochemicalmolStruct1993;22:199.reactionsinresponsetophysiologicneed.MarksF(editor):ProteinPhosphorylation.VCHPublishers,1996.•ThesubstratesformostenzymesareusuallypresentPilkisSJetal:6-Phosphofructo-2-kinase/fructose-2,6-bisphospha-ataconcentrationclosetoKm.Thisfacilitatespassivetase:Ametabolicsignalingenzyme.AnnuRevBiochemcontroloftheratesofproductformationresponseto1995;64:799.changesinlevelsofmetabolicintermediates.ScriverCRetal(editors):TheMetabolicandMolecularBasesofInheritedDisease,8thed.McGraw-Hill,2000.•ActivecontrolofmetabolitefluxinvolveschangesinSitaramayyaA(editor):IntroductiontoCellularSignalTransduction.theconcentration,catalyticactivity,orbothofanen-Birkhauser,1999.zymethatcatalyzesacommitted,rate-limitingreac-StadtmanER,ChockPB(editors):CurrentTopicsinCellularRegu-tion.lation.AcademicPress,1969tothepresent.•Selectiveproteolysisofcatalyticallyinactiveproen-WeberG(editor):AdvancesinEnzymeRegulation.PergamonPress,zymesinitiatesconformationalchangesthatformthe1963tothepresent.
88SECTIONIIBioenergetics&theMetabolismofCarbohydrates&LipidsBioenergetics:TheRoleofATP10PeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DScBIOMEDICALIMPORTANCEthesystemtoanotherormaybetransformedintoan-otherformofenergy.Inlivingsystems,chemicalen-Bioenergetics,orbiochemicalthermodynamics,istheergymaybetransformedintoheatorintoelectrical,ra-studyoftheenergychangesaccompanyingbiochemicaldiant,ormechanicalenergy.reactions.BiologicsystemsareessentiallyisothermicThesecondlawofthermodynamicsstatesthattheandusechemicalenergytopowerlivingprocesses.totalentropyofasystemmustincreaseifaprocessHowananimalobtainssuitablefuelfromitsfoodtoistooccurspontaneously.Entropyistheextentofprovidethisenergyisbasictotheunderstandingofnor-disorderorrandomnessofthesystemandbecomesmalnutritionandmetabolism.Deathfromstarvationmaximumasequilibriumisapproached.Undercondi-occurswhenavailableenergyreservesaredepleted,andtionsofconstanttemperatureandpressure,therela-certainformsofmalnutritionareassociatedwithenergytionshipbetweenthefreeenergychange(ΔG)ofare-imbalance(marasmus).Thyroidhormonescontroltheactingsystemandthechangeinentropy(ΔS)israteofenergyrelease(metabolicrate),anddiseasere-expressedbythefollowingequation,whichcombinessultswhentheymalfunction.Excessstorageofsurplusthetwolawsofthermodynamics:energycausesobesity,oneofthemostcommondis-easesofWesternsociety.ΔG=ΔH−TΔSFREEENERGYISTHEUSEFULENERGYwhereΔHisthechangeinenthalpy(heat)andTistheINASYSTEMabsolutetemperature.Inbiochemicalreactions,becauseΔHisapproxi-Gibbschangeinfreeenergy(ΔG)isthatportionofthematelyequaltoΔE,thetotalchangeininternalenergytotalenergychangeinasystemthatisavailableforofthereaction,theaboverelationshipmaybeexpresseddoingwork—ie,theusefulenergy,alsoknownastheinthefollowingway:chemicalpotential.ΔG=ΔE−TΔSBiologicSystemsConformtotheGeneralIfΔGisnegative,thereactionproceedssponta-LawsofThermodynamicsneouslywithlossoffreeenergy;ie,itisexergonic.If,Thefirstlawofthermodynamicsstatesthatthetotalinaddition,ΔGisofgreatmagnitude,thereactiongoesenergyofasystem,includingitssurroundings,re-virtuallytocompletionandisessentiallyirreversible.mainsconstant.Itimpliesthatwithinthetotalsystem,Ontheotherhand,ifΔGispositive,thereactionpro-energyisneitherlostnorgainedduringanychange.ceedsonlyiffreeenergycanbegained;ie,itisender-However,energymaybetransferredfromonepartofgonic.If,inaddition,themagnitudeofΔGisgreat,the80
89BIOENERGETICS:THEROLEOFATP/81systemisstable,withlittleornotendencyforareactionoccurswithreleaseoffreeenergy.Itiscoupledtoan-tooccur.IfΔGiszero,thesystemisatequilibriumandotherreaction,inwhichfreeenergyisrequiredtocon-nonetchangetakesplace.vertmetaboliteCtometaboliteD.Thetermsexer-Whenthereactantsarepresentinconcentrationsofgonicandendergonicratherthanthenormalchemical01.0mol/L,ΔGisthestandardfreeenergychange.Forterms“exothermic”and“endothermic”areusedtoin-biochemicalreactions,astandardstateisdefinedasdicatethataprocessisaccompaniedbylossorgain,re-havingapHof7.0.Thestandardfreeenergychangeatspectively,offreeenergyinanyform,notnecessarilyas0′thisstandardstateisdenotedbyΔG.heat.Inpractice,anendergonicprocesscannotexistin-Thestandardfreeenergychangecanbecalculateddependentlybutmustbeacomponentofacoupledex-fromtheequilibriumconstantKeq.ergonic-endergonicsystemwheretheoverallnetchangeisexergonic.Theexergonicreactionsaretermedcatab-0′olism(generally,thebreakdownoroxidationoffuelΔG=−RTlnK′eqmolecules),whereasthesyntheticreactionsthatbuildupsubstancesaretermedanabolism.ThecombinedwhereRisthegasconstantandTistheabsolutetem-catabolicandanabolicprocessesconstitutemetabo-perature(Chapter8).Itisimportanttonotethatthelism.0′actualΔGmaybelargerorsmallerthanΔGdepend-IfthereactionshowninFigure10–1istogofromingontheconcentrationsofthevariousreactants,in-lefttoright,thentheoverallprocessmustbeaccompa-cludingthesolvent,variousions,andproteins.niedbylossoffreeenergyasheat.Onepossiblemecha-Inabiochemicalsystem,anenzymeonlyspeedsupnismofcouplingcouldbeenvisagedifacommonoblig-theattainmentofequilibrium;itneveraltersthefinalatoryintermediate(I)tookpartinbothreactions,ie,concentrationsofthereactantsatequilibrium.A+C→I→B+DENDERGONICPROCESSESPROCEEDBYCOUPLINGTOEXERGONICPROCESSESSomeexergonicandendergonicreactionsinbiologicsystemsarecoupledinthisway.ThistypeofsystemhasThevitalprocesses—eg,syntheticreactions,muscularabuilt-inmechanismforbiologiccontroloftherateofcontraction,nerveimpulseconduction,andactiveoxidativeprocessessincethecommonobligatoryinter-transport—obtainenergybychemicallinkage,orcou-mediateallowstherateofutilizationoftheproductofpling,tooxidativereactions.Initssimplestform,thisthesyntheticpath(D)todeterminebymassactionthetypeofcouplingmayberepresentedasshowninFigurerateatwhichAisoxidized.Indeed,theserelationships10–1.TheconversionofmetaboliteAtometaboliteBsupplyabasisfortheconceptofrespiratorycontrol,theprocessthatpreventsanorganismfromburningoutofcontrol.Anextensionofthecouplingconceptispro-videdbydehydrogenationreactions,whicharecoupledtohydrogenationsbyanintermediatecarrier(Figure10–2).Analternativemethodofcouplinganexergonictoanendergonicprocessistosynthesizeacompoundofhigh-energypotentialintheexergonicreactionandtoincorporatethisnewcompoundintotheendergonicre-action,thuseffectingatransferenceoffreeenergyfromtheexergonictotheendergonicpathway(Figure10–3).Thebiologicadvantageofthismechanismisthatthecompoundofhighpotentialenergy,∼E,unlikeIΔG=ΔH−TΔSFigure10–1.Couplingofanexergonictoanender-Figure10–2.Couplingofdehydrogenationandhy-gonicreaction.drogenationreactionsbyanintermediatecarrier.
9082/CHAPTER10Figure10–4.Adenosinetriphosphate(ATP)shownasthemagnesiumcomplex.ADPformsasimilarcom-2+plexwithMg.Figure10–3.Transferoffreeenergyfromanexer-gonictoanendergonicreactionviaahigh-energyin-TheIntermediateValuefortheFreetermediatecompound(∼E).EnergyofHydrolysisofATPHasImportantBioenergeticSignificanceThestandardfreeenergyofhydrolysisofanumberofintheprevioussystem,neednotbestructurallyrelatedtoA,B,C,orD,allowingEtoserveasatransducerofbiochemicallyimportantphosphatesisshowninTable10–1.Anestimateofthecomparativetendencyofeachenergyfromawiderangeofexergonicreactionstoanofthephosphategroupstotransfertoasuitableaccep-equallywiderangeofendergonicreactionsorprocesses,0′tormaybeobtainedfromtheΔGofhydrolysisatsuchasbiosyntheses,muscularcontraction,nervousex-37°C.Thevalueforthehydrolysisoftheterminalcitation,andactivetransport.Inthelivingcell,theprincipalhigh-energyintermediateorcarriercom-pound(designated∼EinFigure10–3)isadenosinetriphosphate(ATP).Table10–1.Standardfreeenergyofhydrolysisofsomeorganophosphatesofbiochemical1,2HIGH-ENERGYPHOSPHATESPLAYAimportance.CENTRALROLEINENERGYCAPTURE0ANDTRANSFERGInordertomaintainlivingprocesses,allorganismsCompoundkJ/molkcal/molmustobtainsuppliesoffreeenergyfromtheirenviron-Phosphoenolpyruvate−61.9−14.8ment.AutotrophicorganismsutilizesimpleexergonicCarbamoylphosphate−51.4−12.3processes;eg,theenergyofsunlight(greenplants),the1,3-Bisphosphoglycerate−49.3−11.8reactionFe2+→Fe3+(somebacteria).Ontheother(to3-phosphoglycerate)hand,heterotrophicorganismsobtainfreeenergybyCreatinephosphate−43.1−10.3couplingtheirmetabolismtothebreakdownofcom-ATP→ADP+Pi−30.5−7.3plexorganicmoleculesintheirenvironment.InallADP→AMP+Pi−27.6−6.6theseorganisms,ATPplaysacentralroleinthetrans-Pyrophosphate−27.6−6.6ferenceoffreeenergyfromtheexergonictotheender-Glucose1-phosphate−20.9−5.0gonicprocesses(Figure10–3).ATPisanucleosideFructose6-phosphate−15.9−3.8triphosphatecontainingadenine,ribose,andthreeAMP−14.2−3.4phosphategroups.Initsreactionsinthecell,itfunc-Glucose6-phosphate−13.8−3.3tionsastheMg2+complex(Figure10–4).Glycerol3-phosphate−9.2−2.2Theimportanceofphosphatesinintermediaryme-1P,inorganicorthophosphate.itabolismbecameevidentwiththediscoveryoftherole2ValuesforATPandmostotherstakenfromKrebsandKornbergofATP,adenosinediphosphate(ADP),andinorganic(1957).Theydifferbetweeninvestigatorsdependingonthepre-phosphate(Pi)inglycolysis(Chapter17).ciseconditionsunderwhichthemeasurementsaremade.
91BIOENERGETICS:THEROLEOFATP/83phosphateofATPdividesthelistintotwogroups.Low-energyphosphates,exemplifiedbytheesterphosphatesfoundintheintermediatesofglycolysis,0′haveΔGvaluessmallerthanthatofATP,whileinhigh-energyphosphatesthevalueishigherthanthatofATP.Thecomponentsofthislattergroup,includingATP,areusuallyanhydrides(eg,the1-phosphateof1,3-bisphosphoglycerate),enolphosphates(eg,phos-phoenolpyruvate),andphosphoguanidines(eg,creatinephosphate,argininephosphate).Theintermediateposi-tionofATPallowsittoplayanimportantroleinen-ergytransfer.ThehighfreeenergychangeonhydrolysisofATPisduetoreliefofchargerepulsionofadjacentnegativelychargedoxygenatomsandtostabilizationofthereactionproducts,especiallyphosphate,asreso-nancehybrids.Other“high-energycompounds”arethiolestersinvolvingcoenzymeA(eg,acetyl-CoA),acylcarrierprotein,aminoacidestersinvolvedinproteinsynthesis,S-adenosylmethionine(activemethionine),UDPGlc(uridinediphosphateglucose),andPRPP(5-phosphoribosyl-1-pyrophosphate).High-EnergyPhosphatesAreDesignatedby~PFigure10–5.StructureofATP,ADP,andAMPshow-Thesymbol∼Pindicatesthatthegroupattachedtoingthepositionandthenumberofhigh-energyphos-thebond,ontransfertoanappropriateacceptor,resultsphates(∼P).intransferofthelargerquantityoffreeenergy.Forthisreason,thetermgrouptransferpotentialispreferredbysometo“high-energybond.”Thus,ATPcontainstwohigh-energyphosphategroupsandADPcontainsone,whereasthephosphateinAMP(adenosinemono-phosphate)isofthelow-energytype,sinceitisanor-malesterlink(Figure10–5).HIGH-ENERGYPHOSPHATESACTASTHE“ENERGYCURRENCY”OFTHECELLATPisabletoactasadonorofhigh-energyphosphatetoformthosecompoundsbelowitinTable10–1.Like-wise,withthenecessaryenzymes,ADPcanaccepthigh-energyphosphatetoformATPfromthosecom-poundsaboveATPinthetable.Ineffect,anATP/ADPcycleconnectsthoseprocessesthatgenerate∼Ptothoseprocessesthatutilize∼P(Figure10–6),con-tinuouslyconsumingandregeneratingATP.Thisoc-cursataveryrapidrate,sincethetotalATP/ADPpoolisextremelysmallandsufficienttomaintainanactivetissueforonlyafewseconds.Therearethreemajorsourcesof∼Ptakingpartinenergyconservationorenergycapture:(1)Oxidativephosphorylation:Thegreatestquan-Figure10–6.RoleofATP/ADPcycleintransferoftitativesourceof∼Pinaerobicorganisms.Freeenergyhigh-energyphosphate.
9284/CHAPTER10(1)Glucose+Pi→Glucose6-phosphate+H2O(ΔG0′=+13.8kJ/mol)Totakeplace,thereactionmustbecoupledwithan-other—moreexergonic—reactionsuchasthehydroly-sisoftheterminalphosphateofATP.0′(2)ATP→ADP+Pi(ΔG=−30.5kJ/mol)Figure10–7.Transferofhigh-energyphosphatebe-tweenATPandcreatine.When(1)and(2)arecoupledinareactioncatalyzedbyhexokinase,phosphorylationofglucosereadilypro-ceedsinahighlyexergonicreactionthatunderphysio-logicconditionsisirreversible.Many“activation”reac-comesfromrespiratorychainoxidationusingmoleculartionsfollowthispattern.O2withinmitochondria(Chapter11).(2)Glycolysis:Anetformationoftwo∼Presultsfromtheformationoflactatefromonemoleculeofglu-AdenylylKinase(Myokinase)cose,generatedintworeactionscatalyzedbyphospho-InterconvertsAdenineNucleotidesglyceratekinaseandpyruvatekinase,respectively(Fig-Thisenzymeispresentinmostcells.Itcatalyzesthefol-ure17–2).lowingreaction:(3)Thecitricacidcycle:One∼Pisgenerateddi-rectlyinthecycleatthesuccinylthiokinasestep(Figure16–3).Phosphagensactasstorageformsofhigh-energyphosphateandincludecreatinephosphate,occurringinvertebrateskeletalmuscle,heart,spermatozoa,andThisallows:brain;andargininephosphate,occurringininverte-(1)High-energyphosphateinADPtobeusedinbratemuscle.WhenATPisrapidlybeingutilizedasathesynthesisofATP.sourceofenergyformuscularcontraction,phosphagens(2)AMP,formedasaconsequenceofseveralacti-permititsconcentrationstobemaintained,butwhenvatingreactionsinvolvingATP,toberecoveredbytheATP/ADPratioishigh,theirconcentrationcanin-rephosphorylationtoADP.creasetoactasastoreofhigh-energyphosphate(Figure(3)AMPtoincreaseinconcentrationwhenATP10–7).becomesdepletedandactasametabolic(allosteric)sig-WhenATPactsasaphosphatedonortoformthosenaltoincreasetherateofcatabolicreactions,whichincompoundsoflowerfreeenergyofhydrolysis(TableturnleadtothegenerationofmoreATP(Chapter19).10–1),thephosphategroupisinvariablyconvertedtooneoflowenergy,eg,WhenATPFormsAMP,InorganicPyrophosphate(PPi)IsProducedThisoccurs,forexample,intheactivationoflong-chainfattyacids(Chapter22):ATPAllowstheCouplingofThermodynamicallyUnfavorableReactionstoFavorableOnesThisreactionisaccompaniedbylossoffreeenergyasheat,whichensuresthattheactivationreactionwillThephosphorylationofglucosetoglucose6-phos-gototheright;andisfurtheraidedbythehydrolyticphate,thefirstreactionofglycolysis(Figure17–2),issplittingofPPi,catalyzedbyinorganicpyrophospha-highlyendergonicandcannotproceedunderphysio-tase,areactionthatitselfhasalargeΔG0′of−27.6kJ/logicconditions.
93BIOENERGETICS:THEROLEOFATP/85Thus,adenylylkinaseisaspecializedmonophosphatekinase.SUMMARY•Biologicsystemsusechemicalenergytopowerthelivingprocesses.•Exergonicreactionstakeplacespontaneouslywithlossoffreeenergy(ΔGisnegative).Endergonicreac-tionsrequirethegainoffreeenergy(ΔGispositive)andonlyoccurwhencoupledtoexergonicreactions.•ATPactsasthe“energycurrency”ofthecell,trans-Figure10–8.Phosphatecyclesandinterchangeofferringfreeenergyderivedfromsubstancesofhigheradeninenucleotides.energypotentialtothoseoflowerenergypotential.REFERENCESmol.Notethatactivationsviathepyrophosphatepath-wayresultinthelossoftwo∼Pratherthanone∼PasdeMeisL:Theconceptofenergy-richphosphatecompounds:occurswhenADPandPareformed.Water,transportATPases,andentropyenergy.ArchBio-ichemBiophys1993;306:287.ErnsterL(editor):Bioenergetics.Elsevier,1984.HaroldFM:TheVitalForce:AStudyofBioenergetics.Freeman,1986.KlotzIM:IntroductiontoBiomolecularEnergetics.AcademicPress,1986.Acombinationoftheabovereactionsmakesitpos-KrebsHA,KornbergHL:EnergyTransformationsinLivingMatter.sibleforphosphatetoberecycledandtheadeninenu-Springer,1957.cleotidestointerchange(Figure10–8).OtherNucleosideTriphosphatesParticipateintheTransferofHigh-EnergyPhosphateBymeansoftheenzymenucleosidediphosphateki-nase,UTP,GTP,andCTPcanbesynthesizedfromtheirdiphosphates,eg,Allofthesetriphosphatestakepartinphosphoryla-tionsinthecell.Similarly,specificnucleosidemono-phosphatekinasescatalyzetheformationofnucleosidediphosphatesfromthecorrespondingmonophosphates.
94BiologicOxidation11PeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DScBIOMEDICALIMPORTANCEgroups:oxidases,dehydrogenases,hydroperoxidases,andoxygenases.Chemically,oxidationisdefinedastheremovalofelec-tronsandreductionasthegainofelectrons.Thus,oxi-dationisalwaysaccompaniedbyreductionofanelec-OXIDASESUSEOXYGENASAtronacceptor.Thisprincipleofoxidation-reductionHYDROGENACCEPTORappliesequallytobiochemicalsystemsandisanimpor-tantconceptunderlyingunderstandingofthenatureofOxidasescatalyzetheremovalofhydrogenfromasub-biologicoxidation.Notethatmanybiologicoxidationsstrateusingoxygenasahydrogenacceptor.*Theyformcantakeplacewithouttheparticipationofmolecularwaterorhydrogenperoxideasareactionproduct(Fig-oxygen,eg,dehydrogenations.Thelifeofhigherani-ure11–1).malsisabsolutelydependentuponasupplyofoxygenforrespiration,theprocessbywhichcellsderiveenergySomeOxidasesContainCopperintheformofATPfromthecontrolledreactionofhy-drogenwithoxygentoformwater.Inaddition,molec-Cytochromeoxidaseisahemoproteinwidelydistrib-ularoxygenisincorporatedintoavarietyofsubstratesutedinmanytissues,havingthetypicalhemepros-byenzymesdesignatedasoxygenases;manydrugs,pol-theticgrouppresentinmyoglobin,hemoglobin,andlutants,andchemicalcarcinogens(xenobiotics)areme-othercytochromes(Chapter6).Itistheterminalcom-tabolizedbyenzymesofthisclass,knownasthecy-ponentofthechainofrespiratorycarriersfoundinmi-tochromeP450system.Administrationofoxygencantochondriaandtransferselectronsresultingfromthebelifesavinginthetreatmentofpatientswithrespira-oxidationofsubstratemoleculesbydehydrogenasestotoryorcirculatoryfailure.theirfinalacceptor,oxygen.Theenzymeispoisonedbycarbonmonoxide,cyanide,andhydrogensulfide.Ithasalsobeentermedcytochromea3.ItisnowknownthatFREEENERGYCHANGESCANcytochromesaanda3arecombinedinasingleprotein,BEEXPRESSEDINTERMSandthecomplexisknownascytochromeaa3.Itcon-OFREDOXPOTENTIALtainstwomoleculesofheme,eachhavingoneFeatom3+2+thatoscillatesbetweenFeandFeduringoxidationInreactionsinvolvingoxidationandreduction,thefreeandreduction.Furthermore,twoatomsofCuarepre-energychangeisproportionatetothetendencyofreac-sent,eachassociatedwithahemeunit.tantstodonateoracceptelectrons.Thus,inadditionto0′expressingfreeenergychangeintermsofΔG(Chapter10),itispossible,inananalogousmanner,toexpressitOtherOxidasesAreFlavoproteinsnumericallyasanoxidation-reductionorredoxpo-Flavoproteinenzymescontainflavinmononucleotidetential(E′0).Theredoxpotentialofasystem(E0)is(FMN)orflavinadeninedinucleotide(FAD)aspros-usuallycomparedwiththepotentialofthehydrogentheticgroups.FMNandFADareformedinthebodyelectrode(0.0voltsatpH0.0).However,forbiologicfromthevitaminriboflavin(Chapter45).FMNandsystems,theredoxpotential(E′0)isnormallyexpressedFADareusuallytightly—butnotcovalently—boundtoatpH7.0,atwhichpHtheelectrodepotentialofthetheirrespectiveapoenzymeproteins.Metalloflavopro-hydrogenelectrodeis−0.42volts.Theredoxpotentialsteinscontainoneormoremetalsasessentialcofactors.ofsomeredoxsystemsofspecialinterestinmammalianExamplesofflavoproteinenzymesincludeL-aminobiochemistryareshowninTable11–1.Therelativepo-acidoxidase,anFMN-linkedenzymefoundinkidneysitionsofredoxsystemsinthetableallowspredictionofwithgeneralspecificityfortheoxidativedeaminationofthedirectionofflowofelectronsfromoneredoxcoupletoanother.Enzymesinvolvedinoxidationandreductionare*Theterm“oxidase”issometimesusedcollectivelytodenoteallcalledoxidoreductasesandareclassifiedintofourenzymesthatcatalyzereactionsinvolvingmolecularoxygen.86
95BIOLOGICOXIDATION/87Table11–1.Someredoxpotentialsofspecialversible,thesepropertiesenablereducingequivalentstointerestinmammalianoxidationsystems.befreelytransferredwithinthecell.Thistypeofreac-tion,whichenablesonesubstratetobeoxidizedattheexpenseofanother,isparticularlyusefulinenablingox-SystemE0Voltsidativeprocessestooccurintheabsenceofoxygen,H+/H2−0.42suchasduringtheanaerobicphaseofglycolysis(FigureNAD+/NADH−0.3217–2).Lipoate;ox/red−0.29(2)Ascomponentsintherespiratorychainofelec-Acetoacetate/3-hydroxybutyrate−0.27trontransportfromsubstratetooxygen(Figure12–3).Pyruvate/lactate−0.19Oxaloacetate/malate−0.17Fumarate/succinate+0.03ManyDehydrogenasesDepend3+2+Cytochromeb;Fe/Fe+0.08onNicotinamideCoenzymesUbiquinone;ox/red+0.103+2+Cytochromec1;Fe/Fe+0.22Thesedehydrogenasesusenicotinamideadeninedi-Cytochromea;Fe3+/Fe2++0.29+nucleotide(NAD)ornicotinamideadeninedinu-Oxygen/water+0.82cleotidephosphate(NADP+)—orboth—andareformedinthebodyfromthevitaminniacin(Chapter45).Thecoenzymesarereducedbythespecificsub-strateofthedehydrogenaseandreoxidizedbyasuitablethenaturallyoccurringL-aminoacids;xanthineoxi-electronacceptor(Figure11–4).Theymayfreelyanddase,whichcontainsmolybdenumandplaysanimpor-reversiblydissociatefromtheirrespectiveapoenzymes.tantroleintheconversionofpurinebasestouricacidGenerally,NAD-linkeddehydrogenasescatalyzeox-(Chapter34),andisofparticularsignificanceinuri-idoreductionreactionsintheoxidativepathwaysofme-cotelicanimals(Chapter29);andaldehydedehydro-tabolism,particularlyinglycolysis,inthecitricacidgenase,anFAD-linkedenzymepresentinmammaliancycle,andintherespiratorychainofmitochondria.livers,whichcontainsmolybdenumandnonhemeironNADP-linkeddehydrogenasesarefoundcharacteristi-andactsuponaldehydesandN-heterocyclicsubstrates.callyinreductivesyntheses,asintheextramitochon-Themechanismsofoxidationandreductionofthesedrialpathwayoffattyacidsynthesisandsteroidsynthe-enzymesarecomplex.Evidencesuggestsatwo-stepre-sis—andalsointhepentosephosphatepathway.actionasshowninFigure11–2.OtherDehydrogenasesDependDEHYDROGENASESCANNOTUSEonRiboflavinOXYGENASAHYDROGENACCEPTORTheflavingroupsassociatedwiththesedehydrogenasesTherearealargenumberofenzymesinthisclass.TheyaresimilartoFMNandFADoccurringinoxidases.performtwomainfunctions:Theyaregenerallymoretightlyboundtotheirapoen-zymesthanarethenicotinamidecoenzymes.Mostof(1)Transferofhydrogenfromonesubstratetoan-theriboflavin-linkeddehydrogenasesareconcernedotherinacoupledoxidation-reductionreaction(Figurewithelectrontransportin(orto)therespiratorychain11–3).Thesedehydrogenasesarespecificfortheirsub-(Chapter12).NADHdehydrogenaseactsasacarrierstratesbutoftenutilizecommoncoenzymesorhydro-+ofelectronsbetweenNADHandthecomponentsofgencarriers,eg,NAD.Sincethereactionsarere-higherredoxpotential(Figure12–3).Otherdehydro-genasessuchassuccinatedehydrogenase,acyl-CoAdehydrogenase,andmitochondrialglycerol-3-phos-phatedehydrogenasetransferreducingequivalentsdi-1/AH22O2AH2O2rectlyfromthesubstratetotherespiratorychain(Fig-(Red)ure12–4).Anotherroleoftheflavin-dependentOXIDASEOXIDASEdehydrogenasesisinthedehydrogenation(bydihy-drolipoyldehydrogenase)ofreducedlipoate,aninter-AH2OAH2O2mediateintheoxidativedecarboxylationofpyruvate(Ox)andα-ketoglutarate(Figures12–4and17–5).TheABelectron-transferringflavoproteinisanintermediaryFigure11–1.Oxidationofametabolitecatalyzedbycarrierofelectronsbetweenacyl-CoAdehydrogenaseanoxidase(A)formingH2O,(B)formingH2O2.andtherespiratorychain(Figure12–4).
9688/CHAPTER11RRRHHH3CNNOH3CNNOH3CNNONHNHNHH3CNH3CNH3CNOOHOHH(H++e–)(H++e–)Figure11–2.Oxidoreductionofisoalloxazineringinflavinnucleotidesviaasemi-quinone(freeradical)intermediate(center).CytochromesMayAlsoBeRegardedPeroxidasesReducePeroxidesUsingasDehydrogenasesVariousElectronAcceptorsThecytochromesareiron-containinghemoproteinsinPeroxidasesarefoundinmilkandinleukocytes,3+2+whichtheironatomoscillatesbetweenFeandFeplatelets,andothertissuesinvolvedineicosanoidme-duringoxidationandreduction.Exceptforcytochrometabolism(Chapter23).Theprostheticgroupisproto-oxidase(previouslydescribed),theyareclassifiedasde-heme.Inthereactioncatalyzedbyperoxidase,hydro-hydrogenases.Intherespiratorychain,theyarein-genperoxideisreducedattheexpenseofseveralvolvedascarriersofelectronsfromflavoproteinsonthesubstancesthatwillactaselectronacceptors,suchasonehandtocytochromeoxidaseontheother(Figureascorbate,quinones,andcytochromec.Thereaction12–4).Severalidentifiablecytochromesoccurinthecatalyzedbyperoxidaseiscomplex,buttheoverallreac-respiratorychain,ie,cytochromesb,c1,c,a,anda3(cy-tionisasfollows:tochromeoxidase).Cytochromesarealsofoundinotherlocations,eg,theendoplasmicreticulum(cy-PEROXIDASEtochromesP450andb5),andinplantcells,bacteria,H2O2+AH22H2O+Aandyeasts.Inerythrocytesandothertissues,theenzymeglu-HYDROPEROXIDASESUSEHYDROGENtathioneperoxidase,containingseleniumasapros-PEROXIDEORANORGANICPEROXIDEtheticgroup,catalyzesthedestructionofH2O2andlipidhydroperoxidesbyreducedglutathione,protectingASSUBSTRATEmembranelipidsandhemoglobinagainstoxidationbyTwotypeofenzymesfoundbothinanimalsandplantsperoxides(Chapter20).fallintothiscategory:peroxidasesandcatalase.HydroperoxidasesprotectthebodyagainstharmfulCatalaseUsesHydrogenPeroxideasperoxides.Accumulationofperoxidescanleadtogen-erationoffreeradicals,whichinturncandisruptmem-ElectronDonor&ElectronAcceptorbranesandperhapscausecancerandatherosclerosis.Catalaseisahemoproteincontainingfourhemegroups.(SeeChapters14and45.)Inadditiontopossessingperoxidaseactivity,itisabletouseonemoleculeofH2O2asasubstrateelectrondonorandanothermoleculeofH2O2asanoxidantorAHCarrierBHelectronacceptor.22(Red)(Ox)(Red)CATALASE2H2O22H2O+O2ACarrier–H2B(Ox)(Red)(Ox)Undermostconditionsinvivo,theperoxidaseactivityDEHYDROGENASEDEHYDROGENASEofcatalaseseemstobefavored.CatalaseisfoundinSPECIFICFORASPECIFICFORBblood,bonemarrow,mucousmembranes,kidney,andFigure11–3.Oxidationofametabolitecatalyzedbyliver.Itsfunctionisassumedtobethedestructionofcoupleddehydrogenases.hydrogenperoxideformedbytheactionofoxidases.
97BIOLOGICOXIDATION/89HH4Figure11–4.MechanismofoxidationDEHYDROGENASECONH2SPECIFICFORAandreductionofnicotinamidecoen-zymes.ThereisstereospecificityaboutAH2AFormNposition4ofnicotinamidewhenitisre-HducedbyasubstrateAH.Oneofthehy-R2drogenatomsisremovedfromthesub-4CONH2+strateasahydrogennucleuswithtwoA+H−electrons(hydrideion,H)andistrans-+ferredtothe4position,whereitmaybeNHHattachedineithertheAortheBpositionRAH4accordingtothespecificitydetermined2CONH2bytheparticulardehydrogenasecatalyz-DEHYDROGENASESPECIFICFORBingthereaction.Theremaininghydro-NBFormgenofthehydrogenpairremovedfromRthesubstrateremainsfreeasahydro-NAD++AHNADH+H++Agenion.2Peroxisomesarefoundinmanytissues,includingliver.Monooxygenases(Mixed-FunctionTheyarerichinoxidasesandincatalase,Thus,theen-Oxidases,Hydroxylases)IncorporatezymesthatproduceH2O2aregroupedwiththeenzymeOnlyOneAtomofMolecularOxygenthatdestroysit.However,mitochondrialandmicroso-IntotheSubstratemalelectrontransportsystemsaswellasxanthineoxi-dasemustbeconsideredasadditionalsourcesofH2O2.Theotheroxygenatomisreducedtowater,anaddi-tionalelectrondonororcosubstrate(Z)beingnecessaryforthispurpose.OXYGENASESCATALYZETHEDIRECTTRANSFER&INCORPORATIONA——HO++→22ZHAOHHOZ++2OFOXYGENINTOASUBSTRATEMOLECULECytochromesP450AreMonooxygenasesOxygenasesareconcernedwiththesynthesisordegra-ImportantfortheDetoxificationofManydationofmanydifferenttypesofmetabolites.Theycat-Drugs&fortheHydroxylationofSteroidsalyzetheincorporationofoxygenintoasubstratemole-culeintwosteps:(1)oxygenisboundtotheenzymeatCytochromesP450areanimportantsuperfamilyoftheactivesite,and(2)theboundoxygenisreducedorheme-containingmonooxgenases,andmorethan1000transferredtothesubstrate.Oxygenasesmaybedividedsuchenzymesareknown.BothNADHandNADPHintotwosubgroups,asfollows.donatereducingequivalentsforthereductionofthesecytochromes(Figure11–5),whichinturnareoxidizedbysubstratesinaseriesofenzymaticreactionscollectivelyDioxygenasesIncorporateBothAtomsknownasthehydroxylasecycle(Figure11–6).InliverofMolecularOxygenIntotheSubstratemicrosomes,cytochromesP450arefoundtogetherwithThebasicreactionisshownbelow:cytochromeb5andhaveanimportantroleindetoxifica-tion.Benzpyrene,aminopyrine,aniline,morphine,andAOAO+→22benzphetaminearehydroxylated,increasingtheirsolubil-ityandaidingtheirexcretion.Manydrugssuchasphe-Examplesincludetheliverenzymes,homogentisatenobarbitalhavetheabilitytoinducetheformationofmi-dioxygenase(oxidase)and3-hydroxyanthranilatecrosomalenzymesandofcytochromesP450.dioxygenase(oxidase),thatcontainiron;andL-trypto-MitochondrialcytochromeP450systemsarefoundphandioxygenase(tryptophanpyrrolase)(Chapterinsteroidogenictissuessuchasadrenalcortex,testis,30),thatutilizesheme.ovary,andplacentaandareconcernedwiththebiosyn-
9890/CHAPTER11CN–NADHFlavoprotein2Cytb5Stearyl-CoAdesaturase–Amineoxidase,etcFlavoprotein3NADPHFlavoprotein1CytP450HydroxylationLipidperoxidationHemeoxygenase−Figure11–5.Electrontransportchaininmicrosomes.Cyanide(CN)inhibitstheindicatedstep.thesisofsteroidhormonesfromcholesterol(hydroxyla-ingrisetofreeradicalchainreactions(Chapter14).tionatC22andC20inside-chaincleavageandattheTheeasewithwhichsuperoxidecanbeformedfrom11βand18positions).Inaddition,renalsystemscat-oxygenintissuesandtheoccurrenceofsuperoxidedis-alyzing1α-and24-hydroxylationsof25-hydroxychole-mutase,theenzymeresponsibleforitsremovalinallcalciferolinvitaminDmetabolism—andcholesterolaerobicorganisms(althoughnotinobligateanaerobes)7α-hydroxylaseandsterol27-hydroxylaseinvolvedinindicatethatthepotentialtoxicityofoxygenisduetobileacidbiosynthesisintheliver(Chapter26)—areitsconversiontosuperoxide.P450enzymes.Superoxideisformedwhenreducedflavins—pre-sent,forexample,inxanthineoxidase—arereoxidizedSUPEROXIDEDISMUTASEPROTECTSunivalentlybymolecularoxygen.AEROBICORGANISMSAGAINST−EnzFlavinH−−+→OEnzFlavinHO−−++⋅H+OXYGENTOXICITY222TransferofasingleelectrontoO2generatesthepoten-Superoxidecanreduceoxidizedcytochromec−tiallydamagingsuperoxideanionfreeradical(O2⋅),thedestructiveeffectsofwhichareamplifiedbyitsgiv-O−⋅+→Cytcc()Fe3++O+Cyt()Fe222SubstrateA-HP450-A-H3+Fee–P450P450-A-HNADPH-CYTP450REDUCTASE2+Fe3+FeNADP+FADH2FeS3+222O2e––NADPH+H+FAD2FeS2+22CO2H+P450-A-HFe2+O2H2OP450-A-HFe2+O–2A-OHFigure11–6.CytochromeP450hydroxylasecycleinmicrosomes.Thesystemshownistypicalofsteroidhydroxylasesoftheadrenalcortex.LivermicrosomalcytochromeP450hydroxylasedoesnotrequiretheiron-sulfurproteinFe2S2.Carbonmonoxide(CO)inhibitstheindicatedstep.
99BIOLOGICOXIDATION/91orberemovedbysuperoxidedismutase.REFERENCESSUPEROXIDEBabcockGT,WikstromM:Oxygenactivationandtheconserva-DISMUTASEtionofenergyincellrespiration.Nature1992;356:301.O−.+O−.+2H+HO+OCoonMJetal:CytochromeP450:Progressandpredictions.22222FASEBJ1992;6:669.Inthisreaction,superoxideactsasbothoxidantandErnsterL(editor):Bioenergetics.Elsevier,1984.reductant.Thus,superoxidedismutaseprotectsaerobicMammaertsGP,VanVeldhovenPP:Roleofperoxisomesinmam-organismsagainstthepotentialdeleteriouseffectsofsu-malianmetabolism.CellBiochemFunct1992;10:141.peroxide.Theenzymeoccursinallmajoraerobictis-NichollsDG:CytochromesandCellRespiration.CarolinaBiologicalsuesinthemitochondriaandthecytosol.Althoughex-SupplyCompany,1984.posureofanimalstoanatmosphereof100%oxygenRahaS,RobinsonBH:Mitochondria,oxygenfreeradicals,diseasecausesanadaptiveincreaseinsuperoxidedismutase,andaging.TrendsBiochemSci2000;25:502.particularlyinthelungs,prolongedexposureleadstoTylerDD:TheMitochondrioninHealthandDisease.VCHPub-lungdamageanddeath.Antioxidants,eg,α-tocopherollishers,1992.(vitaminE),actasscavengersoffreeradicalsandreduceTylerDD,SuttonCM:Respiratoryenzymesystemsinmitochon-thetoxicityofoxygen(Chapter45).drialmembranes.In:MembraneStructureandFunction,vol5.BittarEE(editor).Wiley,1984.YangCS,BradyJF,HongJY:DietaryeffectsoncytochromesSUMMARYP450,xenobioticmetabolism,andtoxicity.FASEBJ1992;•Inbiologicsystems,asinchemicalsystems,oxidation6:737.(lossofelectrons)isalwaysaccompaniedbyreduc-tionofanelectronacceptor.•Oxidoreductaseshaveavarietyoffunctionsinme-tabolism;oxidasesanddehydrogenasesplaymajorrolesinrespiration;hydroperoxidasesprotectthebodyagainstdamagebyfreeradicals;andoxygenasesmediatethehydroxylationofdrugsandsteroids.•Tissuesareprotectedfromoxygentoxicitycausedbythesuperoxidefreeradicalbythespecificenzymesu-peroxidedismutase.
100TheRespiratoryChain&OxidativePhosphorylation12PeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DScBIOMEDICALIMPORTANCEtrappingtheliberatedfreeenergyashigh-energyphos-phate,andtheenzymesofβ-oxidationandofthecitricAerobicorganismsareabletocaptureafargreaterpro-acidcycle(Chapters22and16)thatproducemostofportionoftheavailablefreeenergyofrespiratorysub-thereducingequivalents.stratesthananaerobicorganisms.Mostofthistakesplaceinsidemitochondria,whichhavebeentermedthe“powerhouses”ofthecell.RespirationiscoupledtotheComponentsoftheRespiratoryChaingenerationofthehigh-energyintermediate,ATP,byAreArrangedinOrderofIncreasingoxidativephosphorylation,andthechemiosmoticRedoxPotentialtheoryoffersinsightintohowthisisaccomplished.AHydrogenandelectronsflowthroughtherespiratorynumberofdrugs(eg,amobarbital)andpoisons(eg,chain(Figure12–3)througharedoxspanof1.1Vcyanide,carbonmonoxide)inhibitoxidativephos-+phorylation,usuallywithfatalconsequences.Severalin-fromNAD/NADHtoO2/2H2O(Table11–1).TherespiratorychainconsistsofanumberofredoxcarriersheriteddefectsofmitochondriainvolvingcomponentsthatproceedfromtheNAD-linkeddehydrogenasesys-oftherespiratorychainandoxidativephosphorylationtems,throughflavoproteinsandcytochromes,tomole-havebeenreported.Patientspresentwithmyopathycularoxygen.Notallsubstratesarelinkedtotherespi-andencephalopathyandoftenhavelacticacidosis.ratorychainthroughNAD-specificdehydrogenases;some,becausetheirredoxpotentialsaremorepositiveSPECIFICENZYMESACTASMARKERS(eg,fumarate/succinate;Table11–1),arelinkeddi-rectlytoflavoproteindehydrogenases,whichinturnareOFCOMPARTMENTSSEPARATEDBYlinkedtothecytochromesoftherespiratorychain(Fig-THEMITOCHONDRIALMEMBRANESure12–4).Mitochondriahaveanoutermembranethatisperme-UbiquinoneorQ(coenzymeQ)(Figure12–5)abletomostmetabolites,aninnermembranethatislinkstheflavoproteinstocytochromeb,thememberofselectivelypermeable,andamatrixwithin(Figurethecytochromechainoflowestredoxpotential.Qex-12–1).Theoutermembraneischaracterizedbytheistsintheoxidizedquinoneorreducedquinolformpresenceofvariousenzymes,includingacyl-CoAsyn-underaerobicoranaerobicconditions,respectively.thetaseandglycerolphosphateacyltransferase.AdenylylThestructureofQisverysimilartothatofvitaminKkinaseandcreatinekinasearefoundintheintermem-andvitaminE(Chapter45)andofplastoquinone,branespace.Thephospholipidcardiolipinisconcen-foundinchloroplasts.Qactsasamobilecomponentoftratedintheinnermembranetogetherwiththeen-therespiratorychainthatcollectsreducingequivalentszymesoftherespiratorychain.fromthemorefixedflavoproteincomplexesandpassesthemontothecytochromes.Anadditionalcomponentistheiron-sulfurproteinTHERESPIRATORYCHAINCOLLECTS(FeS;nonhemeiron)(Figure12–6).Itisassociated&OXIDIZESREDUCINGEQUIVALENTSwiththeflavoproteins(metalloflavoproteins)andwithcytochromeb.ThesulfurandironarethoughttotakeMostoftheenergyliberatedduringtheoxidationofpartintheoxidoreductionmechanismbetweenflavin−carbohydrate,fattyacids,andaminoacidsismadeandQ,whichinvolvesonlyasingleechange,theiron2+availablewithinmitochondriaasreducingequivalentsatomundergoingoxidoreductionbetweenFeand3+(⎯Horelectrons)(Figure12–2).Mitochondriacon-Fe.taintherespiratorychain,whichcollectsandtrans-Pyruvateandα-ketoglutaratedehydrogenasehaveportsreducingequivalentsdirectingthemtotheirfinalcomplexsystemsinvolvinglipoateandFADpriortoreactionwithoxygentoformwater,themachineryforthepassageofelectronstoNAD,whileelectrontrans-92
101THERESPIRATORYCHAIN&OXIDATIVEPHOSPHORYLATION/93ElectronsflowfromQthroughtheseriesofcyto-chromesinorderofincreasingredoxpotentialtomole-cularoxygen(Figure12–4).Theterminalcytochromeaa3(cytochromeoxidase),responsibleforthefinalcom-Phosphorylatingbinationofreducingequivalentswithmolecularoxy-complexesgen,hasaveryhighaffinityforoxygen,allowingtherespiratorychaintofunctionatmaximumrateuntilthetissuehasbecomedepletedofO2.Sincethisisanirre-MATRIXversiblereaction(theonlyoneinthechain),itgivesdi-rectiontothemovementofreducingequivalentsandtotheproductionofATP,towhichitiscoupled.Functionallyandstructurally,thecomponentsofCristaetherespiratorychainarepresentintheinnermitochon-drialmembraneasfourprotein-lipidrespiratorychaincomplexesthatspanthemembrane.Cytochromecistheonlysolublecytochromeand,togetherwithQ,INNERseemstobeamoremobilecomponentoftherespira-MEMBRANEtorychainconnectingthefixedcomplexes(Figures12–7and12–8).OUTERMEMBRANETHERESPIRATORYCHAINPROVIDESMOSTOFTHEENERGYCAPTUREDDURINGCATABOLISMADPcaptures,intheformofhigh-energyphosphate,aFigure12–1.Structureofthemitochondrialmem-significantproportionofthefreeenergyreleasedbybranes.Notethattheinnermembranecontainsmanycatabolicprocesses.TheresultingATPhasbeencalledfolds,orcristae.theenergy“currency”ofthecellbecauseitpassesonthisfreeenergytodrivethoseprocessesrequiringen-ergy(Figure10–6).fersfromotherdehydrogenases,eg,L(+)-3-hydroxyacyl-Thereisanetdirectcaptureoftwohigh-energyCoAdehydrogenase,coupledirectlywithNAD.phosphategroupsintheglycolyticreactions(TableThereducedNADHoftherespiratorychainisin17–1),equivalenttoapproximately103.2kJ/molofturnoxidizedbyametalloflavoproteinenzyme—NADHglucose.(Invivo,ΔGforthesynthesisofATPfromdehydrogenase.ThisenzymecontainsFeSandFMN,ADPhasbeencalculatedasapproximately51.6kJ/mol.0′istightlyboundtotherespiratorychain,andpassesre-(ItisgreaterthanΔGforthehydrolysisofATPasducingequivalentsontoQ.giveninTable10–1,whichisobtainedunderstandardFOODATPFatFattyacids+β-OxidationGlycerolO2CitricCarbohydrateGlucose,etcAcetyl–CoAacid2HH2OcycleRespiratorychainProteinAminoacidsDigestionandabsorptionMITOCHONDRIONADPExtramitochondrialsourcesofreducingequivalentsFigure12–2.RoleoftherespiratorychainofmitochondriaintheconversionoffoodenergytoATP.Oxidationofthemajorfoodstuffsleadstothegenerationofreducingequivalents(2H)thatarecollectedbytherespiratorychainforoxidationandcoupledgenerationofATP.
10294/CHAPTER12AHNAD+FpH2Fe3+22H2OSubstrateFlavoproteinCytochromes2+1Figure12–3.TransportofreducingANADHFp2Fe/2O2equivalentsthroughtherespiratoryH+H+2H+2H+chain.concentrationsof1.0mol/L.)Since1molofglucosedentthattherespiratorychainisresponsibleforalargeyieldsapproximately2870kJoncompletecombustion,proportionoftotalATPformation.theenergycapturedbyphosphorylationinglycolysisissmall.Twomorehigh-energyphosphatespermoleofRespiratoryControlEnsuresglucosearecapturedinthecitricacidcycleduringtheaConstantSupplyofATPconversionofsuccinylCoAtosuccinate.Allofthesephosphorylationsoccuratthesubstratelevel.WhenTherateofrespirationofmitochondriacanbecon-substratesareoxidizedviaanNAD-linkeddehydrogen-trolledbytheavailabilityofADP.Thisisbecauseoxi-aseandtherespiratorychain,approximately3molofdationandphosphorylationaretightlycoupled;ie,oxi-inorganicphosphateareincorporatedinto3molofdationcannotproceedviatherespiratorychainwithoutADPtoform3molofATPperhalfmolofO2con-concomitantphosphorylationofADP.Table12–1sumed;ie,theP:Oratio=3(Figure12–7).Ontheshowsthefiveconditionscontrollingtherateofrespira-otherhand,whenasubstrateisoxidizedviaaflavopro-tioninmitochondria.Mostcellsintherestingstatearetein-linkeddehydrogenase,only2molofATPareinstate4,andrespirationiscontrolledbytheavailabil-formed;ie,P:O=2.Thesereactionsareknownasox-ityofADP.Whenworkisperformed,ATPiscon-idativephosphorylationattherespiratorychainvertedtoADP,allowingmorerespirationtooccur,level.SuchdehydrogenationsplusphosphorylationsatwhichinturnreplenishesthestoreofATP.Undercer-thesubstratelevelcannowaccountfor68%ofthefreetainconditions,theconcentrationofinorganicphos-energyresultingfromthecombustionofglucose,cap-phatecanalsoaffecttherateoffunctioningoftherespi-turedintheformofhigh-energyphosphate.Itisevi-ratorychain.Asrespirationincreases(asinexercise),SuccinateCholineProline3-Hydroxyacyl-CoA3-HydroxybutyrateGlutamateFpMalate(FAD)IsocitrateFeSPyruvateFpLipoateFpNAD(FMN)QCytbCytc1CytcCytaa3O2(FAD)FeSFeSCuα-KetoglutarateFpFeS(FAD)ETFFeS(FAD)FpFeS:Iron-sulfurprotein(FAD)ETF:Electron-transferringflavoproteinFp:FlavoproteinQ:UbiquinoneCyt:CytochromeGlycerol3-phosphateAcyl-CoASarcosineDimethylglycineFigure12–4.Componentsoftherespiratorychaininmitochondria,showingthecollectingpointsforreduc-ingequivalentsfromimportantsubstrates.FeSoccursinthesequencesontheO2sideofFporCytb.
103THERESPIRATORYCHAIN&OXIDATIVEPHOSPHORYLATION/95OHOHHOH(H++e–)(H++e–)CH3OCH3CH3CH3O[CH2CHCCH2]nHO•OOHFullyoxidizedorSemiquinoneformReducedorquinolformquinoneform(freeradical)(hydroquinone)Figure12–5.Structureofubiquinone(Q).n=Numberofisoprenoidunits,whichis10inhigheranimals,ie,Q10.thecellapproachesstate3orstate5wheneithertheca-MANYPOISONSINHIBITTHEpacityoftherespiratorychainbecomessaturatedortheRESPIRATORYCHAINPO2decreasesbelowtheKmforcytochromea3.ThereisalsothepossibilitythattheADP/ATPtransporter(Fig-Muchinformationabouttherespiratorychainhasbeenure12–9),whichfacilitatesentryofcytosolicADPintoobtainedbytheuseofinhibitors,and,conversely,thisandATPoutofthemitochondrion,becomesrate-hasprovidedknowledgeaboutthemechanismofactionlimiting.ofseveralpoisons(Figure12–7).TheymaybeclassifiedThus,themannerinwhichbiologicoxidativeasinhibitorsoftherespiratorychain,inhibitorsofox-processesallowthefreeenergyresultingfromtheoxida-idativephosphorylation,anduncouplersofoxidativetionoffoodstuffstobecomeavailableandtobecap-phosphorylation.turedisstepwise,efficient(approximately68%),andBarbituratessuchasamobarbitalinhibitNAD-controlled—ratherthanexplosive,inefficient,andun-linkeddehydrogenasesbyblockingthetransferfromcontrolled,asinmanynonbiologicprocesses.There-FeStoQ.Atsufficientdosage,theyarefatalinvivo.mainingfreeenergythatisnotcapturedashigh-energyAntimycinAanddimercaprolinhibittherespiratoryphosphateisliberatedasheat.Thisneednotbeconsid-chainbetweencytochromebandcytochromec.Theered“wasted,”sinceitensuresthattherespiratorysys-classicpoisonsH2S,carbonmonoxide,andcyanidetemasawholeissufficientlyexergonictoberemovedinhibitcytochromeoxidaseandcanthereforetotallyar-fromequilibrium,allowingcontinuousunidirectionalrestrespiration.MalonateisacompetitiveinhibitorofflowandconstantprovisionofATP.Italsocontributessuccinatedehydrogenase.tomaintenanceofbodytemperature.AtractylosideinhibitsoxidativephosphorylationbyinhibitingthetransporterofADPintoandATPoutofthemitochondrion(Figure12–10).PrTheactionofuncouplersistodissociateoxidationintherespiratorychainfromphosphorylation.TheseCyscompoundsaretoxicinvivo,causingrespirationtobe-Scomeuncontrolled,sincetherateisnolongerlimitedSFebytheconcentrationofADPorP.Theuncouplerthatihasbeenusedmostfrequentlyis2,4-dinitrophenol,PrCysSFeSbutothercompoundsactinasimilarmanner.Thean-tibioticoligomycincompletelyblocksoxidationandphosphorylationbyactingonastepinphosphorylationFeS(Figures12–7and12–8).SCysSFeTHECHEMIOSMOTICTHEORYEXPLAINSSTHEMECHANISMOFOXIDATIVEPrCysPHOSPHORYLATIONPrMitchell’schemiosmotictheorypostulatesthattheenergyfromoxidationofcomponentsintherespiratoryFigure12–6.Iron-sulfur-proteincomplex(Fe4S4).S,chainiscoupledtothetranslocationofhydrogenionsacid-labilesulfur;Pr,apoprotein;Cys,cysteineresidue.(protons,H+)fromtheinsidetotheoutsideoftheSomeiron-sulfurproteinscontaintwoironatomsandinnermitochondrialmembrane.Theelectrochemicaltwosulfuratoms(Fe2S2).potentialdifferenceresultingfromtheasymmetricdis-
10496/CHAPTER12MalonateComplexIIFADSuccinateFeS–CarboxinTTFAH2SCOBAL––CNAntimycinA–ComplexIVComplexIComplexIIICytaCyta3NADHFMN,FeSQCytb,FeS,Cytc1CytcO2CuCu––PiericidinAUncouplers–Amobarbital–Uncouplers–RotenoneOligomycin––Oligomycin–ADP+PiATPADP+PiATPADP+PiATPFigure12–7.Proposedsitesofinhibition(−)oftherespiratorychainbyspecificdrugs,chemicals,andantibi-otics.Thesitesthatappeartosupportphosphorylationareindicated.BAL,dimercaprol.TTFA,anFe-chelatingagent.ComplexI,NADH:ubiquinoneoxidoreductase;complexII,succinate:ubiquinoneoxidoreductase;complexIII,ubiquinol:ferricytochromecoxidoreductase;complexIV,ferrocytochromec:oxygenoxidoreductase.Otherab-breviationsasinFigure12–4.tributionofthehydrogenionsisusedtodrivetheunitsareattachedtoamembraneproteincomplexmechanismresponsiblefortheformationofATP(Fig-knownasF0,whichalsoconsistsofseveralproteinsub-ure12–8).units.F0spansthemembraneandformstheprotonchannel.TheflowofprotonsthroughF0causesittoro-tate,drivingtheproductionofATPintheF1complexTheRespiratoryChainIsaProtonPump(Figure12–9).EstimatessuggestthatforeachNADHoxidized,complexItranslocatesfourprotonsandcom-EachoftherespiratorychaincomplexesI,III,andIVplexesIIIandIVtranslocate6betweenthem.Asfour(Figures12–7and12–8)actsasaprotonpump.TheprotonsaretakenintothemitochondrionforeachATPinnermembraneisimpermeabletoionsingeneralbutexported,theP:Oratiowouldnotnecessarilybeacom-particularlytoprotons,whichaccumulateoutsidethepleteinteger,ie,3,butpossibly2.5.However,forsim-membrane,creatinganelectrochemicalpotentialdif-ferenceacrossthemembrane(Δμ+).Thisconsistsofaplicity,avalueof3fortheoxidationofNADH+H+Hchemicalpotential(differenceinpH)andanelectricaland2fortheoxidationofFADH2willcontinuetobeusedthroughoutthistext.potential.ExperimentalFindingsSupportAMembrane-LocatedATPSynthasetheChemiosmoticTheoryFunctionsasaRotaryMotortoFormATP(1)Additionofprotons(acid)totheexternalTheelectrochemicalpotentialdifferenceisusedtodrivemediumofintactmitochondrialeadstothegenerationamembrane-locatedATPsynthasewhichinthepres-ofATP.enceofPi+ADPformsATP(Figure12–8).Scattered(2)Oxidativephosphorylationdoesnotoccurinsolu-overthesurfaceoftheinnermembranearethephos-blesystemswherethereisnopossibilityofavectorialphorylatingcomplexes,ATPsynthase,responsibleforATPsynthase.AclosedmembranemustbepresentintheproductionofATP(Figure12–1).Theseconsistofordertoachieveoxidativephosphorylation(Figure12–8).severalproteinsubunits,collectivelyknownasF1,(3)Therespiratorychaincontainscomponentsor-whichprojectintothematrixandwhichcontaintheganizedinasidedmanner(transverseasymmetry)asre-phosphorylationmechanism(Figure12–8).Thesesub-quiredbythechemiosmotictheory.
105THERESPIRATORYCHAIN&OXIDATIVEPHOSPHORYLATION/97H+ProtoncircOligomycinuit–F0PhospholipidbilayerF1H+IATPSYNTHASENADH+H+ADP+PATP+HOi2Q+tNADrMitochondrialanPRespiratoryinner(coupling)+sloroIII+(electronmembraneHctoHantransport)toichainn1/+2OH2CHOIVUncouplingagents2+HINSIDE+HpHgradient(ΔpH)–ElectricalpotentialOUTSIDE+(ΔΨ)Figure12–8.Principlesofthechemiosmotictheoryofoxidativephosphorylation.Themainprotoncircuitiscreatedbythecouplingofoxidationintherespiratorychaintoprotontranslocationfromtheinsidetotheoutsideofthemembrane,drivenbytherespiratorychaincomplexesI,III,andIV,eachofwhichactsasapro-tonpump.Q,ubiquinone;C,cytochromec;F1,F0,proteinsubunitswhichutilizeenergyfromtheprotongra-dienttopromotephosphorylation.UncouplingagentssuchasdinitrophenolallowleakageofH+acrossthemembrane,thuscollapsingtheelectrochemicalprotongradient.OligomycinspecificallyblocksconductionofH+throughF0.TheChemiosmoticTheoryCanAccountforRespiratoryControlandtheActionofUncouplersTheelectrochemicalpotentialdifferenceacrossthemem-brane,onceestablishedasaresultofprotontransloca-Table12–1.Statesofrespiratorycontrol.tion,inhibitsfurthertransportofreducingequivalentsthroughtherespiratorychainunlessdischargedbyback-translocationofprotonsacrossthemembranethroughConditionsLimitingtheRateofRespirationthevectorialATPsynthase.ThisinturndependsonState1AvailabilityofADPandsubstrateavailabilityofADPandPi.State2AvailabilityofsubstrateonlyUncouplers(eg,dinitrophenol)areamphipathicState3Thecapacityoftherespiratorychainitself,when(Chapter14)andincreasethepermeabilityofthelipoidallsubstratesandcomponentsarepresentininnermitochondrialmembranetoprotons(Figuresaturatingamounts12–8),thusreducingtheelectrochemicalpotentialandState4AvailabilityofADPonlyshort-circuitingtheATPsynthase.Inthisway,oxida-State5Availabilityofoxygenonlytioncanproceedwithoutphosphorylation.
10698/CHAPTER12ingelectricalandosmoticequilibrium.TheinnerβbilipoidmitochondrialmembraneisfreelypermeableATPααtounchargedsmallmolecules,suchasoxygen,water,ADPββγCO2,andNH3,andtomonocarboxylicacids,suchas+αATP3-hydroxybutyric,acetoacetic,andacetic.Long-chainPifattyacidsaretransportedintomitochondriaviathecarnitinesystem(Figure22–1),andthereisalsoaspe-cialcarrierforpyruvateinvolvingasymportthatutilizestheH+gradientfromoutsidetoinsidethemitochon-drion(Figure12–10).However,dicarboxylateandtri-γH+InsideInnerOUTSIDEmitochondrialINSIDEmembraneMitochondrialinnerN-EthylmaleimidemembraneOH–CC1OutsideCCHPO–24CCN-Ethylmaleimide–HydroxycinnamatePyruvate–2H+H+–Figure12–9.HPO2–MechanismofATPproductionbyATP4synthase.TheenzymecomplexconsistsofanF0sub-3Malate2–complexwhichisadiskof“C”proteinsubunits.At-tachedisaγ-subunitintheformofa“bentaxle.”Pro-Malate2–tonspassingthroughthediskof“C”unitscauseitandtheattachedγ-subunittorotate.Theγ-subunitfitsin-4Citrate3–sidetheF1subcomplexofthreeα-andthreeβ-sub-+H+units,whicharefixedtothemembraneanddonotro-Malate2–tate.ADPandPiaretakenupsequentiallybytheβ-subunitstoformATP,whichisexpelledastherotat-5α-Ketoglutarate2–ingγ-subunitsqueezeseachβ-subunitinturn.Thus,–threeATPmoleculesaregeneratedperrevolution.ForADP3–clarity,notallthesubunitsthathavebeenidentifiedare6shown—eg,the“axle”alsocontainsanε-subunit.ATP4–AtractylosideFigure12–10.Transportersystemsintheinnermi-THERELATIVEIMPERMEABILITYtochondrialmembrane.1,phosphatetransporter;OFTHEINNERMITOCHONDRIAL2,pyruvatesymport;3,dicarboxylatetransporter;4,tricarboxylatetransporter;5,α-ketoglutaratetrans-MEMBRANENECESSITATESporter;6,adeninenucleotidetransporter.N-Ethyl-EXCHANGETRANSPORTERSmaleimide,hydroxycinnamate,andatractylosideinhibitExchangediffusionsystemsarepresentinthemem-(−)theindicatedsystems.Alsopresent(butnotbraneforexchangeofanionsagainstOH−ionsandshown)aretransportersystemsforglutamate/aspar-cationsagainstH+ions.Suchsystemsarenecessaryfortate(Figure12–13),glutamine,ornithine,neutralaminouptakeandoutputofionizedmetaboliteswhilepreserv-acids,andcarnitine(Figure22–1).
107THERESPIRATORYCHAIN&OXIDATIVEPHOSPHORYLATION/99carboxylateanionsandaminoacidsrequirespecificIonophoresPermitSpecificCationstransporterorcarriersystemstofacilitatetheirpassagetoPenetrateMembranesacrossthemembrane.Monocarboxylicacidspenetratemorereadilyintheirundissociatedandmorelipid-solu-Ionophoresarelipophilicmoleculesthatcomplexspe-bleform.cificcationsandfacilitatetheirtransportthroughbio-Thetransportofdi-andtricarboxylateanionsislogicmembranes,eg,valinomycin(K+).Theclassiccloselylinkedtothatofinorganicphosphate,whichuncouplerssuchasdinitrophenolare,infact,protonpenetratesreadilyastheHPO−ioninexchangeforionophores.24−OH.ThenetuptakeofmalatebythedicarboxylatetransporterrequiresinorganicphosphateforexchangeAProton-TranslocatingTranshydrogenaseintheoppositedirection.Thenetuptakeofcitrate,IsaSourceofIntramitochondrialNADPHisocitrate,orcis-aconitatebythetricarboxylatetrans-Energy-linkedtranshydrogenase,aproteinintheinnerporterrequiresmalateinexchange.α-Ketoglutaratemitochondrialmembrane,couplesthepassageofpro-transportalsorequiresanexchangewithmalate.ThetonsdowntheelectrochemicalgradientfromoutsidetoadeninenucleotidetransporterallowstheexchangeofinsidethemitochondrionwiththetransferofHfromATPandADPbutnotAMP.ItisvitalinallowingintramitochondrialNADHtoNADPHforintramito-ATPexitfrommitochondriatothesitesofextramito-chondrialenzymessuchasglutamatedehydrogenasechondrialutilizationandinallowingthereturnofADPandhydroxylasesinvolvedinsteroidsynthesis.forATPproductionwithinthemitochondrion(Figure12–11).Na+canbeexchangedforH+,drivenbytheprotongradient.ItisbelievedthatactiveuptakeofCa2+OxidationofExtramitochondrialNADHbymitochondriaoccurswithanetchargetransferof1IsMediatedbySubstrateShuttles(Ca+uniport),possiblythroughaCa2+/H+antiport.NADHcannotpenetratethemitochondrialmem-Calciumreleasefrommitochondriaisfacilitatedbyex-brane,butitisproducedcontinuouslyinthecytosolbychangewithNa+.3-phosphoglyceraldehydedehydrogenase,anenzymeintheglycolysissequence(Figure17–2).However,underaerobicconditions,extramitochondrialNADHdoesnotaccumulateandispresumedtobeoxidizedbytherespi-ratorychaininmitochondria.Thetransferofreducingequivalentsthroughthemitochondrialmembranere-InnerOUTSIDEmitochondrialINSIDEquiressubstratepairs,linkedbysuitabledehydrogen-membraneasesoneachsideofthemitochondrialmembrane.TheFmechanismoftransferusingtheglycerophosphate1ATPSYNTHASEshuttleisshowninFigure12–12).Sincethemitochon-drialenzymeislinkedtotherespiratorychainviaa3H+flavoproteinratherthanNAD,only2molratherthan3molofATPareformedperatomofoxygencon-sumed.Althoughthisshuttleispresentinsometissues4–(eg,brain,whitemuscle),inothers(eg,heartmuscle)itATPisdeficient.Itisthereforebelievedthatthemalateshuttlesystem(Figure12–13)isofmoreuniversal23–utility.Thecomplexityofthissystemisduetotheim-ADPpermeabilityofthemitochondrialmembranetooxalo-P–acetate,whichmustreactwithglutamateandtransami-i1+natetoaspartateandα-ketoglutaratebeforetransportHthroughthemitochondrialmembraneandreconstitu-tiontooxaloacetateinthecytosol.Figure12–11.Combinationofphosphatetrans-porter(1)withtheadeninenucleotidetransporter(2)+IonTransportinMitochondriainATPsynthesis.TheH/Pisymportshownisequiva-−IsEnergy-LinkedlenttothePi/OHantiportshowninFigure12–10.FourprotonsaretakenintothemitochondrionforeachATPMitochondriamaintainoraccumulatecationssuchasexported.However,onelessprotonwouldbetakeninK+,Na+,Ca2+,andMg2+,andP.ItisassumedthataiwhenATPisusedinsidethemitochondrion.primaryprotonpumpdrivescationexchange.
108100/CHAPTER12OUTERINNERMEMBRANEMEMBRANECYTOSOLMITOCHONDRIONNAD+Glycerol3-phosphateGlycerol3-phosphateFADGLYCEROL-3-PHOSPHATEGLYCEROL-3-PHOSPHATEDEHYDROGENASEDEHYDROGENASE(CYTOSOLIC)(MITOCHONDRIAL)NADH+H+DihydroxyacetoneDihydroxyacetoneFADH2phosphatephosphateRespiratorychainFigure12–12.Glycerophosphateshuttlefortransferofreducingequivalentsfromthecytosolintothemitochondrion.TheCreatinePhosphateShuttleportedintothecytosolviaproteinporesintheouterFacilitatesTransportofHigh-Energymitochondrialmembrane,becomingavailableforgen-PhosphateFromMitochondriaerationofextramitochondrialATP.Thisshuttle(Figure12–14)augmentsthefunctionsofCLINICALASPECTScreatinephosphateasanenergybufferbyactingasadynamicsystemfortransferofhigh-energyphosphateTheconditionknownasfatalinfantilemitochondrialfrommitochondriainactivetissuessuchasheartandmyopathyandrenaldysfunctioninvolvesseveredim-skeletalmuscle.Anisoenzymeofcreatinekinase(CKm)inutionorabsenceofmostoxidoreductasesoftherespi-isfoundinthemitochondrialintermembranespace,ratorychain.MELAS(mitochondrialencephalopathy,catalyzingthetransferofhigh-energyphosphatetocre-lacticacidosis,andstroke)isaninheritedconditiondueatinefromATPemergingfromtheadeninenucleotidetoNADH:ubiquinoneoxidoreductase(complexI)ortransporter.Inturn,thecreatinephosphateistrans-cytochromeoxidasedeficiency.Itiscausedbyamuta-INNERCYTOSOLMEMBRANEMITOCHONDRIONNAD+MalateMalateNAD+1MALATEDEHYDROGENASEMALATEDEHYDROGENASENADHOxaloacetateα-KGα-KGOxaloacetateNADH+H++H+TRANSAMINASETRANSAMINASEGlutamateAspAspGlutamate2H+H+Figure12–13.Malateshuttlefortransferofreducingequivalentsfromthecytosolintothemitochondrion.1Ketoglutaratetransporter;2,glutamate/aspartatetransporter(notetheprotonsymportwithglutamate).
109THERESPIRATORYCHAIN&OXIDATIVEPHOSPHORYLATION/101Energy-requiringprocesses(eg,musclecontraction)ATPADPCKaATPADPCreatineCreatine-PCKcCKgATPADPGlycolysisOutermitochondrialCytosolmembranePFigure12–14.ThecreatinephosphateshuttleofPheartandskeletalmuscle.TheshuttleallowsrapidCKmtransportofhigh-energyphosphatefromthemito-chondrialmatrixintothecytosol.CKa,creatinekinaseInter-membraneconcernedwithlargerequirementsforATP,eg,mus-ATPADPspacecularcontraction;CKc,creatinekinaseformaintainingAdenineequilibriumbetweencreatineandcreatinephosphatenucleotideandATP/ADP;CKg,creatinekinasecouplingglycolysistransportermitInmocnetocreatinephosphatesynthesis;CKm,mitochondrialehormbnrdcreatinekinasemediatingcreatinephosphateproduc-OxidativearniationfromATPformedinoxidativephosphorylation;P,phosphorylationelporeproteininoutermitochondrialmembrane.MatrixtioninmitochondrialDNAandmaybeinvolvedin•Becausetheinnermitochondrialmembraneisimper-Alzheimer’sdiseaseanddiabetesmellitus.Anumberofmeabletoprotonsandotherions,specialexchangedrugsandpoisonsactbyinhibitionofoxidativephos-transportersspanthemembranetoallowpassageof–−4−3−phorylation(seeabove).ionssuchasOH,Pi,ATP,ADP,andmetabo-lites,withoutdischargingtheelectrochemicalgradi-entacrossthemembrane.SUMMARY•Manywell-knownpoisonssuchascyanidearrestres-pirationbyinhibitionoftherespiratorychain.•Virtuallyallenergyreleasedfromtheoxidationofcarbohydrate,fat,andproteinismadeavailablein−REFERENCESmitochondriaasreducingequivalents(⎯Hore).Thesearefunneledintotherespiratorychain,whereBalabanRS:Regulationofoxidativephosphorylationinthemam-theyarepasseddownaredoxgradientofcarrierstomaliancell.AmJPhysiol1990;258:C377.theirfinalreactionwithoxygentoformwater.HinklePCetal:Mechanisticstoichiometryofmitochondrialox-•Theredoxcarriersaregroupedintorespiratorychainidativephosphorylation.Biochemistry1991;30:3576.complexesintheinnermitochondrialmembrane.MitchellP:Keilin’srespiratorychainconceptanditschemiosmoticTheseusetheenergyreleasedintheredoxgradienttoconsequences.Science1979;206:1148.pumpprotonstotheoutsideofthemembrane,creat-SmeitinkJetal:Thegeneticsandpathologyofoxidativephosphor-ylation.NatRevGenet2001;2:342.inganelectrochemicalpotentialacrossthemembrane.TylerDD:TheMitochondrioninHealthandDisease.VCHPub-•SpanningthemembraneareATPsynthasecom-lishers,1992.plexesthatusethepotentialenergyoftheprotongra-WallaceDC:MitochondrialDNAinaginganddisease.SciAmdienttosynthesizeATPfromADPandPi.Inthis1997;277(2):22.way,oxidationiscloselycoupledtophosphorylationYoshidaMetal:ATPsynthase—amarvellousrotaryengineofthetomeettheenergyneedsofthecell.cell.NatRevMolCellBiol2001;2:669.
110CarbohydratesofPhysiologicSignificance13PeterA.Mayes,PhD,DSc,&DavidA.Bender,PhDBIOMEDICALIMPORTANCE(4)Polysaccharidesarecondensationproductsofmorethantenmonosaccharideunits;examplesaretheCarbohydratesarewidelydistributedinplantsandani-starchesanddextrins,whichmaybelinearorbranchedmals;theyhaveimportantstructuralandmetabolicpolymers.Polysaccharidesaresometimesclassifiedasroles.Inplants,glucoseissynthesizedfromcarbonhexosansorpentosans,dependingupontheidentityofdioxideandwaterbyphotosynthesisandstoredastheconstituentmonosaccharides.starchorusedtosynthesizecelluloseoftheplantframe-work.Animalscansynthesizecarbohydratefromlipidglycerolandaminoacids,butmostanimalcarbohy-BIOMEDICALLY,GLUCOSEISTHEMOSTdrateisderivedultimatelyfromplants.GlucoseistheIMPORTANTMONOSACCHARIDEmostimportantcarbohydrate;mostdietarycarbohy-drateisabsorbedintothebloodstreamasglucose,andTheStructureofGlucoseCanBeothersugarsareconvertedintoglucoseintheliver.RepresentedinThreeWaysGlucoseisthemajormetabolicfuelofmammals(ex-ceptruminants)andauniversalfuelofthefetus.ItisThestraight-chainstructuralformula(aldohexose;theprecursorforsynthesisofalltheothercarbohy-Figure13–1A)canaccountforsomeofthepropertiesdratesinthebody,includingglycogenforstorage;ri-ofglucose,butacyclicstructureisfavoredonthermo-boseanddeoxyriboseinnucleicacids;andgalactosedynamicgroundsandaccountsfortheremainderofitsinlactoseofmilk,inglycolipids,andincombinationchemicalproperties.Formostpurposes,thestructuralwithproteininglycoproteinsandproteoglycans.Dis-formulaisrepresentedasasimpleringinperspectiveaseasesassociatedwithcarbohydratemetabolismincludeproposedbyHaworth(Figure13–1B).Inthisrepresen-diabetesmellitus,galactosemia,glycogenstoragetation,themoleculeisviewedfromthesideandabovediseases,andlactoseintolerance.theplaneofthering.Byconvention,thebondsnearesttotheviewerareboldandthickened.Thesix-mem-beredringcontainingoneoxygenatomisintheformCARBOHYDRATESAREALDEHYDEofachair(Figure13–1C).ORKETONEDERIVATIVESOFPOLYHYDRICALCOHOLSSugarsExhibitVariousFormsofIsomerism(1)Monosaccharidesarethosecarbohydratesthatcannotbehydrolyzedintosimplercarbohydrates:TheyGlucose,withfourasymmetriccarbonatoms,canformmaybeclassifiedastrioses,tetroses,pentoses,hex-16isomers.Themoreimportanttypesofisomerismoses,orheptoses,dependinguponthenumberofcar-foundwithglucoseareasfollows.bonatoms;andasaldosesorketosesdependingupon(1)DandLisomerism:Thedesignationofasugarwhethertheyhaveanaldehydeorketonegroup.Exam-isomerastheDformorofitsmirrorimageastheLformplesarelistedinTable13–1.(2)Disaccharidesarecondensationproductsoftwomonosaccharideunits.Examplesaremaltoseandsu-Table13–1.Classificationofimportantsugars.crose.(3)Oligosaccharidesarecondensationproductsoftwototenmonosaccharides;maltotriose*isanexam-AldosesKetosesple.Trioses(C3H6O3)GlyceroseDihydroxyacetoneTetroses(C4H8O4)ErythroseErythrulosePentoses(C5H10O5)RiboseRibulose*Notethatthisisnotatruetriosebutatrisaccharidecontainingthreeα-glucoseresidues.Hexoses(C6H12O6)GlucoseFructose102
111CARBOHYDRATESOFPHYSIOLOGICSIGNIFICANCE/103AOOO1CHH2COHHO3CHPyranFuranH4COHH5COH6CHOHHOCH22HOCH26HCOHOBHOCH2OHHHH5OHHHHOOHHOHHOHHOH41HOOHHOHHOHHOH32HOHα-D-Glucopyranoseα-D-GlucofuranoseCHFigure13–3.Pyranoseandfuranoseformsofglu-6HOCHOcose.2HO45HH2HHOOH13OHHFigure13–1.D-Glucose.A:straightchainform.B:α-D-glucose;Haworthprojection.C:α-D-glucose;chairform.1OOHHOCH2H16O6OCHCHHHHHOH2CCHOHHOH52523CHOHCHOHHOHHOOHHOHHO221L-GlyceroseD-Glycerose4343COH(L-glyceraldehyde)(D-glyceraldehyde)OHHOHHH2α-D-Fructopyranoseβ-D-FructopyranoseOO1CCHH6162HOCH2HOCH2HOCH2OHHOCHHCOHOO3CCHOHHOH5252HO4CHHCOHHHHOOHHHHO5HOCHHCOH43431COH6OHHOHHHCH2OHCH2OH2L-GlucoseD-Glucoseα-D-Fructofuranoseβ-D-FructofuranoseFigure13–2.D-andL-isomerismofglyceroseandFigure13–4.Pyranoseandfuranoseformsoffruc-glucose.tose.
112104/CHAPTER13HOCH2HOCH2HOCH2OOOHOHHHHHHHH44HOHHOHOHOHHOHOHOHHOOH22HOHHOHHHFigure13–5.Epimerizationofα-D-Galactoseα-D-Glucoseα-D-Mannoseglucose.isdeterminedbyitsspatialrelationshiptotheparent(3)Alphaandbetaanomers:Theringstructureofcompoundofthecarbohydrates,thethree-carbonanaldoseisahemiacetal,sinceitisformedbycombina-sugarglycerose(glyceraldehyde).TheLandDformsoftionofanaldehydeandanalcoholgroup.Similarly,thethissugar,andofglucose,areshowninFigure13–2.ringstructureofaketoseisahemiketal.Crystallineglu-Theorientationofthe⎯Hand⎯OHgroupsaroundcoseisα-D-glucopyranose.Thecyclicstructureisre-thecarbonatomadjacenttotheterminalprimaryalco-tainedinsolution,butisomerismoccursaboutpositionholcarbon(carbon5inglucose)determineswhether1,thecarbonyloranomericcarbonatom,togiveathesugarbelongstotheDorLseries.Whenthe⎯OHmixtureofα-glucopyranose(38%)andβ-glucopyra-grouponthiscarbonisontheright(asseeninFigurenose(62%).Lessthan0.3%isrepresentedbyαandβ13–2),thesugaristheD-isomer;whenitisontheanomersofglucofuranose.left,itistheL-isomer.Mostofthemonosaccharides(4)Epimers:Isomersdifferingasaresultofvaria-occurringinmammalsareDsugars,andtheenzymestionsinconfigurationofthe⎯OHand⎯Honcar-responsiblefortheirmetabolismarespecificforthisbonatoms2,3,and4ofglucoseareknownasepimers.configuration.Insolution,glucoseisdextrorotatory—Biologically,themostimportantepimersofglucosearehencethealternativenamedextrose,oftenusedinmannoseandgalactose,formedbyepimerizationatcar-clinicalpractice.bons2and4,respectively(Figure13–5).Thepresenceofasymmetriccarbonatomsalsocon-(5)Aldose-ketoseisomerism:Fructosehasthefersopticalactivityonthecompound.Whenabeamsamemolecularformulaasglucosebutdiffersinitsofplane-polarizedlightispassedthroughasolutionofstructuralformula,sincethereisapotentialketogroupanopticalisomer,itwillberotatedeithertotheright,inposition2,theanomericcarbonoffructose(Figuresdextrorotatory(+);ortotheleft,levorotatory(−).The13–4and13–7),whereasthereisapotentialaldehydedirectionofrotationisindependentofthestereochem-groupinposition1,theanomericcarbonofglucoseistryofthesugar,soitmaybedesignatedD(−),D(+),(Figures13–2and13–6).L(−),orL(+).Forexample,thenaturallyoccurringformoffructoseistheD(−)isomer.ManyMonosaccharidesAre(2)Pyranoseandfuranoseringstructures:ThePhysiologicallyImportantstableringstructuresofmonosaccharidesaresimilartotheringstructuresofeitherpyran(asix-memberedDerivativesoftrioses,tetroses,andpentosesandofaring)orfuran(afive-memberedring)(Figures13–3seven-carbonsugar(sedoheptulose)areformedasmeta-and13–4).Forglucoseinsolution,morethan99%isbolicintermediatesinglycolysisandthepentosephos-inthepyranoseform.phatepathway.Pentosesareimportantinnucleotides,CHOCHOCHOCHOCHOCHOCHOHCOHHOCHHCOHCHOHOCHHCOHHOCHHCOHHOCHHOCHHOCHCHOHCOHHOCHHOCHHCOHHCOHHOCHHCOHHCOHHCOHHCOHHCOHHCOHHCOHHCOHHCOHHCOHHCOHCH2OHD-GlyceroseCH2OHCH2OHCH2OHCH2OHCH2OHCH2OHCH2OHCH2OH(D-glyceraldehyde)D-ErythroseD-LyxoseD-XyloseD-ArabinoseD-RiboseD-GalactoseD-MannoseD-GlucoseFigure13–6.Examplesofaldosesofphysiologicsignificance.
113CARBOHYDRATESOFPHYSIOLOGICSIGNIFICANCE/105Table13–2.Pentosesofphysiologicimportance.SugarWhereFoundBiochemicalImportanceClinicalSignificanceD-RiboseNucleicacids.Structuralelementsofnucleicacidsandcoenzymes,eg,ATP,NAD,NADP,flavo-proteins.Ribosephosphatesareinter-mediatesinpentosephosphatepathway.D-RibuloseFormedinmetabolicprocesses.Ribulosephosphateisanintermediateinpentosephosphatepathway.D-ArabinoseGumarabic.Plumandcherrygums.Constituentofglycoproteins.D-XyloseWoodgums,proteoglycans,Constituentofglycoproteins.glycosaminoglycans.D-LyxoseHeartmuscle.Aconstituentofalyxoflavinisolatedfromhumanheartmuscle.L-XyluloseIntermediateinuronicacidpathway.Foundinurineinessentialpentosuria.nucleicacids,andseveralcoenzymes(Table13–2).SugarsFormGlycosidesWithOtherGlucose,galactose,fructose,andmannosearephysio-Compounds&WithEachOtherlogicallythemostimportanthexoses(Table13–3).ThebiochemicallyimportantaldosesareshowninFigureGlycosidesareformedbycondensationbetweenthehy-13–6,andimportantketosesinFigure13–7.droxylgroupoftheanomericcarbonofamonosaccha-Inaddition,carboxylicacidderivativesofglucoseareride,ormonosaccharideresidue,andasecondcompoundimportant,includingD-glucuronate(forglucuronidethatmay—ormaynot(inthecaseofanaglycone)—beformationandinglycosaminoglycans)anditsmeta-anothermonosaccharide.Ifthesecondgroupisahy-bolicderivative,L-iduronate(inglycosaminoglycans)droxyl,theO-glycosidicbondisanacetallinkbecauseit(Figure13–8)andL-gulonate(anintermediateintheresultsfromareactionbetweenahemiacetalgroupuronicacidpathway;seeFigure20–4).(formedfromanaldehydeandan⎯OHgroup)andan-Table13–3.Hexosesofphysiologicimportance.SugarSourceImportanceClinicalSignificanceD-GlucoseFruitjuices.Hydrolysisofstarch,caneThe“sugar”ofthebody.ThesugarcarriedPresentintheurine(glycosuria)sugar,maltose,andlactose.bytheblood,andtheprincipaloneusedindiabetesmellitusowingtobythetissues.raisedbloodglucose(hyper-glycemia).D-FructoseFruitjuices.Honey.HydrolysisofCanbechangedtoglucoseintheliverHereditaryfructoseintolerancecanesugarandofinulin(fromtheandsousedinthebody.leadstofructoseaccumulationJerusalemartichoke).andhypoglycemia.D-GalactoseHydrolysisoflactose.CanbechangedtoglucoseintheliverFailuretometabolizeleadsandmetabolized.Synthesizedinthetogalactosemiaandcataract.mammaryglandtomakethelactoseofmilk.Aconstituentofglycolipidsandglycoproteins.D-MannoseHydrolysisofplantmannansandAconstituentofmanyglycoproteins.gums.
114106/CHAPTER13CH2OHCH2OHCOCH2OHCH2OHCOHOCHCOCOHOCHHCOHCH2OHHOCHHCOHHCOHHCOHCOHCOHHCOHHCOHHCOHCH2OHCH2OHCH2OHCH2OHCH2OHDihydroxyacetoneD-XyluloseD-RibuloseD-FructoseD-SedoheptuloseFigure13–7.Examplesofketosesofphysiologicsignificance.other⎯OHgroup.Ifthehemiacetalportionisglucose,COO–Htheresultingcompoundisaglucoside;ifgalactose,aOOgalactoside;andsoon.Ifthesecondgroupisanamine,HHHHCOO–HanN-glycosidicbondisformed,eg,betweenadenineandriboseinnucleotidessuchasATP(Figure10–4).HOOHHOHHOOHHOHGlycosidesarewidelydistributedinnature;theagly-conemaybemethanol,glycerol,asterol,aphenol,orabasesuchasadenine.TheglycosidesthatareimportantHOHHOHinmedicinebecauseoftheiractionontheheart(car-Figure13–8.α-D-Glucuronate(left)anddiacglycosides)allcontainsteroidsastheaglycone.β-L-iduronate(right).Theseincludederivativesofdigitalisandstrophanthussuchasouabain,aninhibitoroftheNa+-K+ATPaseofcellmembranes.Otherglycosidesincludeantibioticssuchasstreptomycin.5HOCH2DeoxySugarsLackanOxygenAtomOOHDeoxysugarsarethoseinwhichahydroxylgrouphas41beenreplacedbyhydrogen.AnexampleisdeoxyriboseHHHH(Figure13–9)inDNA.ThedeoxysugarL-fucose(Figure3213–15)occursinglycoproteins;2-deoxyglucoseisusedOHHexperimentallyasaninhibitorofglucosemetabolism.Figure13–9.2-Deoxy-D-ribofuranose(βform).AminoSugars(Hexosamines)AreComponentsofGlycoproteins,Gangliosides,&GlycosaminoglycansTheaminosugarsincludeD-glucosamine,aconstituentHOCH2ofhyaluronicacid(Figure13–10),D-galactosamineO(chondrosamine),aconstituentofchondroitin;andHHHD-mannosamine.Severalantibiotics(eg,erythromycin)containaminosugarsbelievedtobeimportantfortheirHOOHHOHantibioticactivity.H+NH3MALTOSE,SUCROSE,&LACTOSEAREFigure13–10.Glucosamine(2-amino-D-glucopyra-IMPORTANTDISACCHARIDESnose)(αform).Galactosamineis2-amino-D-galactopy-Thephysiologicallyimportantdisaccharidesaremal-ranose.Bothglucosamineandgalactosamineoccurastose,sucrose,andlactose(Table13–4;Figure13–11).N-acetylderivativesinmorecomplexcarbohydrates,Hydrolysisofsucroseyieldsamixtureofglucoseandeg,glycoproteins.
115CARBOHYDRATESOFPHYSIOLOGICSIGNIFICANCE/107Table13–4.Disaccharides.SugarSourceClinicalSignificanceMaltoseDigestionbyamylaseorhydrolysisofstarch.Germinatingcerealsandmalt.LactoseMilk.Mayoccurinurineduringpregnancy.Inlactasedeficiency,malabsorptionleadstodiarrheaandflatulence.SucroseCaneandbeetsugar.Sorghum.Pineapple.Insucrasedeficiency,malabsorptionleadstodiarrheaandflatulence.Carrotroots.1TrehaloseFungiandyeasts.Themajorsugarofinsecthemolymph.1O-α-D-Glucopyranosyl-(1→1)-α-D-glucopyranoside.fructosewhichiscalled“invertsugar”becausethetoes,legumes,andothervegetables.Thetwomaincon-stronglylevorotatoryfructosechanges(inverts)thepre-stituentsareamylose(15–20%),whichhasanon-viousdextrorotatoryactionofsucrose.branchinghelicalstructure(Figure13–12);andamy-lopectin(80–85%),whichconsistsofbranchedchainsPOLYSACCHARIDESSERVESTORAGEcomposedof24–30glucoseresiduesunitedby1→4&STRUCTURALFUNCTIONSlinkagesinthechainsandby1→6linkagesatthebranchpoints.PolysaccharidesincludethefollowingphysiologicallyGlycogen(Figure13–13)isthestoragepolysaccha-importantcarbohydrates.rideinanimals.ItisamorehighlybranchedstructureStarchisahomopolymerofglucoseforminganα-thanamylopectin,withchainsof12–14α-D-glucopyra-glucosidicchain,calledaglucosanorglucan.Itisthenoseresidues(inα[1→4]-glucosidiclinkage),withmostabundantdietarycarbohydrateincereals,pota-branchingbymeansofα(1→6)-glucosidicbonds.MaltoseLactose6666HOCH2HOCH2HOCH2HOCH25O5O5O5OHHHHHHHOHHHOH41**4141*O41*HOOHHOHHOHHOHHHOHHH32323232OHOHHOHHOHHOHO-α-D-Glucopyranosyl-(1→4)-α-D-glucopyranoseO-β-D-Galactopyranosyl-(1→4)-β-D-glucopyranoseSucroseFigure13–11.Structuresofimportantdisaccharides.Theαandβ61HOCH2HOCH2refertotheconfigurationattheanomericcarbonatom(asterisk).When5OtheanomericcarbonofthesecondresiduetakespartintheformationOHHHHoftheglycosidicbond,asinsucrose,theresiduebecomesaglycoside4125knownasafuranosideorpyranoside.Asthedisaccharidenolongerhas**6HOOHHHHOCOHananomericcarbonwithafreepotentialaldehydeorketonegroup,it3234H2Onolongerexhibitsreducingproperties.TheconfigurationoftheHOHOHHβ-fructofuranoseresidueinsucroseresultsfromturningtheβ-fructofu-ranosemoleculedepictedinFigure13–4through180degreesandin-O-α-D-Glucopyranosyl-(1→2)-β-D-fructofuranosidevertingit.
116108/CHAPTER13HOCH624O1HOCH624OO1ABOOOOOOOOO6666HOCH2HOCH2CH2HOCH2OOOOOO41414141OOOOOOOFigure13–12.Structureofstarch.A:Amylose,showinghelicalcoilstructure.B:Amylopectin,showing1→6branchpoint.OHOO4CH264OO11OO4HOCH2O164CH2OHOCH21O12GO3HO44CH26O1OABFigure13–13.Theglycogenmolecule.A:Generalstructure.B:Enlargementofstructureatabranchpoint.Themoleculeisasphereapproximately21nmindiameterthatcanbevisualizedinelectronmicrographs.Ithasamo-7lecularmassof10Daandconsistsofpolysaccharidechainseachcontainingabout13glucoseresidues.Thechainsareeitherbranchedorunbranchedandarearrangedin12concentriclayers(onlyfourareshowninthefigure).Thebranchedchains(eachhastwobranches)arefoundintheinnerlayersandtheunbranchedchainsintheouterlayer.(G,glycogenin,theprimermoleculeforglycogensynthesis.)
117CARBOHYDRATESOFPHYSIOLOGICSIGNIFICANCE/109ChitinInulinisapolysaccharideoffructose(andhenceafruc-tosan)foundintubersandrootsofdahlias,artichokes,HOCH2HOCH2anddandelions.ItisreadilysolubleinwaterandisusedOOtodeterminetheglomerularfiltrationrate.DextrinsareHHHHintermediatesinthehydrolysisofstarch.CelluloseisO14OOthechiefconstituentoftheframeworkofplants.Itisin-OHHHOHHHsolubleandconsistsofβ-D-glucopyranoseunitslinkedbyβ(1→4)bondstoformlong,straightchainsHHNCOCH3HHNCOCH3strengthenedbycross-linkedhydrogenbonds.CellulosencannotbedigestedbymammalsbecauseoftheabsenceN-AcetylglucosamineN-Acetylglucosamineofanenzymethathydrolyzestheβlinkage.Itisanim-portantsourceof“bulk”inthediet.MicroorganismsinthegutofruminantsandotherherbivorescanhydrolyzeHyaluronicacidtheβlinkageandfermenttheproductstoshort-chainHOCH2fattyacidsasamajorenergysource.ThereislimitedObacterialmetabolismofcelluloseinthehumancolon.COO–HHChitinisastructuralpolysaccharideintheexoskeletonO1Oofcrustaceansandinsectsandalsoinmushrooms.ItHHHOOHHconsistsofN-acetyl-D-glucosamineunitsjoinedby3O41β(1→4)-glycosidiclinkages(Figure13–14).OHHHGlycosaminoglycans(mucopolysaccharides)areHHNCOCH3complexcarbohydratescharacterizedbytheircontentofaminosugarsanduronicacids.WhenthesechainsHOHnareattachedtoaproteinmolecule,theresultisapro-β-GlucuronicacidN-Acetylglucosamineteoglycan.Proteoglycansprovidethegroundorpack-ingsubstanceofconnectivetissues.Theirpropertyofholdinglargequantitiesofwaterandoccupyingspace,Chondroitin4-sulfate(Note:Thereisalsoa6-sulfate)thuscushioningorlubricatingotherstructures,isduetothelargenumberof⎯OHgroupsandnegativeHOCH2chargesonthemolecules,which,byrepulsion,keeptheOcarbohydratechainsapart.ExamplesarehyaluronicCOO––SOOHacid,chondroitinsulfate,andheparin(Figure3O1O13–14).HHHOHHGlycoproteins(mucoproteins)occurinmanydif-3O41ferentsituationsinfluidsandtissues,includingthecellOHHHHHNCOCHmembranes(Chapters41and47).Theyareproteins3HOHnβ-GlucuronicacidN-AcetylgalactosaminesulfateTable13–5.Carbohydratesfoundinglycoproteins.HeparinHexosesMannose(Man)COSO–HGalactose(Gal)3OO–AcetylhexosaminesN-Acetylglucosamine(GlcNAc)HHHHCOON-Acetylgalactosamine(GalNAc)O14OOHHOHHHPentosesArabinose(Ara)Xylose(Xyl)OHNHSO3–HOSO3–MethylpentoseL-Fucose(Fuc;seeFigure13–15)nSialicacidsN-Acylderivativesofneuraminicacid,SulfatedglucosamineSulfatediduronicacideg,N-acetylneuraminicacid(NeuAc;seeFigure13–14.Structureofsomecomplexpolysac-Figure13–16),thepredominantsialicacid.charidesandglycosaminoglycans.
118110/CHAPTER13Houtsideboththeexternalandinternal(cytoplasmic)Osurfaces.CarbohydratechainsareonlyattachedtotheHCH3Haminoterminalportionoutsidetheexternalsurface(Chapter41).HOHHOOHSUMMARYOHH•CarbohydratesaremajorconstituentsofanimalfoodFigure13–15.β-L-Fucose(6-deoxy-β-L-galactose).andanimaltissues.Theyarecharacterizedbythetypeandnumberofmonosaccharideresiduesintheirmolecules.•Glucoseisthemostimportantcarbohydrateinmam-malianbiochemistrybecausenearlyallcarbohydratecontainingbranchedorunbranchedoligosaccharideinfoodisconvertedtoglucoseformetabolism.chains(seeTable13–5).ThesialicacidsareN-orO-acylderivativesofneuraminicacid(Figure13–16).•SugarshavelargenumbersofstereoisomersbecauseNeuraminicacidisanine-carbonsugarderivedfromtheycontainseveralasymmetriccarbonatoms.mannosamine(anepimerofglucosamine)andpyru-•Themonosaccharidesincludeglucose,the“bloodvate.Sialicacidsareconstituentsofbothglycoproteinssugar”;andribose,animportantconstituentofnu-andgangliosides(Chapters14and47).cleotidesandnucleicacids.•Thedisaccharidesincludemaltose(glucosylglucose),CARBOHYDRATESOCCURINCELLanintermediateinthedigestionofstarch;sucroseMEMBRANES&INLIPOPROTEINS(glucosylfructose),importantasadietaryconstituentcontainingfructose;andlactose(galactosylglucose),Inadditiontothelipidofcellmembranes(seeChaptersinmilk.14and41),approximately5%iscarbohydrateinglyco-•Starchandglycogenarestoragepolymersofglucoseproteinsandglycolipids.Carbohydratesarealsopresentinplantsandanimals,respectively.StarchistheinapoBoflipoproteins.Theirpresenceontheoutermajorsourceofenergyinthediet.surfaceoftheplasmamembrane(theglycocalyx)has•Complexcarbohydratescontainothersugarderiva-beenshownwiththeuseofplantlectins,proteinagglu-tivessuchasaminosugars,uronicacids,andsialictininsthatbindwithspecificglycosylresidues.Foracids.Theyincludeproteoglycansandglycosamino-example,concanavalinAbindsα-glucosylandα-man-glycans,associatedwithstructuralelementsofthetis-nosylresidues.Glycophorinisamajorintegralmem-sues;andglycoproteins,proteinscontainingattachedbraneglycoproteinofhumanerythrocytesandspansoligosaccharidechains.Theyarefoundinmanysitu-thelipidmembrane,havingfreepolypeptideportionsationsincludingthecellmembrane.REFERENCESHBinkleyRW:ModernCarbohydrateChemistry.MarcelDekker,O—1988.AcNHCHOHCOOCollinsPM(editor):Carbohydrates.Chapman&Hall,1988.CHOHEl-KhademHS:CarbohydrateChemistry:MonosaccharidesandCHOHTheirOligomers.AcademicPress,1988.2HHHOHLehmanJ(editor)(translatedbyHainesA.):Carbohydrates:Struc-tureandBiology.Thieme,1998.LindahlU,HöökM:Glycosaminoglycansandtheirbindingtobio-OHHlogicalmacromolecules.AnnuRevBiochem1978;47:385.Melendes-HeviaE,WaddellTG,SheltonED:OptimizationofFigure13–16.StructureofN-acetylneuraminicacid,moleculardesignintheevolutionofmetabolism:theglyco-asialicacid(Ac=CH3⎯CO⎯).genmolecule.BiochemJ1993;295:477.
119LipidsofPhysiologicSignificance14PeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DScBIOMEDICALIMPORTANCEc.Othercomplexlipids:Lipidssuchassul-folipidsandaminolipids.LipoproteinsmayThelipidsareaheterogeneousgroupofcompounds,alsobeplacedinthiscategory.includingfats,oils,steroids,waxes,andrelatedcom-3.Precursorandderivedlipids:Theseincludefattypounds,whicharerelatedmorebytheirphysicalthanacids,glycerol,steroids,otheralcohols,fattyalde-bytheirchemicalproperties.Theyhavethecommonhydes,andketonebodies(Chapter22),hydrocar-propertyofbeing(1)relativelyinsolubleinwaterandbons,lipid-solublevitamins,andhormones.(2)solubleinnonpolarsolventssuchasetherandchloroform.TheyareimportantdietaryconstituentsBecausetheyareuncharged,acylglycerols(glyc-notonlybecauseoftheirhighenergyvaluebutalsobe-erides),cholesterol,andcholesterylestersaretermedcauseofthefat-solublevitaminsandtheessentialfattyneutrallipids.acidscontainedinthefatofnaturalfoods.Fatisstoredinadiposetissue,whereitalsoservesasathermalinsu-latorinthesubcutaneoustissuesandaroundcertainor-FATTYACIDSAREALIPHATICgans.Nonpolarlipidsactaselectricalinsulators,al-CARBOXYLICACIDSlowingrapidpropagationofdepolarizationwavesalongmyelinatednerves.CombinationsoflipidandproteinFattyacidsoccurmainlyasestersinnaturalfatsandoils(lipoproteins)areimportantcellularconstituents,oc-butdooccurintheunesterifiedformasfreefattycurringbothinthecellmembraneandinthemito-acids,atransportformfoundintheplasma.Fattyacidschondria,andservingalsoasthemeansoftransportingthatoccurinnaturalfatsareusuallystraight-chainde-lipidsintheblood.Knowledgeoflipidbiochemistryisrivativescontaininganevennumberofcarbonatoms.necessaryinunderstandingmanyimportantbiomedicalThechainmaybesaturated(containingnodoubleareas,eg,obesity,diabetesmellitus,atherosclerosis,bonds)orunsaturated(containingoneormoredoubleandtheroleofvariouspolyunsaturatedfattyacidsinbonds).nutritionandhealth.FattyAcidsAreNamedAfterLIPIDSARECLASSIFIEDASSIMPLECorrespondingHydrocarbonsORCOMPLEXThemostfrequentlyusedsystematicnomenclature1.Simplelipids:Estersoffattyacidswithvariousal-namesthefattyacidafterthehydrocarbonwiththecohols.samenumberandarrangementofcarbonatoms,witha.Fats:Estersoffattyacidswithglycerol.Oils-oicbeingsubstitutedforthefinal-e(Genevansys-arefatsintheliquidstate.tem).Thus,saturatedacidsendin-anoic,eg,octanoicb.Waxes:Estersoffattyacidswithhighermole-acid,andunsaturatedacidswithdoublebondsendincularweightmonohydricalcohols.-enoic,eg,octadecenoicacid(oleicacid).2.Complexlipids:EstersoffattyacidscontainingCarbonatomsarenumberedfromthecarboxylcar-groupsinadditiontoanalcoholandafattyacid.bon(carbonNo.1).Thecarbonatomsadjacenttothea.Phospholipids:Lipidscontaining,inadditioncarboxylcarbon(Nos.2,3,and4)arealsoknownastofattyacidsandanalcohol,aphosphorictheα,β,andγcarbons,respectively,andtheterminalacidresidue.Theyfrequentlyhavenitrogen-methylcarbonisknownastheωorn-carbon.containingbasesandothersubstituents,eg,inVariousconventionsuseΔforindicatingthenum-glycerophospholipidsthealcoholisglycerolberandpositionofthedoublebonds(Figure14–1);eg,9andinsphingophospholipidsthealcoholisΔindicatesadoublebondbetweencarbons9and10sphingosine.ofthefattyacid;ω9indicatesadoublebondontheb.Glycolipids(glycosphingolipids):Lipidsninthcarboncountingfromtheω-carbon.Inanimals,containingafattyacid,sphingosine,andcar-additionaldoublebondsareintroducedonlybetweenbohydrate.theexistingdoublebond(eg,ω9,ω6,orω3)andthe111
120112/CHAPTER1418:1;9orΔ918:1UnsaturatedFattyAcidsContainOne181091orMoreDoubleBondsCH3(CH2)7CHCH(CH2)7COOH(Table14–2)orFattyacidsmaybefurthersubdividedasfollows:ω9,C18:1orn–9,18:1ω234567891018(1)Monounsaturated(monoethenoid,monoenoic)CH3CH2CH2CH2CH2CH2CH2CH2CHCH(CH2)7COOHacids,containingonedoublebond.n171091(2)Polyunsaturated(polyethenoid,polyenoic)Figure14–1.Oleicacid.n−9(nminus9)isequiva-acids,containingtwoormoredoublebonds.lenttoω9.(3)Eicosanoids:Thesecompounds,derivedfromeicosa-(20-carbon)polyenoicfattyacids,com-prisetheprostanoids,leukotrienes(LTs),andcarboxylcarbon,leadingtothreeseriesoffattyacidslipoxins(LXs).Prostanoidsincludeprosta-knownastheω9,ω6,andω3families,respectively.glandins(PGs),prostacyclins(PGIs),andthromboxanes(TXs).SaturatedFattyAcidsContainProstaglandinsexistinvirtuallyeverymammalianNoDoubleBondstissue,actingaslocalhormones;theyhaveimportantphysiologicandpharmacologicactivities.Theyaresyn-Saturatedfattyacidsmaybeenvisagedasbasedonthesizedinvivobycyclizationofthecenterofthecar-aceticacid(CH3⎯COOH)asthefirstmemberofthebonchainof20-carbon(eicosanoic)polyunsaturatedseriesinwhich⎯CH2⎯isprogressivelyaddedbe-fattyacids(eg,arachidonicacid)toformacyclopentanetweentheterminalCH3⎯and⎯COOHgroups.Ex-ring(Figure14–2).Arelatedseriesofcompounds,theamplesareshowninTable14–1.Otherhighermem-thromboxanes,havethecyclopentaneringinterruptedbersoftheseriesareknowntooccur,particularlyinwithanoxygenatom(oxanering)(Figure14–3).Threewaxes.Afewbranched-chainfattyacidshavealsobeendifferenteicosanoicfattyacidsgiverisetothreegroupsisolatedfrombothplantandanimalsources.ofeicosanoidscharacterizedbythenumberofdoublebondsinthesidechains,eg,PG1,PG2,PG3.Differentsubstituentgroupsattachedtotheringsgiverisetose-Table14–1.Saturatedfattyacids.riesofprostaglandinsandthromboxanes,labeledA,B,etc—eg,the“E”typeofprostaglandin(asinPGE2)hasaketogroupinposition9,whereasthe“F”typehasaCommonNumberofhydroxylgroupinthisposition.TheleukotrienesandNameCAtomslipoxinsareathirdgroupofeicosanoidderivativesAcetic2Majorendproductofcarbohy-formedviathelipoxygenasepathway(Figure14–4).dratefermentationbyrumenTheyarecharacterizedbythepresenceofthreeorfour1organismsconjugateddoublebonds,respectively.LeukotrienesPropionic3Anendproductofcarbohydratecausebronchoconstrictionaswellasbeingpotentfermentationbyrumenproinflammatoryagentsandplayapartinasthma.1organismsButyric4IncertainfatsinsmallamountsMostNaturallyOccurringUnsaturatedValeric5(especiallybutter).AnendproductFattyAcidsHavecisDoubleBondsofcarbohydratefermentationbyCaproic6rumenorganisms1Thecarbonchainsofsaturatedfattyacidsformazigzagpatternwhenextended,asatlowtemperatures.AtLauric12Spermaceti,cinnamon,palmker-highertemperatures,somebondsrotate,causingchainnel,coconutoils,laurels,buttershortening,whichexplainswhybiomembranesbecomeMyristic14Nutmeg,palmkernel,coconutoils,thinnerwithincreasesintemperature.Atypeofgeo-myrtles,buttermetricisomerismoccursinunsaturatedfattyacids,de-Palmitic16Commoninallanimalandplantpendingontheorientationofatomsorgroupsaroundfatstheaxesofdoublebonds,whichdonotallowrotation.Stearic18Iftheacylchainsareonthesamesideofthebond,it1Alsoformedinthececumofherbivoresandtoalesserextentiniscis-,asinoleicacid;ifonoppositesides,itistrans-,asthecolonofhumans.inelaidicacid,thetransisomerofoleicacid(Fig-
121LIPIDSOFPHYSIOLOGICSIGNIFICANCE/113Table14–2.Unsaturatedfattyacidsofphysiologicandnutritionalsignificance.NumberofCAtomsandNumberandPositionofCommonDoubleBondsFamilyNameSystematicNameOccurrenceMonoenoicacids(onedoublebond)16:1;9ω7Palmitoleiccis-9-HexadecenoicInnearlyallfats.18:1;9ω9Oleiccis-9-OctadecenoicPossiblythemostcommonfattyacidinnaturalfats.18:1;9ω9Elaidictrans-9-OctadecenoicHydrogenatedandruminantfats.Dienoicacids(twodoublebonds)18:2;9,12ω6Linoleicall-cis-9,12-OctadecadienoicCorn,peanut,cottonseed,soybean,andmanyplantoils.Trienoicacids(threedoublebonds)18:3;6,9,12ω6γ-Linolenicall-cis-6,9,12-OctadecatrienoicSomeplants,eg,oilofeveningprim-rose,borageoil;minorfattyacidinanimals.18:3;9,12,15ω3α-Linolenicall-cis-9,12,15-OctadecatrienoicFrequentlyfoundwithlinoleicacidbutparticularlyinlinseedoil.Tetraenoicacids(fourdoublebonds)20:4;5,8,11,14ω6Arachidonicall-cis-5,8,11,14-EicosatetraenoicFoundinanimalfatsandinpeanutoil;importantcomponentofphospho-lipidsinanimals.Pentaenoicacids(fivedoublebonds)20:5;5,8,11,14,17ω3Timnodonicall-cis-5,8,11,14,17-EicosapentaenoicImportantcomponentoffishoils,eg,codliver,mackerel,menhaden,salmonoils.Hexaenoicacids(sixdoublebonds)22:6;4,7,10,13,16,19ω3Cervonicall-cis-4,7,10,13,16,19-DocosahexaenoicFishoils,phospholipidsinbrain.ure14–5).Naturallyoccurringunsaturatedlong-chainUshape.Thishasprofoundsignificanceonmolecularfattyacidsarenearlyallofthecisconfiguration,thepackinginmembranesandonthepositionsoccupiedmoleculesbeing“bent”120degreesatthedoublebyfattyacidsinmorecomplexmoleculessuchasphos-bond.Thus,oleicacidhasanLshape,whereaselaidicpholipids.Transdoublebondsalterthesespatialrela-acidremains“straight.”Increaseinthenumberofcistionships.Transfattyacidsarepresentincertainfoods,doublebondsinafattyacidleadstoavarietyofpossi-arisingasaby-productofthesaturationoffattyacidsblespatialconfigurationsofthemolecule—eg,arachi-duringhydrogenation,or“hardening,”ofnaturaloilsdonicacid,withfourcisdoublebonds,has“kinks”orainthemanufactureofmargarine.AnadditionalsmallO59COO—COO—10O11OOHOHOHFigure14–2.ProstaglandinE2(PGE2).Figure14–3.ThromboxaneA2(TXA2).
122114/CHAPTER14Omoreunsaturatedthanstoragelipids.Lipidsintissues–thataresubjecttocooling,eg,inhibernatorsorintheCOOextremitiesofanimals,aremoreunsaturated.TRIACYLGLYCEROLS(TRIGLYCERIDES)*ARETHEMAINSTORAGEFORMSOFFigure14–4.LeukotrieneA4(LTA4).FATTYACIDSThetriacylglycerols(Figure14–6)areestersofthetri-hydricalcoholglycerolandfattyacids.Mono-anddi-contributioncomesfromtheingestionofruminantfatacylglycerolswhereinoneortwofattyacidsareesteri-thatcontainstransfattyacidsarisingfromtheactionoffiedwithglycerolarealsofoundinthetissues.Thesemicroorganismsintherumen.areofparticularsignificanceinthesynthesisandhy-drolysisoftriacylglycerols.PhysicalandPhysiologicPropertiesofFattyAcidsReflectChainLengthCarbons1&3ofGlycerolAreandDegreeofUnsaturationNotIdenticalThemeltingpointsofeven-numbered-carbonfattyTonumberthecarbonatomsofglycerolunambigu-acidsincreasewithchainlengthanddecreaseaccordingously,the-sn-(stereochemicalnumbering)systemistounsaturation.Atriacylglycerolcontainingthreesatu-used.Itisimportanttorealizethatcarbons1and3ofratedfattyacidsof12carbonsormoreissolidatbodyglycerolarenotidenticalwhenviewedinthreedimen-temperature,whereasifthefattyacidresiduesare18:2,sions(shownasaprojectionformulainFigure14–7).itisliquidtobelow0°C.Inpractice,naturalacylglyc-Enzymesreadilydistinguishbetweenthemandareerolscontainamixtureoffattyacidstailoredtosuitnearlyalwaysspecificforoneortheothercarbon;eg,theirfunctionalroles.Themembranelipids,whichglycerolisalwaysphosphorylatedonsn-3byglycerolmustbefluidatallenvironmentaltemperatures,arekinasetogiveglycerol3-phosphateandnotglycerol1-phosphate.18CH3CH3PHOSPHOLIPIDSARETHEMAINLIPIDCONSTITUENTSOFMEMBRANESPhospholipidsmayberegardedasderivativesofphos-Transform(elaidicacid)phatidicacid(Figure14–8),inwhichthephosphateisesterifiedwiththe⎯OHofasuitablealcohol.Phos-phatidicacidisimportantasanintermediateinthesyn-thesisoftriacylglycerolsaswellasphosphoglycerolsbut12010HHisnotfoundinanygreatquantityintissues.CCCisform(oleicacid)CCPhosphatidylcholines(Lecithins)9HHOccurinCellMembranes110Phosphoacylglycerolscontainingcholine(Figure14–8)arethemostabundantphospholipidsofthecellmem-*AccordingtothestandardizedterminologyoftheInternationalUnionofPureandAppliedChemistry(IUPAC)andtheInterna-1tionalUnionofBiochemistry(IUB),themonoglycerides,diglyc-––COOCOOerides,andtriglyceridesshouldbedesignatedmonoacylglycerols,diacylglycerols,andtriacylglycerols,respectively.However,the9Figure14–5.GeometricisomerismofΔ,18:1fattyolderterminologyisstillwidelyused,particularlyinclinicalmedi-acids(oleicandelaidicacids).cine.
123LIPIDSOFPHYSIOLOGICSIGNIFICANCE/115OO1RO1CHOCROCH2OC12122R2COCHOR2COCHO3CH2OCR23CH2OPO––OFigure14–6.Triacylglycerol.Phosphatidicacidbraneandrepresentalargeproportionofthebody’sstoreofcholine.Cholineisimportantinnervoustrans-CH3+mission,asacetylcholine,andasastoreoflabilemethylAOCH2CH2NCH3groups.Dipalmitoyllecithinisaveryeffectivesurface-CH3activeagentandamajorconstituentofthesurfactantpreventingadherence,duetosurfacetension,oftheCholineinnersurfacesofthelungs.Itsabsencefromthelungs+ofprematureinfantscausesrespiratorydistresssyn-OCH2CH2NH3drome.Mostphospholipidshaveasaturatedacylradi-Bcalinthesn-1positionbutanunsaturatedradicalintheEthanolaminesn-2positionofglycerol.Phosphatidylethanolamine(cephalin)andphos-+NH3phatidylserine(foundinmosttissues)differfromphosphatidylcholineonlyinthatethanolamineorser-COCH2CHCOO–ine,respectively,replacescholine(Figure14–8).SerinePhosphatidylinositolIsaPrecursorOHOHofSecondMessengers23OHHHTheinositolispresentinphosphatidylinositolasthe14stereoisomer,myoinositol(Figure14–8).Phosphati-dylinositol4,5-bisphosphateisanimportantcon-DHHOHOH65stituentofcellmembranephospholipids;uponstimula-tionbyasuitablehormoneagonist,itiscleavedintoOHHdiacylglycerolandinositoltrisphosphate,bothofwhichactasinternalsignalsorsecondmessengers.Myoinositol–OCardiolipinIsaMajorLipidofMitochondrialMembranesCH2OOPCH2OOPhosphatidicacidisaprecursorofphosphatidylglyc-EHCOHOHCOCR3erolwhich,inturn,givesrisetocardiolipin(FigureOCH2R4COCH214–8).OPhosphatidylglycerol1H2COCR1Figure14–8.Phosphatidicacidanditsderivatives.OTheO−shownshadedinphosphatidicacidissubsti-R2CO2CHtutedbythesubstituentsshowntoformin(A)3-phos-phatidylcholine,(B)3-phosphatidylethanolamine,O(C)3-phosphatidylserine,(D)3-phosphatidylinositol,3HCOCR3and(E)cardiolipin(diphosphatidylglycerol).2Figure14–7.Triacyl-sn-glycerol.
124116/CHAPTER14LysophospholipidsAreIntermediatesinO1CHOCHCHR21theMetabolismofPhosphoglycerols2R2COCHOThesearephosphoacylglycerolscontainingonlyone3OOPCHCHNH+acylradical,eg,lysophosphatidylcholine(lysoleci-CH2223–thin),importantinthemetabolismandinterconver-Osionofphospholipids(Figure14–9).ItisalsofoundinEthanolamineoxidizedlipoproteinsandhasbeenimplicatedinsomeFigure14–10.Plasmalogen.oftheireffectsinpromotingatherosclerosis.PlasmalogensOccurinBrain&Musclesphingolipidofbrainandothernervoustissue,foundinThesecompoundsconstituteasmuchas10%oftherelativelylowamountselsewhere.Itcontainsanumberphospholipidsofbrainandmuscle.Structurally,theofcharacteristicC24fattyacids,eg,cerebronicacid.plasmalogensresemblephosphatidylethanolaminebutGalactosylceramide(Figure14–12)canbeconvertedtopossessanetherlinkonthesn-1carboninsteadofthesulfogalactosylceramide(sulfatide),presentinhighesterlinkfoundinacylglycerols.Typically,thealkylamountsinmyelin.Glucosylceramideisthepredomi-radicalisanunsaturatedalcohol(Figure14–10).Innantsimpleglycosphingolipidofextraneuraltissues,someinstances,choline,serine,orinositolmaybesub-alsooccurringinthebraininsmallamounts.Ganglio-stitutedforethanolamine.sidesarecomplexglycosphingolipidsderivedfromglu-cosylceramidethatcontaininadditiononeormoreSphingomyelinsAreFoundmoleculesofasialicacid.Neuraminicacid(NeuAc;intheNervousSystemseeChapter13)istheprincipalsialicacidfoundinhumantissues.GangliosidesarealsopresentinnervousSphingomyelinsarefoundinlargequantitiesinbraintissuesinhighconcentration.Theyappeartohavere-andnervetissue.Onhydrolysis,thesphingomyelinsceptorandotherfunctions.Thesimplestgangliosideyieldafattyacid,phosphoricacid,choline,andacom-foundintissuesisGM3,whichcontainsceramide,oneplexaminoalcohol,sphingosine(Figure14–11).Nomoleculeofglucose,onemoleculeofgalactose,andoneglycerolispresent.ThecombinationofsphingosinemoleculeofNeuAc.Intheshorthandnomenclatureplusfattyacidisknownasceramide,astructurealsoused,Grepresentsganglioside;Misamonosialo-foundintheglycosphingolipids(seebelow).containingspecies;andthesubscript3isanumberas-signedonthebasisofchromatographicmigration.GM1GLYCOLIPIDS(GLYCOSPHINGOLIPIDS)(Figure14–13),amorecomplexgangliosidederivedAREIMPORTANTINNERVETISSUESfromGM3,isofconsiderablebiologicinterest,asitis&INTHECELLMEMBRANEknowntobethereceptorinhumanintestineforcholeratoxin.OthergangliosidescancontainanywhereGlycolipidsarewidelydistributedineverytissueofthefromonetofivemoleculesofsialicacid,givingrisetobody,particularlyinnervoustissuesuchasbrain.Theydi-,trisialogangliosides,etc.occurparticularlyintheouterleafletoftheplasmamembrane,wheretheycontributetocellsurfacecar-bohydrates.ThemajorglycolipidsfoundinanimaltissuesareCeramideglycosphingolipids.TheycontainceramideandoneorSphingosinemoresugars.Galactosylceramideisamajorglyco-OHOHOCH3(CH2)12CHCHCHCHNCR1CHOCRCH2Fattyacid22OHOCHOCH3Phosphoricacid+–3OPOCH2OOPCH2CH2NCH3+O–CH3OCH2CH2N(CH3)3CholineCholineFigure14–9.Lysophosphatidylcholine(lysolecithin).Figure14–11.Asphingomyelin.
125LIPIDSOFPHYSIOLOGICSIGNIFICANCE/117CeramideSphingosineOHOHCH3(CH2)12CHCHCHCHNCCH(OH)(CH2)21CH3CHOHFattyacid2(eg,cerebronicacid)OHOHOCHGalactose2HORHHFigure14–12.Structureofgalactosylcer-amide(galactocerebroside,R=H),andsul-3fogalactosylceramide(asulfatide,R=SO2−).HOH4STEROIDSPLAYMANYgroupsandnocarbonylorcarboxylgroups,itisaPHYSIOLOGICALLYIMPORTANTROLESsterol,andthenameterminatesin-ol.CholesterolisprobablythebestknownsteroidbecauseBecauseofAsymmetryintheSteroidofitsassociationwithatherosclerosis.However,bio-Molecule,ManyStereoisomerschemicallyitisalsoofsignificancebecauseitisthepre-ArePossiblecursorofalargenumberofequallyimportantsteroidsthatincludethebileacids,adrenocorticalhormones,Eachofthesix-carbonringsofthesteroidnucleusisca-sexhormones,Dvitamins,cardiacglycosides,sitos-pableofexistinginthethree-dimensionalconformationterolsoftheplantkingdom,andsomealkaloids.eitherofa“chair”ora“boat”(Figure14–15).Innatu-Allofthesteroidshaveasimilarcyclicnucleusre-rallyoccurringsteroids,virtuallyalltheringsareinthesemblingphenanthrene(ringsA,B,andC)towhicha“chair”form,whichisthemorestableconformation.cyclopentanering(D)isattached.ThecarbonpositionsWithrespecttoeachother,theringscanbeeithercisoronthesteroidnucleusarenumberedasshowninFiguretrans(Figure14–16).ThejunctionbetweentheAand14–14.Itisimportanttorealizethatinstructuralfor-Bringscanbecisortransinnaturallyoccurringmulasofsteroids,asimplehexagonalringdenotesasteroids.ThatbetweenBandCistrans,asisusuallythecompletelysaturatedsix-carbonringwithallvalencesC/Djunction.Bondsattachingsubstituentgroupssatisfiedbyhydrogenbondsunlessshownotherwise;ie,abovetheplaneoftherings(βbonds)areshownwithitisnotabenzenering.Alldoublebondsareshownasboldsolidlines,whereasthosebondsattachinggroupssuch.Methylsidechainsareshownassinglebondsun-below(αbonds)areindicatedwithbrokenlines.TheAattachedatthefarther(methyl)end.Theseoccurtypi-ringofa5αsteroidisalwaystranstotheBring,callyatpositions10and13(constitutingCatoms19whereasitiscisina5βsteroid.Themethylgroupsat-and18).Asidechainatposition17isusual(asincho-tachedtoC10andC13areinvariablyintheβconfigura-lesterol).Ifthecompoundhasoneormorehydroxyltion.CeramideGlucoseGalactoseN-Acetylgalactosamine(Acyl-sphingo-NeuAcGalactose18sine)1217or11161319CD19CerGlcGalGalNAcGal15142108ABNeuAc37546Figure14–13.GM1ganglioside,amonosialoganglio-side,thereceptorinhumanintestineforcholeratoxin.Figure14–14.Thesteroidnucleus.
126118/CHAPTER14chainalcoholdolichol(Figure14–20),whichtakespartinglycoproteinsynthesisbytransferringcarbohy-drateresiduestoasparagineresiduesofthepolypeptide(Chapter47).Plant-derivedisoprenoidcompoundsin-“Chair”form“Boat”formcluderubber,camphor,thefat-solublevitaminsA,D,E,andK,andβ-carotene(provitaminA).Figure14–15.Conformationsofstereoisomersofthesteroidnucleus.LIPIDPEROXIDATIONISASOURCEOFFREERADICALSCholesterolIsaSignificantConstituentofManyTissuesPeroxidation(auto-oxidation)oflipidsexposedtooxygenisresponsiblenotonlyfordeteriorationoffoodsCholesterol(Figure14–17)iswidelydistributedinall(rancidity)butalsofordamagetotissuesinvivo,cellsofthebodybutparticularlyinnervoustissue.Itiswhereitmaybeacauseofcancer,inflammatorydis-amajorconstituentoftheplasmamembraneandofeases,atherosclerosis,andaging.Thedeleteriouseffectsplasmalipoproteins.Itisoftenfoundascholesterylareconsideredtobecausedbyfreeradicals(ROO•,ester,wherethehydroxylgrouponposition3isesteri-RO•,OH•)producedduringperoxideformationfromfiedwithalong-chainfattyacid.Itoccursinanimalsfattyacidscontainingmethylene-interrupteddoublebutnotinplants.bonds,ie,thosefoundinthenaturallyoccurringpolyunsaturatedfattyacids(Figure14–21).Lipidper-ErgosterolIsaPrecursorofVitaminDoxidationisachainreactionprovidingacontinuoussupplyoffreeradicalsthatinitiatefurtherperoxidation.ErgosteroloccursinplantsandyeastandisimportantThewholeprocesscanbedepictedasfollows:asaprecursorofvitaminD(Figure14–18).Whenirra-(1)Initiation:diatedwithultravioletlight,itacquiresantirachiticpropertiesconsequenttotheopeningofringB.ROOHMetal+→()nn++ROO•++Metal(–)1H+PolyprenoidsSharetheSameParentXR••+→+HRXHCompoundasCholesterolAlthoughnotsteroids,thesecompoundsarerelatedbe-(2)Propagation:causetheyaresynthesized,likecholesterol(Figure26–2),fromfive-carbonisopreneunits(Figure14–19).R••+→OROOTheyincludeubiquinone(Chapter12),amemberof2therespiratorychaininmitochondria,andthelong-ROO••+→RHROOHR+,etcABH13H10DB10C59814BAA53HH3Hor17or13CD19H11410H8HA105BAB53H3HFigure14–16.Generalizedsteroidnucleus,showing(A)anall-transconfigurationbe-tweenadjacentringsand(B)acisconfigurationbetweenringsAandB.
127LIPIDSOFPHYSIOLOGICSIGNIFICANCE/119CH3CHCCHCH17Figure14–19.Isopreneunit.3HO56Peroxidationisalsocatalyzedinvivobyhemecom-Figure14–17.Cholesterol,3-hydroxy-5,6-poundsandbylipoxygenasesfoundinplateletsandcholestene.leukocytes.Otherproductsofauto-oxidationoren-zymicoxidationofphysiologicsignificanceincludeoxysterols(formedfromcholesterol)andisoprostanes(3)Termination:(prostanoids).ROO••+→+ROOROOROAMPHIPATHICLIPIDSSELF-ORIENT2••ATOIL:WATERINTERFACESROO+→RROORRRR••+→RTheyFormMembranes,Micelles,Liposomes,&EmulsionsSincethemolecularprecursorfortheinitiationIngeneral,lipidsareinsolubleinwatersincetheyprocessisgenerallythehydroperoxideproductROOH,containapredominanceofnonpolar(hydrocarbon)lipidperoxidationisachainreactionwithpotentiallygroups.However,fattyacids,phospholipids,sphin-devastatingeffects.Tocontrolandreducelipidperoxi-golipids,bilesalts,and,toalesserextent,cholesteroldation,bothhumansintheiractivitiesandnaturein-containpolargroups.Therefore,partofthemoleculeisvoketheuseofantioxidants.Propylgallate,butylatedhydrophobic,orwater-insoluble;andpartishydro-hydroxyanisole(BHA),andbutylatedhydroxytoluenephilic,orwater-soluble.Suchmoleculesaredescribed(BHT)areantioxidantsusedasfoodadditives.Natu-asamphipathic(Figure14–22).TheybecomeorientedrallyoccurringantioxidantsincludevitaminE(tocoph-atoil:waterinterfaceswiththepolargroupinthewatererol),whichislipid-soluble,andurateandvitaminC,phaseandthenonpolargroupintheoilphase.Abi-whicharewater-soluble.Beta-caroteneisanantioxidantlayerofsuchamphipathiclipidshasbeenregardedasaatlowPO2.Antioxidantsfallintotwoclasses:(1)pre-basicstructureinbiologicmembranes(Chapter41).ventiveantioxidants,whichreducetherateofchainini-Whenacriticalconcentrationoftheselipidsispresenttiation;and(2)chain-breakingantioxidants,whichin-inanaqueousmedium,theyformmicelles.Aggrega-terferewithchainpropagation.Preventiveantioxidantstionsofbilesaltsintomicellesandliposomesandtheincludecatalaseandotherperoxidasesthatreactwithformationofmixedmicelleswiththeproductsoffatdi-ROOHandchelatorsofmetalionssuchasEDTAgestionareimportantinfacilitatingabsorptionoflipids(ethylenediaminetetraacetate)andDTPA(diethylene-fromtheintestine.Liposomesmaybeformedbysoni-triaminepentaacetate).Invivo,theprincipalchain-catinganamphipathiclipidinanaqueousmedium.breakingantioxidantsaresuperoxidedismutase,whichTheyconsistofspheresoflipidbilayersthatencloseactsintheaqueousphasetotrapsuperoxidefreeradi-partoftheaqueousmedium.Theyareofpotentialclin-cals(O−•);perhapsurate;andvitaminE,whichactsinicaluse—particularlywhencombinedwithtissue-2•thelipidphasetotrapROOradicals(Figure45–6).specificantibodies—ascarriersofdrugsinthecircula-tion,targetedtospecificorgans,eg,incancertherapy.Inaddition,theyarebeingusedforgenetransferintovascularcellsandascarriersfortopicalandtransdermalCH2OHBHO16Figure14–18.Ergosterol.Figure14–20.Dolichol—aC95alcohol.
128120/CHAPTER14RHR•R•ROO•X•XHHO2HOO•••HHHRHOOOOHOOH•+R•HHMalondialdehydeEndoperoxideHydroperoxideROOH•Figure14–21.Lipidperoxidation.Thereactionisinitiatedbyanexistingfreeradical(X),bylight,orbymetalions.Malondialdehydeisonlyformedbyfattyacidswiththreeormoredoublebondsandisusedasameasureoflipidperoxidationtogetherwithethanefromtheterminaltwocarbonsofω3fattyacidsandpen-tanefromtheterminalfivecarbonsofω6fattyacids.AMPHIPATHICLIPIDAPolarorhydrophiIicgroupsNonpolarorhydrophobicgroupsAqueousphaseAqueousphaseAqueousphase“Oil”ornonpolarphaseNonpolarphase“Oil”ornonpolarphaseAqueousphaseLIPIDBILAYERMICELLEOILINWATEREMULSIONBCDNonpolarphaseAqueousAqueousphasephaseLipidAqueousLipidbilayercompartmentsbilayersLIPOSOMELIPOSOME(UNILAMELLAR)(MULTILAMELLAR)EFFigure14–22.Formationoflipidmembranes,micelles,emulsions,andliposomesfromam-phipathiclipids,eg,phospholipids.
129LIPIDSOFPHYSIOLOGICSIGNIFICANCE/121deliveryofdrugsandcosmetics.Emulsionsaremuchareamphipathiclipidsandhaveimportantroles—aslargerparticles,formedusuallybynonpolarlipidsinanmajorconstituentsofmembranesandtheouterlayeraqueousmedium.Thesearestabilizedbyemulsifyingoflipoproteins,assurfactantinthelung,asprecur-agentssuchasamphipathiclipids(eg,lecithin),whichsorsofsecondmessengers,andasconstituentsofner-formasurfacelayerseparatingthemainbulkofthevoustissue.nonpolarmaterialfromtheaqueousphase(Figure•Glycolipidsarealsoimportantconstituentsofner-14–22).voustissuesuchasbrainandtheouterleafletofthecellmembrane,wheretheycontributetothecarbo-SUMMARYhydratesonthecellsurface.•Cholesterol,anamphipathiclipid,isanimportant•Lipidshavethecommonpropertyofbeingrelativelycomponentofmembranes.Itistheparentmoleculeinsolubleinwater(hydrophobic)butsolubleinnon-fromwhichallothersteroidsinthebody,includingpolarsolvents.Amphipathiclipidsalsocontainonemajorhormonessuchastheadrenocorticalandsexormorepolargroups,makingthemsuitableascon-hormones,Dvitamins,andbileacids,aresynthe-stituentsofmembranesatlipid:waterinterfaces.sized.•Thelipidsofmajorphysiologicsignificancearefatty•Peroxidationoflipidscontainingpolyunsaturatedacidsandtheiresters,togetherwithcholesterolandfattyacidsleadstogenerationoffreeradicalsthatothersteroids.maydamagetissuesandcausedisease.•Long-chainfattyacidsmaybesaturated,monounsat-urated,orpolyunsaturated,accordingtothenumberofdoublebondspresent.TheirfluiditydecreasesREFERENCESwithchainlengthandincreasesaccordingtodegreeofunsaturation.BenzieIFF:Lipidperoxidation:areviewofcauses,consequences,•Eicosanoidsareformedfrom20-carbonpolyunsatu-measurementanddietaryinfluences.IntJFoodSciNutrratedfattyacidsandmakeupanimportantgroupof1996;47:233.physiologicallyandpharmacologicallyactivecom-ChristieWW:LipidAnalysis,2nded.PergamonPress,1982.poundsknownasprostaglandins,thromboxanes,CullisPR,FenskeDB,HopeMJ:Physicalpropertiesandfunc-leukotrienes,andlipoxins.tionalrolesoflipidsinmembranes.In:BiochemistryofLipids,LipoproteinsandMembranes.VanceDE,VanceJE(editors).•Theestersofglycerolarequantitativelythemostsig-Elsevier,1996.nificantlipids,representedbytriacylglycerol(“fat”),GunstoneFD,HarwoodJL,PadleyFB:TheLipidHandbook.amajorconstituentoflipoproteinsandthestorageChapman&Hall,1986.formoflipidinadiposetissue.PhosphoacylglycerolsGurrMI,HarwoodJL:LipidBiochemistry:AnIntroduction,4thed.Chapman&Hall,1991.
130OverviewofMetabolism15PeterA.Mayes,PhD,DSc,&DavidA.Bender,PhDBIOMEDICALIMPORTANCECarbohydrateMetabolismIsCenteredontheProvision&FateofGlucoseThefateofdietarycomponentsafterdigestionandab-(Figure15–2)sorptionconstitutesmetabolism—themetabolicpath-waystakenbyindividualmolecules,theirinterrelation-Glucoseismetabolizedtopyruvatebythepathwayofships,andthemechanismsthatregulatetheflowofglycolysis,whichcanoccuranaerobically(intheab-metabolitesthroughthepathways.Metabolicpathwayssenceofoxygen),whentheendproductislactate.Aero-fallintothreecategories:(1)Anabolicpathwaysarebictissuesmetabolizepyruvatetoacetyl-CoA,whichthoseinvolvedinthesynthesisofcompounds.Proteincanenterthecitricacidcycleforcompleteoxidationsynthesisissuchapathway,asisthesynthesisoffueltoCO2andH2O,linkedtotheformationofATPreservesoftriacylglycerolandglycogen.Anabolicpath-intheprocessofoxidativephosphorylation(Figurewaysareendergonic.(2)Catabolicpathwaysarein-16–2).Glucoseisthemajorfuelofmosttissues.volvedinthebreakdownoflargermolecules,com-monlyinvolvingoxidativereactions;theyareexergonic,producingreducingequivalentsand,mainlyviatheres-piratorychain,ATP.(3)AmphibolicpathwaysoccurCarbohydrateProteinFatatthe“crossroads”ofmetabolism,actingaslinksbe-tweentheanabolicandcatabolicpathways,eg,thecit-ricacidcycle.DigestionandabsorptionAknowledgeofnormalmetabolismisessentialforanunderstandingofabnormalitiesunderlyingdisease.NormalmetabolismincludesadaptationtoperiodsofSimplesugarsFattyacidsAminoacidsstarvation,exercise,pregnancy,andlactation.Abnor-(mainlyglucose)+glycerolmalmetabolismmayresultfromnutritionaldeficiency,enzymedeficiency,abnormalsecretionofhormones,ortheactionsofdrugsandtoxins.AnimportantexampleCatabolismofametabolicdiseaseisdiabetesmellitus.Acetyl-CoAPATHWAYSTHATPROCESSTHEMAJORPRODUCTSOFDIGESTIONThenatureofthedietsetsthebasicpatternofmetabo-Citriclism.Thereisaneedtoprocesstheproductsofdiges-acid2HATPtionofdietarycarbohydrate,lipid,andprotein.Thesecyclearemainlyglucose,fattyacidsandglycerol,andaminoacids,respectively.Inruminants(andtoalesserextentinotherherbivores),dietarycelluloseisfermentedby2CO2symbioticmicroorganismstoshort-chainfattyacids(acetic,propionic,butyric),andmetabolismintheseFigure15–1.Outlineofthepathwaysforthecatab-animalsisadaptedtousethesefattyacidsasmajorsub-olismofdietarycarbohydrate,protein,andfat.Allthestrates.Alltheproductsofdigestionaremetabolizedtopathwaysleadtotheproductionofacetyl-CoA,whichisacommonproduct,acetyl-CoA,whichisthenoxi-oxidizedinthecitricacidcycle,ultimatelyyieldingATPdizedbythecitricacidcycle(Figure15–1).intheprocessofoxidativephosphorylation.122
131OVERVIEWOFMETABOLISM/123Dietcursoroffattyacidsandcholesterol(andhenceofallsteroidssynthesizedinthebody).GluconeogenesisistheprocessofformingglucosefromnoncarbohydrateGlucoseGlycogenprecursors,eg,lactate,aminoacids,andglycerol.GlucoseLipidMetabolismIsConcernedMainly3CO2phosphatesWithFattyAcids&Cholesterol(Figure15–3)PentosephosphatepathwayThesourceoflong-chainfattyacidsiseitherdietaryislipidordenovosynthesisfromacetyl-CoAderivedfromsTriosecarbohydrate.Fattyacidsmaybeoxidizedtoacetyl-RiboseRNAphosphatesphosphateDNACoA(β-oxidation)oresterifiedwithglycerol,formingGlycolytriacylglycerol(fat)asthebody’smainfuelreserve.Acetyl-CoAformedbyβ-oxidationmayundergoseveralfates:(1)Aswithacetyl-CoAarisingfromglycolysis,itisPyruvateLactateoxidizedtoCO2+H2Oviathecitricacidcycle.Acylglycerols(fat)CO2AminoacidsTriacylglycerolSteroidsAcetyl-CoAFattyn(fat)oacidsistiasProteinicylfCholesteroliorAminopacidseitsLEDietFattyacidsCitricsacidinSteroidogenesiscyclesoietneadgioxpCholesteroliOL-βCarbohydrate2CO2Acetyl-CoAAminoacidsCholesterologenesisFigure15–2.OverviewofcarbohydratemetabolismKetogenesisshowingthemajorpathwaysandendproducts.Gluco-neogenesisisnotshown.KetonebodiesCitricGlucoseanditsmetabolitesalsotakepartinotheracidprocesses.Examples:(1)Conversiontothestoragecyclepolymerglycogeninskeletalmuscleandliver.(2)Thepentosephosphatepathway,analternativetopartofthepathwayofglycolysis,isasourceofreducingequiv-alents(NADPH)forbiosynthesisandthesourceofri-2CO2bosefornucleotideandnucleicacidsynthesis.(3)TriosephosphategivesrisetotheglycerolmoietyFigure15–3.Overviewoffattyacidmetabolismoftriacylglycerols.(4)Pyruvateandintermediatesofshowingthemajorpathwaysandendproducts.Ketonethecitricacidcycleprovidethecarbonskeletonsforbodiescomprisethesubstancesacetoacetate,3-hy-thesynthesisofaminoacids;andacetyl-CoA,thepre-droxybutyrate,andacetone.
132124/CHAPTER15(2)ItistheprecursorforsynthesisofcholesterolandMETABOLICPATHWAYSMAYBEothersteroids.STUDIEDATDIFFERENTLEVELS(3)Intheliver,itformsketonebodies(acetone,ace-OFORGANIZATIONtoacetate,and3-hydroxybutyrate)thatareimpor-tantfuelsinprolongedstarvation.Inadditiontostudiesinthewholeorganism,theloca-tionandintegrationofmetabolicpathwaysisrevealedbystudiesatseverallevelsoforganization.Atthetissueandorganlevel,thenatureofthesubstratesenteringMuchofAminoAcidMetabolismandmetabolitesleavingtissuesandorgansisdefined.AtInvolvesTransaminationthesubcellularlevel,eachcellorganelle(eg,themito-(Figure15–4)chondrion)orcompartment(eg,thecytosol)hasspe-cificrolesthatformpartofasubcellularpatternofTheaminoacidsarerequiredforproteinsynthesis.metabolicpathways.Somemustbesuppliedinthediet(theessentialaminoacids)sincetheycannotbesynthesizedinthebody.TheremainderarenonessentialaminoacidsthatareAttheTissueandOrganLevel,theBloodsuppliedinthedietbutcanbeformedfrommetabolicCirculationIntegratesMetabolismintermediatesbytransamination,usingtheaminoni-trogenfromotheraminoacids.Afterdeamination,Aminoacidsresultingfromthedigestionofdietaryaminonitrogenisexcretedasurea,andthecarbonproteinandglucoseresultingfromthedigestionofcar-skeletonsthatremainaftertransamination(1)areoxi-bohydrateareabsorbedanddirectedtotheliverviathedizedtoCO2viathecitricacidcycle,(2)formglucosehepaticportalvein.Theliverhastheroleofregulating(gluconeogenesis),or(3)formketonebodies.thebloodconcentrationofmostwater-solublemetabo-Severalaminoacidsarealsotheprecursorsofotherlites(Figure15–5).Inthecaseofglucose,thisiscompounds,eg,purines,pyrimidines,hormonessuchachievedbytakingupglucoseinexcessofimmediateasepinephrineandthyroxine,andneurotransmitters.requirementsandconvertingittoglycogen(glycogene-DietproteinNonproteinTissueproteinAminoacidsnitrogenderivativesTRANSAMINATIONCarbohydrateKetonebodies(glucose)AminonitrogeninAcetyl-CoAglutamateDEAMINATIONNHCitric3acidcycleUrea2CO2Figure15–4.Overviewofaminoacidmetabolismshowingthemajorpathwaysandendproducts.
133OVERVIEWOFMETABOLISM/125PlasmaproteinsLIVERUreaProteinAminoacidsCO2GlucoseAminoacidsGlycogenProteinLactateUreaAminoacidsAlanine,etcHERYTHROCYTESGlucoseeCO2pphosphateaGlucosetiKIDNEYUrinecpoGlycogenrtaDietlveinCarbohydrateGlucoseBLOODPLASMAAminoacidsProteinMUSCLESMALLINTESTINEFigure15–5.Transportandfateofmajorcarbohydrateandaminoacidsubstratesandmetabolites.Notethatthereislittlefreeglucoseinmuscle,sinceitisrapidlyphosphorylateduponentry.sis)ortofat(lipogenesis).Betweenmeals,thelivermucosa.Heretheyarepackagedwithproteinandse-actstomaintainthebloodglucoseconcentrationfromcretedintothelymphaticsystemandthenceintotheglycogen(glycogenolysis)and,togetherwiththekid-bloodstreamaschylomicrons,thelargestoftheplasmaney,byconvertingnoncarbohydratemetabolitessuchlipoproteins.Chylomicronsalsocontainotherlipid-aslactate,glycerol,andaminoacidstoglucose(gluco-solublenutrients,eg,vitamins.Unlikeglucoseandneogenesis).Maintenanceofanadequateconcentra-aminoacids,chylomicrontriacylglycerolisnottakenuptionofbloodglucoseisvitalforthosetissuesinwhichitdirectlybytheliver.Itisfirstmetabolizedbytissuesthatisthemajorfuel(thebrain)ortheonlyfuel(theeryth-havelipoproteinlipase,whichhydrolyzesthetriacyl-rocytes).Theliveralsosynthesizesthemajorplasmaglycerol,releasingfattyacidsthatareincorporatedintoproteins(eg,albumin)anddeaminatesaminoacidstissuelipidsoroxidizedasfuel.Theothermajorsourcethatareinexcessofrequirements,formingurea,whichoflong-chainfattyacidissynthesis(lipogenesis)fromistransportedtothekidneyandexcreted.carbohydrate,mainlyinadiposetissueandtheliver.Skeletalmuscleutilizesglucoseasafuel,formingAdiposetissuetriacylglycerolisthemainfuelreservebothlactateandCO2.Itstoresglycogenasafuelforitsofthebody.Onhydrolysis(lipolysis)freefattyacidsareuseinmuscularcontractionandsynthesizesmusclereleasedintothecirculation.Thesearetakenupbymostproteinfromplasmaaminoacids.Muscleaccountsfortissues(butnotbrainorerythrocytes)andesterifiedtoapproximately50%ofbodymassandconsequentlyacylglycerolsoroxidizedasafuel.Intheliver,triacyl-representsaconsiderablestoreofproteinthatcanbeglycerolarisingfromlipogenesis,freefattyacids,anddrawnupontosupplyaminoacidsforgluconeogenesischylomicronremnants(seeFigures25–3and25–4)isse-instarvation.cretedintothecirculationasverylowdensitylipopro-Lipidsinthediet(Figure15–6)aremainlytriacyl-tein(VLDL).Thistriacylglycerolundergoesafatesimi-glycerolandarehydrolyzedtomonoacylglycerolsandlartothatofchylomicrons.Partialoxidationoffattyfattyacidsinthegut,thenreesterifiedintheintestinalacidsintheliverleadstoketonebodyproduction(keto-
134126/CHAPTER15FFACO2GlucoseFattyacidsKetoneEbodiesstesrsiifyilcaotpiioLnBLOODTGPLASMACO2LIVERLPLFattyVLacidsDLEstesrsiifyilcLipoproteinaFattyotTGpiGlucoseacidsLPLioLnECshyteloTGsmriisificyrlcoanoMUSCLEtspiioLnTGDietMG+ADIPOSETGfattyacidsTGTISSUESMALLINTESTINEFigure15–6.Transportandfateofmajorlipidsubstratesandmetabolites.(FFA,freefattyacids;LPL,lipopro-teinlipase;MG,monoacylglycerol;TG,triacylglycerol;VLDL,verylowdensitylipoprotein.)genesis).KetonebodiesaretransportedtoextrahepaticGlycolysis,thepentosephosphatepathway,andfattytissues,wheretheyactasafuelsourceinstarvation.acidsynthesisareallfoundinthecytosol.Ingluconeo-genesis,substratessuchaslactateandpyruvate,whichAttheSubcellularLevel,GlycolysisOccursareformedinthecytosol,enterthemitochondriontoyieldoxaloacetatebeforeformationofglucose.intheCytosol&theCitricAcidCycleThemembranesoftheendoplasmicreticulumcon-intheMitochondriataintheenzymesystemforacylglycerolsynthesis,andCompartmentationofpathwaysinseparatesubcellulartheribosomesareresponsibleforproteinsynthesis.compartmentsororganellespermitsintegrationandregulationofmetabolism.NotallpathwaysareofequalTHEFLUXOFMETABOLITESINimportanceinallcells.Figure15–7depictsthesubcel-METABOLICPATHWAYSMUSTBElularcompartmentationofmetabolicpathwaysinahe-REGULATEDINACONCERTEDMANNERpaticparenchymalcell.Thecentralroleofthemitochondrionisimmedi-Regulationoftheoverallfluxthroughapathwayisim-atelyapparent,sinceitactsasthefocusofcarbohydrate,portanttoensureanappropriatesupply,whenre-lipid,andaminoacidmetabolism.Itcontainstheen-quired,oftheproductsofthatpathway.Regulationiszymesofthecitricacidcycle,β-oxidationoffattyacids,achievedbycontrolofoneormorekeyreactionsinandketogenesis,aswellastherespiratorychainandthepathway,catalyzedby“regulatoryenzymes.”TheATPsynthase.physicochemicalfactorsthatcontroltherateofan
135OVERVIEWOFMETABOLISM/127CYTOSOLGlycogenAAProteinRibosomeENDOPLASMICPentoseRETICULUMGlucosephosphatepathwayTriosephosphateGlycerolphosphateTriacylglycerolFattyacidsGlycerolGlycolysisPhosphoenolpyruvateLactatePyruvate-OxidationβAAAAPyruvateCO2GluconeogenesisLipogenesisOxaloacetateAcetyl-CoAKetonebodiesAAFumarateAACitrateCitricacidcycleAACO2AASuccinyl-CoAα-KetoglutarateCO2AAMITOCHONDRIONAAAAFigure15–7.Intracellularlocationandoverviewofmajormetabolicpathwaysinaliverparenchymalcell.(AA→,metabolismofoneormoreessentialaminoacids;AA↔,metabolismofoneormorenonessentialaminoacids.)
136128/CHAPTER15enzyme-catalyzedreaction,eg,substrateconcentration,Invivo,under“steady-state”conditions,thereisanetareofprimaryimportanceinthecontroloftheoverallfluxfromlefttorightbecausethereisacontinuoussup-rateofametabolicpathway(Chapter9).plyofAandremovalofD.Inpractice,thereareinvari-ablyoneormorenonequilibriumreactionsinameta-“Nonequilibrium”ReactionsArebolicpathway,wherethereactantsarepresentinPotentialControlPointsconcentrationsthatarefarfromequilibrium.Inat-temptingtoreachequilibrium,largelossesoffreeen-Inareactionatequilibrium,theforwardandreversere-ergyoccurasheat,makingthistypeofreactionessen-actionsoccuratequalrates,andthereisthereforenotiallyirreversible,eg,netfluxineitherdirection:HeatABCD↔↔↔A↔↔BD→CInactiveEnz122++2+/calmodulincAMPCaCellmembraneXYActiveEnz1AABCDEnz2+–1NegativeallostericPositiveallostericfeed-backfeed-forwardinhibitionactivation+or–+or–Ribosomalsynthesisofnewenzymeprotein3NuclearproductionofmRNA+–45InductionRepressionFigure15–8.Mechanismsofcontrolofanenzyme-catalyzedreaction.Circlednumbersindicatepossiblesitesofactionofhormones.1,Alterationofmem-branepermeability;2,conversionofaninactivetoanactiveenzyme,usuallyin-volvingphosphorylation/dephosphorylationreactions;3,alterationoftherateoftranslationofmRNAattheribosomallevel;4,inductionofnewmRNAforma-tion;and5,repressionofmRNAformation.1and2arerapid,whereas3–5areslowerwaysofregulatingenzymeactivity.
137OVERVIEWOFMETABOLISM/129Suchapathwayhasbothflowanddirection.Theactivityofexistingenzymemolecules,orslowly,byal-enzymescatalyzingnonequilibriumreactionsareusu-teringtherateofenzymesynthesis.allypresentinlowconcentrationsandaresubjecttoavarietyofregulatorymechanisms.However,manyofthereactionsinmetabolicpathwayscannotbeclassifiedSUMMARYasequilibriumornonequilibriumbutfallsomewhere•Theproductsofdigestionprovidethetissueswithbetweenthetwoextremes.thebuildingblocksforthebiosynthesisofcomplexmoleculesandalsowiththefueltopowerthelivingTheFlux-GeneratingReactionprocesses.IstheFirstReactioninaPathway•Nearlyallproductsofdigestionofcarbohydrate,fat,ThatIsSaturatedWithSubstrateandproteinaremetabolizedtoacommonmetabo-Itmaybeidentifiedasanonequilibriumreactioninlite,acetyl-CoA,beforefinaloxidationtoCO2inthecitricacidcycle.whichtheKmoftheenzymeisconsiderablylowerthanthenormalsubstrateconcentration.Thefirstreaction•Acetyl-CoAisalsousedastheprecursorforbiosyn-inglycolysis,catalyzedbyhexokinase(Figure17–2),isthesisoflong-chainfattyacids;steroids,includingsuchaflux-generatingstepbecauseitsKmforglucoseofcholesterol;andketonebodies.0.05mmol/Liswellbelowthenormalbloodglucose•Glucoseprovidescarbonskeletonsfortheglycerolconcentrationof5mmol/L.moietyoffatandofseveralnonessentialaminoacids.•Water-solubleproductsofdigestionaretransportedALLOSTERIC&HORMONALdirectlytotheliverviathehepaticportalvein.TheMECHANISMSAREIMPORTANTliverregulatesthebloodconcentrationsofglucoseandaminoacids.INTHEMETABOLICCONTROLOF•Pathwaysarecompartmentalizedwithinthecell.ENZYME-CATALYZEDREACTIONSGlycolysis,glycogenesis,glycogenolysis,thepentoseAhypotheticalmetabolicpathwayisshowninFigurephosphatepathway,andlipogenesisoccurinthecy-15–8,inwhichreactionsA↔BandC↔Dareequi-tosol.ThemitochondrioncontainstheenzymesoflibriumreactionsandB→Cisanonequilibriumreac-thecitricacidcycle,β-oxidationoffattyacids,andoftion.Thefluxthroughsuchapathwaycanberegulatedoxidativephosphorylation.Theendoplasmicreticu-bytheavailabilityofsubstrateA.Thisdependsonitslumalsocontainstheenzymesformanyothersupplyfromtheblood,whichinturndependsoneitherprocesses,includingproteinsynthesis,glycerolipidfoodintakeorkeyreactionsthatmaintainandreleaseformation,anddrugmetabolism.substratesfromtissuereservestotheblood,eg,the•Metabolicpathwaysareregulatedbyrapidmecha-glycogenphosphorylaseinliver(Figure18–1)andhor-nismsaffectingtheactivityofexistingenzymes,eg,mone-sensitivelipaseinadiposetissue(Figure25–7).allostericandcovalentmodification(ofteninre-ThefluxalsodependsonthetransportofsubstrateAsponsetohormoneaction);andslowmechanismsaf-acrossthecellmembrane.Fluxisalsodeterminedbyfectingthesynthesisofenzymes.theremovaloftheendproductDandtheavailabilityofcosubstrateorcofactorsrepresentedbyXandY.En-zymescatalyzingnonequilibriumreactionsareoftenal-REFERENCESlostericproteinssubjecttotherapidactionsof“feed-CohenP:ControlofEnzymeActivity,2nded.Chapman&Hall,back”or“feed-forward”controlbyallostericmodifiers1983.inimmediateresponsetotheneedsofthecell(Chap-FellD:UnderstandingtheControlofMetabolism.PortlandPress,ter9).Frequently,theproductofabiosyntheticpath-1997.waywillinhibittheenzymecatalyzingthefirstreactionFraynKN:MetabolicRegulation—AHumanPerspective.Portlandinthepathway.OthercontrolmechanismsdependonPress,1996.theactionofhormonesrespondingtotheneedsoftheNewsholmeEA,CrabtreeB:Flux-generatingandregulatorystepsbodyasawhole;theymayactrapidly,byalteringtheinmetaboliccontrol.TrendsBiochemSci1981;6:53.
138TheCitricAcidCycle:TheCatabolismofAcetyl-CoA16PeterA.Mayes,PhD,DSc,&DavidA.Bender,PhDBIOMEDICALIMPORTANCEcatedinthemitochondrialmatrix,eitherfreeorat-tachedtotheinnermitochondrialmembrane,whereThecitricacidcycle(Krebscycle,tricarboxylicacidtheenzymesoftherespiratorychainarealsofound.cycle)isaseriesofreactionsinmitochondriathatoxi-dizeacetylresidues(asacetyl-CoA)andreducecoen-zymesthatuponreoxidationarelinkedtotheforma-REACTIONSOFTHECITRICACIDtionofATP.CYCLELIBERATEREDUCINGThecitricacidcycleisthefinalcommonpathwayEQUIVALENTS&CO2fortheaerobicoxidationofcarbohydrate,lipid,and(Figure16–3)*proteinbecauseglucose,fattyacids,andmostaminoacidsaremetabolizedtoacetyl-CoAorintermediatesofTheinitialreactionbetweenacetyl-CoAandoxaloac-thecycle.Italsohasacentralroleingluconeogenesis,etatetoformcitrateiscatalyzedbycitratesynthaselipogenesis,andinterconversionofaminoacids.Manywhichformsacarbon-carbonbondbetweenthemethyloftheseprocessesoccurinmosttissues,buttheliveriscarbonofacetyl-CoAandthecarbonylcarbonofox-theonlytissueinwhichalloccurtoasignificantextent.aloacetate.Thethioesterbondoftheresultantcitryl-Therepercussionsarethereforeprofoundwhen,forex-CoAishydrolyzed,releasingcitrateandCoASH—anample,largenumbersofhepaticcellsaredamagedasinexergonicreaction.acutehepatitisorreplacedbyconnectivetissue(asinCitrateisisomerizedtoisocitratebytheenzymecirrhosis).Veryfew,ifany,geneticabnormalitiesofaconitase(aconitatehydratase);thereactionoccursincitricacidcycleenzymeshavebeenreported;suchab-twosteps:dehydrationtocis-aconitate,someofwhichnormalitieswouldbeincompatiblewithlifeornormalremainsboundtotheenzyme;andrehydrationtoisoci-development.trate.Althoughcitrateisasymmetricmolecule,aconi-tasereactswithcitrateasymmetrically,sothatthetwocarbonatomsthatarelostinsubsequentreactionsofTHECITRICACIDCYCLEPROVIDESthecyclearenotthosethatwereaddedfromacetyl-SUBSTRATEFORTHECoA.Thisasymmetricbehaviorisduetochanneling—RESPIRATORYCHAINtransferoftheproductofcitratesynthasedirectlyontotheactivesiteofaconitasewithoutenteringfreesolu-Thecyclestartswithreactionbetweentheacetylmoietytion.Thisprovidesintegrationofcitricacidcycleactiv-ofacetyl-CoAandthefour-carbondicarboxylicacidox-ityandtheprovisionofcitrateinthecytosolasasourcealoacetate,formingasix-carbontricarboxylicacid,cit-ofacetyl-CoAforfattyacidsynthesis.Thepoisonfluo-rate.Inthesubsequentreactions,twomoleculesofCO2roacetateistoxicbecausefluoroacetyl-CoAcondensesarereleasedandoxaloacetateisregenerated(Figurewithoxaloacetatetoformfluorocitrate,whichinhibits16–1).Onlyasmallquantityofoxaloacetateisneededaconitase,causingcitratetoaccumulate.fortheoxidationofalargequantityofacetyl-CoA;ox-Isocitrateundergoesdehydrogenationcatalyzedbyaloacetatemaybeconsideredtoplayacatalyticrole.isocitratedehydrogenasetoform,initially,oxalosucci-Thecitricacidcycleisanintegralpartoftheprocessnate,whichremainsenzyme-boundandundergoesde-bywhichmuchofthefreeenergyliberatedduringthecarboxylationtoα-ketoglutarate.Thedecarboxylationoxidationoffuelsismadeavailable.Duringoxidationofacetyl-CoA,coenzymesarereducedandsubsequentlyreoxidizedintherespiratorychain,linkedtotheforma-*FromCircularNo.200oftheCommitteeofEditorsofBiochemi-calJournalsRecommendations(1975):“AccordingtostandardtionofATP(oxidativephosphorylation;seeFigurebiochemicalconvention,theendingatein,eg,palmitate,denotes16–2andalsoChapter12).Thisprocessisaerobic,re-anymixtureoffreeacidandtheionizedform(s)(accordingtopH)quiringoxygenasthefinaloxidantofthereducedinwhichthecationsarenotspecified.”Thesameconventioniscoenzymes.Theenzymesofthecitricacidcyclearelo-adoptedinthistextforallcarboxylicacids.130
139THECITRICACIDCYCLE:THECATABOLISMOFACETYL-CoA/131Acetyl-CoACarbohydrateProteinLipids(C2)CoAAcetyl-CoA(C2)HOCitrateOxaloacetateCitrateOxaloacetate2(C6)(C4)(C6)(C4)H2OCitricacidcycleCis-aconitate(C6)HOMalate2(C4)2HIsocitrateHOCO2CO22(C6)2HCO2Figure16–1.Citricacidcycle,illustratingthecat-Fumarate(C4)α-Ketoglutaratealyticroleofoxaloacetate.(C)5NAD2HCOSuccinate2Succinyl-CoA2+2+(C4)(C)requiresMgorMnions.Therearethreeisoenzymes4ofisocitratedehydrogenase.One,whichusesNAD+,is2HPFp+HOPfoundonlyinmitochondria.TheothertwouseNADP2andarefoundinmitochondriaandthecytosol.Respi-Qratorychain-linkedoxidationofisocitrateproceedsal-mostcompletelythroughtheNAD+-dependenten-zyme.CytbPOxidativeα-Ketoglutarateundergoesoxidativedecarboxyla-phosphorylationtioninareactioncatalyzedbyamulti-enzymecomplexsimilartothatinvolvedintheoxidativedecarboxylationCytcofpyruvate(Figure17–5).The-ketoglutaratedehy-drogenasecomplexrequiresthesamecofactorsasthepyruvatedehydrogenasecomplex—thiamindiphos-Cytaa3P+1/2Ophate,lipoate,NAD,FAD,andCoA—andresultsin2theformationofsuccinyl-CoA.Theequilibriumofthis–Anaerobiosisreactionissomuchinfavorofsuccinyl-CoAformation(hypoxia,anoxia)thatitmustbeconsideredphysiologicallyunidirec-RespiratorychainH2Otional.Asinthecaseofpyruvateoxidation(Chapter17),arseniteinhibitsthereaction,causingthesubstrate,FpFlavoprotein-ketoglutarate,toaccumulate.CytCytochromeSuccinyl-CoAisconvertedtosuccinatebytheen-zymesuccinatethiokinase(succinyl-CoAsynthe-PHigh-energyphosphatetase).ThisistheonlyexampleinthecitricacidcycleofFigure16–2.Thecitricacidcycle:themajorcatabo-substrate-levelphosphorylation.Tissuesinwhichglu-licpathwayforacetyl-CoAinaerobicorganisms.Acetyl-coneogenesisoccurs(theliverandkidney)containtwoCoA,theproductofcarbohydrate,protein,andlipidca-isoenzymesofsuccinatethiokinase,onespecificfortabolism,istakenintothecycle,togetherwithH2O,andGDPandtheotherforADP.TheGTPformedisusedforthedecarboxylationofoxaloacetatetophos-oxidizedtoCO2withthereleaseofreducingequivalentsphoenolpyruvateingluconeogenesisandprovidesa(2H).Subsequentoxidationof2HintherespiratoryregulatorylinkbetweencitricacidcycleactivityandchainleadstocoupledphosphorylationofADPtoATP.thewithdrawalofoxaloacetateforgluconeogenesis.Foroneturnofthecycle,11~Paregeneratedviaox-Nongluconeogenictissueshaveonlytheisoenzymethatidativephosphorylationandone~ParisesatsubstrateusesADP.levelfromtheconversionofsuccinyl-CoAtosuccinate.
140CH3CO*SCoAAcetyl-CoAOCITRATESYNTHASEMALATEDEHYDROGENASECCOO–CoASH–CHCOO*–CH2COO2NADH+H+OxaloacetateH2O–HOCCOOHOCHCOO*–NAD+CHCOO–2CHCOO*–Citrate2L-MalateACONITASEFUMARASEFe2+H2OCHCOO*–Fluoroacetate2H2OCCOO–HCCOO*–CHCOO––OOC*CHCis-aconitateFumarateFADH2HO2SUCCINATEACONITASEFe2+DEHYDROGENASEFADMalonateCHCOO*–2CHCOO*–2CHCOO–CHCOO*–2HOCHCOO–Succinate+IsocitrateATPNADMg2+CoASHNADH+H+ADP+PiISOCITRATESUCCINATE*–DEHYDROGENASETHIOKINASECH2COOCHCOO*–Arsenite2CH2+CHCOO–NADH+HOCSCoACO+2Succinyl-CoANADOCCOO–CHCOO*–2Oxalosuccinateα-KETOGLUTARATEDEHYDROGENASECOMPLEXCH2Mn2+ISOCITRATECoASHDEHYDROGENASEOCCOO–CO2α-KetoglutarateFigure16–3.Reactionsofthecitricacid(Krebs)cycle.OxidationofNADHandFADH2intherespiratorychainleadstothegenerationofATPviaoxidativephosphorylation.Inordertofollowthepassageofacetyl-CoAthroughthecycle,thetwocarbonatomsoftheacetylradicalareshownlabeledonthecarboxylcarbon(designatedbyas-terisk)andonthemethylcarbon(usingthedesignation•).AlthoughtwocarbonatomsarelostasCO2inonerevo-lutionofthecycle,theseatomsarenotderivedfromtheacetyl-CoAthathasimmediatelyenteredthecyclebutfromthatportionofthecitratemoleculethatwasderivedfromoxaloacetate.However,oncompletionofasingleturnofthecycle,theoxaloacetatethatisregeneratedisnowlabeled,whichleadstolabeledCO2beingevolvedduringthesecondturnofthecycle.Becausesuccinateisasymmetriccompoundandbecausesuccinatedehydro-genasedoesnotdifferentiatebetweenitstwocarboxylgroups,“randomization”oflabeloccursatthisstepsuchthatallfourcarbonatomsofoxaloacetateappeartobelabeledafteroneturnofthecycle.Duringgluconeogene-sis,someofthelabelinoxaloacetateisincorporatedintoglucoseandglycogen(Figure19–1).Foradiscussionofthestereochemicalaspectsofthecitricacidcycle,seeGreville(1968).Thesitesofinhibition(−)byfluoroacetate,malonate,andarseniteareindicated.132
141THECITRICACIDCYCLE:THECATABOLISMOFACETYL-CoA/133Whenketonebodiesarebeingmetabolizedinextra-thecoenzymeforthreedehydrogenasesinthecycle—hepatictissuesthereisanalternativereactioncatalyzedisocitratedehydrogenase,α-ketoglutaratedehydrogen-bysuccinyl-CoA–acetoacetate-CoAtransferase(thio-ase,andmalatedehydrogenase;(3)thiamin(vitaminphorase)—involvingtransferofCoAfromsuccinyl-B1),asthiamindiphosphate,thecoenzymefordecar-CoAtoacetoacetate,formingacetoacetyl-CoA(Chap-boxylationintheα-ketoglutaratedehydrogenasereac-ter22).tion;and(4)pantothenicacid,aspartofcoenzymeA,Theonwardmetabolismofsuccinate,leadingtothethecofactorattachedto“active”carboxylicacidresi-regenerationofoxaloacetate,isthesamesequenceofduessuchasacetyl-CoAandsuccinyl-CoA.chemicalreactionsasoccursintheβ-oxidationoffattyacids:dehydrogenationtoformacarbon-carbondoublebond,additionofwatertoformahydroxylgroup,andTHECITRICACIDCYCLEPLAYSAafurtherdehydrogenationtoyieldtheoxo-groupofPIVOTALROLEINMETABOLISMoxaloacetate.ThecitricacidcycleisnotonlyapathwayforoxidationThefirstdehydrogenationreaction,formingfu-oftwo-carbonunits—itisalsoamajorpathwayforin-marate,iscatalyzedbysuccinatedehydrogenase,whichterconversionofmetabolitesarisingfromtransamina-isboundtotheinnersurfaceoftheinnermitochondrialtionanddeaminationofaminoacids.Italsoprovidesmembrane.TheenzymecontainsFADandiron-sulfurthesubstratesforaminoacidsynthesisbytransamina-(Fe:S)proteinanddirectlyreducesubiquinoneinthetion,aswellasforgluconeogenesisandfattyacidsyn-respiratorychain.Fumarase(fumaratehydratase)cat-thesis.Becauseitfunctionsinbothoxidativeandsyn-alyzestheadditionofwateracrossthedoublebondoftheticprocesses,itisamphibolic(Figure16–4).fumarate,yieldingmalate.Malateisconvertedtoox-aloacetatebymalatedehydrogenase,areactionrequir-ingNAD+.AlthoughtheequilibriumofthisreactionTheCitricAcidCycleTakesPartinstronglyfavorsmalate,thenetfluxistowardthedirec-Gluconeogenesis,Transamination,tionofoxaloacetatebecauseofthecontinualremovalof&Deaminationoxaloacetate(eithertoformcitrate,asasubstrateforgluconeogenesis,ortoundergotransaminationtoas-Alltheintermediatesofthecyclearepotentiallygluco-partate)andalsobecauseofthecontinualreoxidationgenic,sincetheycangiverisetooxaloacetateandthusofNADH.netproductionofglucose(intheliverandkidney,theorgansthatcarryoutgluconeogenesis;seeChapter19).ThekeyenzymethatcatalyzesnettransferoutoftheTWELVEATPAREFORMEDPERTURNcycleintogluconeogenesisisphosphoenolpyruvateOFTHECITRICACIDCYCLEcarboxykinase,whichdecarboxylatesoxaloacetatetophosphoenolpyruvate,withGTPactingasthedonorAsaresultofoxidationscatalyzedbythedehydrogen-phosphate(Figure16–4).asesofthecitricacidcycle,threemoleculesofNADHNettransferintothecycleoccursasaresultofsev-andoneofFADH2areproducedforeachmoleculeoferaldifferentreactions.Amongthemostimportantofacetyl-CoAcatabolizedinoneturnofthecycle.Thesesuchanapleroticreactionsistheformationofoxaloac-reducingequivalentsaretransferredtotherespiratoryetatebythecarboxylationofpyruvate,catalyzedbychain(Figure16–2),wherereoxidationofeachNADHpyruvatecarboxylase.Thisreactionisimportantinresultsinformationof3ATPandreoxidationofmaintaininganadequateconcentrationofoxaloacetateFADH2informationof2ATP.Inaddition,1ATPforthecondensationreactionwithacetyl-CoA.Ifacetyl-(orGTP)isformedbysubstrate-levelphosphorylationCoAaccumulates,itactsbothasanallostericactivatorcatalyzedbysuccinatethiokinase.ofpyruvatecarboxylaseandasaninhibitorofpyruvatedehydrogenase,therebyensuringasupplyofoxaloac-etate.Lactate,animportantsubstrateforgluconeogene-VITAMINSPLAYKEYROLESsis,entersthecycleviaoxidationtopyruvateandthenINTHECITRICACIDCYCLEcarboxylationtooxaloacetate.Aminotransferase(transaminase)reactionsformFouroftheBvitaminsareessentialinthecitricacidpyruvatefromalanine,oxaloacetatefromaspartate,andcycleandthereforeinenergy-yieldingmetabolism:(1)α-ketoglutaratefromglutamate.Becausethesereac-riboflavin,intheformofflavinadeninedinucleotidetionsarereversible,thecyclealsoservesasasourceof(FAD),acofactorintheα-ketoglutaratedehydrogenasecarbonskeletonsforthesynthesisoftheseaminoacids.complexandinsuccinatedehydrogenase;(2)niacin,inOtheraminoacidscontributetogluconeogenesisbe-theformofnicotinamideadeninedinucleotide(NAD),causetheircarbonskeletonsgiverisetocitricacidcycle
142134/CHAPTER16HydroxyprolineLactateSerineCysteineThreonineGlycineTRANSAMINASETryptophanAlaninePyruvateAcetyl-CoAPYRUVATECARBOXYLASEPHOSPHOENOLPYRUVATECARBOXYKINASEPhosphoenol-GlucosepyruvateOxaloacetateTyrosineTRANSAMINASEFumaratePhenylalanineAspartateCitrateIsoleucineMethionineSuccinyl-CoAValineCO2α-KetoglutaratePropionateCO2TRANSAMINASEHistidineProlineGlutamateGlutamineArginineFigure16–4.Involvementofthecitricacidcycleintransaminationandgluconeo-genesis.Theboldarrowsindicatethemainpathwayofgluconeogenesis.intermediates.Alanine,cysteine,glycine,hydroxypro-Pyruvatedehydrogenaseisamitochondrialenzyme,line,serine,threonine,andtryptophanyieldpyruvate;andfattyacidsynthesisisacytosolicpathway,butthearginine,histidine,glutamine,andprolineyieldα-ke-mitochondrialmembraneisimpermeabletoacetyl-toglutarate;isoleucine,methionine,andvalineyieldCoA.Acetyl-CoAismadeavailableinthecytosolfromsuccinyl-CoA;andtyrosineandphenylalanineyieldfu-citratesynthesizedinthemitochondrion,transportedmarate(Figure16–4).intothecytosolandcleavedinareactioncatalyzedbyInruminants,whosemainmetabolicfuelisshort-ATP-citratelyase.chainfattyacidsformedbybacterialfermentation,theconversionofpropionate,themajorglucogenicproductofrumenfermentation,tosuccinyl-CoAviatheRegulationoftheCitricAcidCyclemethylmalonyl-CoApathway(Figure19–2)isespe-DependsPrimarilyonaSupplyciallyimportant.ofOxidizedCofactorsTheCitricAcidCycleTakesPartInmosttissues,wheretheprimaryroleofthecitricacidinFattyAcidSynthesiscycleisinenergy-yieldingmetabolism,respiratorycontrolviatherespiratorychainandoxidativephos-(Figure16–5)phorylationregulatescitricacidcycleactivity(Chap-Acetyl-CoA,formedfrompyruvatebytheactionofter14).Thus,activityisimmediatelydependentonthepyruvatedehydrogenase,isthemajorbuildingblockforsupplyofNAD+,whichinturn,becauseofthetightlong-chainfattyacidsynthesisinnonruminants.(Inru-couplingbetweenoxidationandphosphorylation,isde-minants,acetyl-CoAisderiveddirectlyfromacetate.)pendentontheavailabilityofADPandhence,ulti-
143THECITRICACIDCYCLE:THECATABOLISMOFACETYL-CoA/135FattyregulatedinthesamewayasispyruvatedehydrogenasePyruvateGlucoseacids(Figure17–6).Succinatedehydrogenaseisinhibitedbyoxaloacetate,andtheavailabilityofoxaloacetate,ascontrolledbymalatedehydrogenase,dependsonthe[NADH]/[NAD+]ratio.SincetheKmforoxaloacetatePYRUVATEofcitratesynthaseisofthesameorderofmagnitudeasDEHYDROGENASEAcetyl-CoAtheintramitochondrialconcentration,itislikelythatAcetyl-CoAtheconcentrationofoxaloacetatecontrolstherateofcitrateformation.Whichofthesemechanismsareim-Oxaloacetateportantinvivohasstilltoberesolved.CitricATP-CITRATEacidLYASEcycleSUMMARYOxaloacetateCitrateCitrate•Thecitricacidcycleisthefinalpathwayfortheoxi-dationofcarbohydrate,lipid,andproteinwhosecommonend-metabolite,acetyl-CoA,reactswithox-aloacetatetoformcitrate.Byaseriesofdehydrogena-tionsanddecarboxylations,citrateisdegraded,releasingreducedcoenzymesand2CO2andregener-CO2CO2atingoxaloacetate.MITOCHONDRIAL•Thereducedcoenzymesareoxidizedbytherespira-MEMBRANEtorychainlinkedtoformationofATP.Thus,thecycleisthemajorrouteforthegenerationofATPFigure16–5.Participationofthecitricacidcycleinandislocatedinthematrixofmitochondriaadjacentfattyacidsynthesisfromglucose.SeealsoFigure21–5.totheenzymesoftherespiratorychainandoxidativephosphorylation.•Thecitricacidcycleisamphibolic,sinceinadditionmately,ontherateofutilizationofATPinchemicaltooxidationitisimportantintheprovisionofcar-andphysicalwork.Inaddition,individualenzymesofbonskeletonsforgluconeogenesis,fattyacidsynthe-thecycleareregulated.Themostlikelysitesforregula-sis,andinterconversionofaminoacids.tionarethenonequilibriumreactionscatalyzedbypyruvatedehydrogenase,citratesynthase,isocitratede-REFERENCEShydrogenase,andα-ketoglutaratedehydrogenase.ThedehydrogenasesareactivatedbyCa2+,whichincreasesBaldwinJE,KrebsHA:Theevolutionofmetaboliccycles.Natureinconcentrationduringmuscularcontractionandse-1981;291:381.cretion,whenthereisincreasedenergydemand.InaGoodwinTW(editor):TheMetabolicRolesofCitrate.Academictissuesuchasbrain,whichislargelydependentoncar-Press,1968.bohydratetosupplyacetyl-CoA,controlofthecitricGrevilleGD:Vol1,p297,in:CarbohydrateMetabolismandItsDisorders.DickensF,RandlePJ,WhelanWJ(editors).Acad-acidcyclemayoccuratpyruvatedehydrogenase.Sev-emicPress,1968.eralenzymesareresponsivetotheenergystatus,as+KayJ,WeitzmanPDJ(editors):Krebs’CitricAcidCycle—Halfashownbythe[ATP]/[ADP]and[NADH]/[NAD]ra-CenturyandStillTurning.BiochemicalSociety,London,tios.Thus,thereisallostericinhibitionofcitratesyn-1987.thasebyATPandlong-chainfattyacyl-CoA.AllostericSrerePA:Theenzymologyoftheformationandbreakdownofcit-activationofmitochondrialNAD-dependentisocitraterate.AdvEnzymol1975;43:57.dehydrogenasebyADPiscounteractedbyATPandTylerDD:TheMitochondrioninHealthandDisease.VCHPub-NADH.Theα-ketoglutaratedehydrogenasecomplexislishers,1992.
144Glycolysis&theOxidationofPyruvate17PeterA.Mayes,PhD,DSc,&DavidA.Bender,PhDBIOMEDICALIMPORTANCEGLYCOLYSISCANFUNCTIONUNDERMosttissueshaveatleastsomerequirementforglucose.ANAEROBICCONDITIONSInbrain,therequirementissubstantial.Glycolysis,theWhenamusclecontractsinananaerobicmedium,ie,majorpathwayforglucosemetabolism,occursintheonefromwhichoxygenisexcluded,glycogendisap-cytosolofallcells.Itisuniqueinthatitcanfunctionei-pearsandlactateappearsastheprincipalendproduct.theraerobicallyoranaerobically.Erythrocytes,whichWhenoxygenisadmitted,aerobicrecoverytakesplacelackmitochondria,arecompletelyreliantonglucoseasandlactatedisappears.However,ifcontractionoccurstheirmetabolicfuelandmetabolizeitbyanaerobicgly-underaerobicconditions,lactatedoesnotaccumulatecolysis.However,tooxidizeglucosebeyondpyruvateandpyruvateisthemajorendproductofglycolysis.(theendproductofglycolysis)requiresbothoxygenPyruvateisoxidizedfurthertoCO2andwater(Figureandmitochondrialenzymesystemssuchasthepyruvate17–1).Whenoxygenisinshortsupply,mitochondrialdehydrogenasecomplex,thecitricacidcycle,andthereoxidationofNADHformedfromNAD+duringgly-respiratorychain.colysisisimpaired,andNADHisreoxidizedbyreduc-Glycolysisisboththeprincipalrouteforglucoseingpyruvatetolactate,sopermittingglycolysistopro-metabolismandthemainpathwayforthemetabolismceed(Figure17–1).Whileglycolysiscanoccurunderoffructose,galactose,andothercarbohydratesderivedanaerobicconditions,thishasaprice,foritlimitsthefromthediet.TheabilityofglycolysistoprovideATPamountofATPformedpermoleofglucoseoxidized,intheabsenceofoxygenisespeciallyimportantbecausesothatmuchmoreglucosemustbemetabolizedunderitallowsskeletalmuscletoperformatveryhighlevelsanaerobicthanunderaerobicconditions.whenoxygensupplyisinsufficientandbecauseitallowstissuestosurviveanoxicepisodes.However,heartmus-THEREACTIONSOFGLYCOLYSIScle,whichisadaptedforaerobicperformance,hasrela-tivelylowglycolyticactivityandpoorsurvivalunderCONSTITUTETHEMAINPATHWAYconditionsofischemia.DiseasesinwhichenzymesofOFGLUCOSEUTILIZATIONglycolysis(eg,pyruvatekinase)aredeficientaremainlyTheoverallequationforglycolysisfromglucosetolac-seenashemolyticanemiasor,ifthedefectaffectstateisasfollows:skeletalmuscle(eg,phosphofructokinase),asfatigue.Infast-growingcancercells,glycolysisproceedsataGlucose++222ADPPi→L()+−Lactate++22ATPHO2higherratethanisrequiredbythecitricacidcycle,forminglargeamountsofpyruvate,whichisreducedtoAlloftheenzymesofglycolysis(Figure17–2)arelactateandexported.Thisproducesarelativelyacidicfoundinthecytosol.Glucoseentersglycolysisbyphos-localenvironmentinthetumorwhichmayhaveimpli-phorylationtoglucose6-phosphate,catalyzedbyhexo-cationsforcancertherapy.Thelactateisusedforgluco-kinase,usingATPasthephosphatedonor.Underneogenesisintheliver,anenergy-expensiveprocessre-physiologicconditions,thephosphorylationofglucosesponsibleformuchofthehypermetabolismseenintoglucose6-phosphatecanberegardedasirreversible.cancercachexia.LacticacidosisresultsfromseveralHexokinaseisinhibitedallostericallybyitsproduct,causes,includingimpairedactivityofpyruvatedehy-glucose6-phosphate.Intissuesotherthantheliveranddrogenase.pancreaticBisletcells,theavailabilityofglucosefor136
145GLYCOLYSIS&THEOXIDATIONOFPYRUVATE/137GlucoseGlycogenThisreactionisfollowedbyanotherphosphorylationC6(C6)nwithATPcatalyzedbytheenzymephosphofructoki-nase(phosphofructokinase-1),formingfructose1,6-bisphosphate.Thephosphofructokinasereactionmaybeconsideredtobefunctionallyirreversibleunderphysiologicconditions;itisbothinducibleandsubjecttoallostericregulationandhasamajorroleinregulat-Hexosephosphatesingtherateofglycolysis.Fructose1,6-bisphosphateisC6cleavedbyaldolase(fructose1,6-bisphosphatealdolase)intotwotriosephosphates,glyceraldehyde3-phosphateanddihydroxyacetonephosphate.Glyceraldehyde3-phosphateanddihydroxyacetonephosphateareinter-convertedbytheenzymephosphotrioseisomerase.Glycolysiscontinueswiththeoxidationofglycer-TriosephosphateTriosephosphatealdehyde3-phosphateto1,3-bisphosphoglycerate.TheC3C3enzymecatalyzingthisoxidation,glyceraldehydeNAD+HO3-phosphatedehydrogenase,isNAD-dependent.2Structurally,itconsistsoffouridenticalpolypeptides(monomers)formingatetramer.⎯SHgroupsareO2NADH1/+H+2O2presentoneachpolypeptide,derivedfromcysteineresidueswithinthepolypeptidechain.OneoftheCO2PyruvateLactate⎯SHgroupsattheactivesiteoftheenzyme(Figure+C3C317–3)combineswiththesubstrateformingathiohemi-H2Oacetalthatisoxidizedtoathiolester;thehydrogensre-movedinthisoxidationaretransferredtoNAD+.TheFigure17–1.Summaryofglycolysis.−,blockedbythiolesterthenundergoesphosphorolysis;inorganicanaerobicconditionsorbyabsenceofmitochondriaphosphate(Pi)isadded,forming1,3-bisphosphoglycer-containingkeyrespiratoryenzymes,eg,asinerythro-ate,andthe⎯SHgroupisreconstituted.cytes.Inthenextreaction,catalyzedbyphosphoglyceratekinase,phosphateistransferredfrom1,3-bisphospho-glycerateontoADP,formingATP(substrate-levelglycolysis(orglycogensynthesisinmuscleandlipogen-phosphorylation)and3-phosphoglycerate.Sincetwoesisinadiposetissue)iscontrolledbytransportintothemoleculesoftriosephosphateareformedpermoleculecell,whichinturnisregulatedbyinsulin.Hexokinaseofglucose,twomoleculesofATParegeneratedatthishasahighaffinity(lowKm)foritssubstrate,glucose,stagepermoleculeofglucoseundergoingglycolysis.andintheliverandpancreaticBisletcellsissaturatedThetoxicityofarsenicisduetocompetitionofarsenateunderallnormalconditionsandsoactsataconstantwithinorganicphosphate(Pi)intheabovereactionstoratetoprovideglucose6-phosphatetomeetthecell’sgive1-arseno-3-phosphoglycerate,whichhydrolyzesneed.LiverandpancreaticBisletcellsalsocontainanspontaneouslytogive3-phosphoglycerateplusheat,isoenzymeofhexokinase,glucokinase,whichhasaKmwithoutgeneratingATP.3-Phosphoglycerateisisomer-verymuchhigherthanthenormalintracellularconcen-izedto2-phosphoglyceratebyphosphoglyceratemu-trationofglucose.Thefunctionofglucokinaseinthetase.Itislikelythat2,3-bisphosphoglycerate(diphos-liveristoremoveglucosefromthebloodfollowingaphoglycerate;DPG)isanintermediateinthisreaction.meal,providingglucose6-phosphateinexcessofre-Thesubsequentstepiscatalyzedbyenolaseandin-quirementsforglycolysis,whichwillbeusedforglyco-volvesadehydration,formingphosphoenolpyruvate.gensynthesisandlipogenesis.Inthepancreas,theEnolaseisinhibitedbyfluoride.Topreventglycolysisglucose6-phosphateformedbyglucokinasesignalsin-intheestimationofglucose,bloodiscollectedincreasedglucoseavailabilityandleadstothesecretionoftubescontainingfluoride.Theenzymeisalsodepen-2+2+insulin.dentonthepresenceofeitherMgorMn.TheGlucose6-phosphateisanimportantcompoundatphosphateofphosphoenolpyruvateistransferredtothejunctionofseveralmetabolicpathways(glycolysis,ADPbypyruvatekinasetogenerate,atthisstage,gluconeogenesis,thepentosephosphatepathway,gly-twomoleculesofATPpermoleculeofglucoseoxi-cogenesis,andglycogenolysis).Inglycolysis,itiscon-dized.Theproductoftheenzyme-catalyzedreaction,vertedtofructose6-phosphatebyphosphohexose-enolpyruvate,undergoesspontaneous(nonenzymic)isomerase,whichinvolvesanaldose-ketoseisomerization.isomerizationtopyruvateandsoisnotavailableto
146GlycogenGlucose1-phosphateHEXOKINASECH2OHCH2OPGLUCOKINASECH2OPOOPHOSPHOHEXOSEOHHMg2+HHISOMERASECH2OHHHOHHOHHHHOHOOHHOOHHOHHOHHOHOHHATPADPα-D-Glucoseα-D-Glucose6-phosphateD-Fructose6-phosphateATPMg2+PHOSPHOFRUCTO-ADPKINASECH2OPO*CH2OPD-Fructose1,6-bisphosphateHHOHOHALDOLASEIodoacetateHOHCH*2OPPHOSPHOGLYCERATEOGLYCERALDEHYDE-3-PHOSPHATECODEHYDROGENASEKINASECOO–COPHCOCHOH22+MgPiDihydroxyacetonephosphateHCOHHCOHHCOHCH2OPCH2OPCH2OPPHOSPHOTRIOSEATPADPNADHNAD+ISOMERASE+H+3-Phosphoglycerate1,3-BisphosphoglycerateGlyceraldehyde3-phosphate1/2O2PHOSPHOGLYCERATEMUTASEMitochondrialrespiratorychainH2OCOO–3ADP3ATPHCOP2-Phosphoglycerate+PiCH2OHAnaerobiosisFluorideMg2+H2OENOLASECOO–PhosphoenolpyruvateCOPOxidationincitricCH2acidcycleADPMg2+PYRUVATEKINASENADH+H+NAD+ATPCOO––COO–COOSpontaneousCOHCOHOCHLACTATECH2CH3DEHYDROGENASECH3(Enol)(Keto)L(+)-LactatePyruvatePyruvate2−2−Figure17–2.Thepathwayofglycolysis.(P,⎯PO3;Pi,HOPO3;−,inhibition.)Atasterisk:Carbonatoms1–3offructosebisphosphateformdihydroxyacetonephosphate,whereascarbons4–6formglyceraldehyde3-phosphate.Theterm“bis-,”asinbisphosphate,indicatesthatthephosphategroupsareseparated,whereasdiphosphate,asinadenosinediphosphate,indicatesthattheyarejoined.138
147GLYCOLYSIS&THEOXIDATIONOFPYRUVATE/139SEnzHCOHCOHNAD+HCOHHCOHCH2OPCH2OPGlyceraldehyde3-phosphateEnzyme-substratecomplexHSEnzNAD+BoundcoenzymeSubstrateoxidationOPbybound+NADCOHCOHPiCH2OP1,3-BisphosphoglycerateSEnzSEnzCOCO+NADH+HNAD*+HCOHHCOHNADH+H+NAD*+CH2OOPCH2PEnergy-richintermediateFigure17–3.Mechanismofoxidationofglyceraldehyde3-phosphate.(Enz,glycer-aldehyde-3-phosphatedehydrogenase.)Theenzymeisinhibitedbythe⎯SHpoisoniodoacetate,whichisthusabletoinhibitglycolysis.TheNADHproducedontheenzyme+isnotasfirmlyboundtotheenzymeasisNAD.Consequently,NADHiseasilydisplaced+byanothermoleculeofNAD.undergothereversereaction.Thepyruvatekinasere-upintomitochondriaforoxidationviaoneofthetwoactionisthusalsoirreversibleunderphysiologiccon-shuttlesdescribedinChapter12.ditions.Theredoxstateofthetissuenowdetermineswhichoftwopathwaysisfollowed.Underanaerobiccondi-TissuesThatFunctionUnderHypoxictions,thereoxidationofNADHthroughtherespira-CircumstancesTendtoProduceLactatetorychaintooxygenisprevented.Pyruvateisreduced(Figure17–2)bytheNADHtolactate,thereactionbeingcatalyzedbylactatedehydrogenase.Severaltissue-specificisoen-Thisistrueofskeletalmuscle,particularlythewhitezymesofthisenzymehavebeendescribedandhavefibers,wheretherateofworkoutput—andthereforeclinicalsignificance(Chapter7).ThereoxidationoftheneedforATPformation—mayexceedtherateatNADHvialactateformationallowsglycolysistopro-whichoxygencanbetakenupandutilized.Glycolysisceedintheabsenceofoxygenbyregeneratingsufficientinerythrocytes,evenunderaerobicconditions,alwaysNAD+foranothercycleofthereactioncatalyzedbyterminatesinlactate,becausethesubsequentreactionsglyceraldehyde-3-phosphatedehydrogenase.Underaer-ofpyruvatearemitochondrial,anderythrocyteslackobicconditions,pyruvateistakenupintomitochon-mitochondria.Othertissuesthatnormallyderivemuchdriaandafterconversiontoacetyl-CoAisoxidizedtooftheirenergyfromglycolysisandproducelactatein-CO2bythecitricacidcycle.Thereducingequivalentscludebrain,gastrointestinaltract,renalmedulla,retina,fromtheNADH+H+formedinglycolysisaretakenandskin.Theliver,kidneys,andheartusuallytakeup
148140/CHAPTER17lactateandoxidizeitbutwillproduceitunderhypoxicHCOGlucoseconditions.HCOHGlycolysisIsRegulatedatThreeStepsCH2OPInvolvingNonequilibriumReactionsGlyceraldehyde3-phosphatePiNAD+Althoughmostofthereactionsofglycolysisarere-versible,threearemarkedlyexergonicandmustthere-GLYCERALDEHYDE-3-PHOSPHATEforebeconsideredphysiologicallyirreversible.Thesere-DEHYDROGENASEactions,catalyzedbyhexokinase(andglucokinase),NADH+H+phosphofructokinase,andpyruvatekinase,aretheOmajorsitesofregulationofglycolysis.Cellsthatareca-pableofreversingtheglycolyticpathway(gluconeoge-COPnesis)havedifferentenzymesthatcatalyzereactionsBISPHOSPHOGLYCERATEHCOHwhicheffectivelyreversetheseirreversiblereactions.MUTASETheimportanceofthesestepsintheregulationofgly-CH2OPcolysisandgluconeogenesisisdiscussedinChapter19.1,3-BisphosphoglycerateInErythrocytes,theFirstSiteinGlycolysisADPCOO–forATPGenerationMayBeBypassedPHOSPHOGLYCERATEHCOPKINASEIntheerythrocytesofmanymammals,thereactioncat-CH2OPalyzedbyphosphoglyceratekinasemaybebypassedbyaprocessthateffectivelydissipatesasheatthefreeATP2,3-Bisphosphoglycerateenergyassociatedwiththehigh-energyphosphateof–COO1,3-bisphosphoglycerate(Figure17–4).Bisphospho-glyceratemutasecatalyzestheconversionof1,3-bis-HCOHPiphosphoglycerateto2,3-bisphosphoglycerate,whichis2,3-BISPHOSPHOGLYCERATEconvertedto3-phosphoglycerateby2,3-bisphospho-CH2OPPHOSPHATASEglyceratephosphatase(andpossiblyalsophosphoglyc-3-Phosphoglycerateeratemutase).ThisalternativepathwayinvolvesnonetPyruvateyieldofATPfromglycolysis.However,itdoesservetoFigure17–4.2,3-Bisphosphoglyceratepathwayinprovide2,3-bisphosphoglycerate,whichbindstohemo-erythrocytes.globin,decreasingitsaffinityforoxygenandsomakingoxygenmorereadilyavailabletotissues(seeChapter6).THEOXIDATIONOFPYRUVATETOinthiamindeficiencyglucosemetabolismisimpairedandthereissignificant(andpotentiallylife-threatening)ACETYL-CoAISTHEIRREVERSIBLElacticandpyruvicacidosis.AcetyllipoamidereactswithROUTEFROMGLYCOLYSISTOTHEcoenzymeAtoformacetyl-CoAandreducedlipoamide.CITRICACIDCYCLEThecycleofreactioniscompletedwhenthereducedlipoamideisreoxidizedbyaflavoprotein,dihydrolipoylPyruvate,formedinthecytosol,istransportedintothedehydrogenase,containingFAD.Finally,thereducedmitochondrionbyaprotonsymporter(Figure12–10).+flavoproteinisoxidizedbyNAD,whichinturntrans-Insidethemitochondrion,pyruvateisoxidativelydecar-fersreducingequivalentstotherespiratorychain.boxylatedtoacetyl-CoAbyamultienzymecomplexthatisassociatedwiththeinnermitochondrialmembrane.++ThispyruvatedehydrogenasecomplexisanalogoustoPyruvate++→NADCoAAcetylCoANADHH−+++CO2theα-ketoglutaratedehydrogenasecomplexofthecitricacidcycle(Figure16–3).PyruvateisdecarboxylatedbyThepyruvatedehydrogenasecomplexconsistsofathepyruvatedehydrogenasecomponentoftheenzymenumberofpolypeptidechainsofeachofthethreecom-complextoahydroxyethylderivativeofthethiazoleringponentenzymes,allorganizedinaregularspatialcon-ofenzyme-boundthiamindiphosphate,whichinturnfiguration.Movementoftheindividualenzymesap-reactswithoxidizedlipoamide,theprostheticgroupofpearstoberestricted,andthemetabolicintermediatesdihydrolipoyltransacetylase,toformacetyllipoamidedonotdissociatefreelybutremainboundtotheen-(Figure17–5).ThiaminisvitaminB1(Chapter45),andzymes.Suchacomplexofenzymes,inwhichthesub-
149GLYCOLYSIS&THEOXIDATIONOFPYRUVATE/141OCHCCOO–+H+3TDPPyruvateAcetyllipoamideHSCoA-SHCHH3CSPYRUVATE2DEHYDROGENASECHCCOO22CHCHNTDPOH3COCHHydroxyethylHCHH2H2CCNDIHYDROLIPOYLOxidizedlipoamideCTRANSACETYLASESSOLipoicacidLysinesidechain+ONNADHCFADH2HCSHCH2DIHYDROLIPOYLCHCHCOSCoADEHYDROGENASE23Acetyl-CoASHDihydrolipoamide+FADNADH+HFigure17–5.Oxidativedecarboxylationofpyruvatebythepyruvatedehydrogenasecomplex.Lipoicacidisjoinedbyanamidelinktoalysineresidueofthetransacetylasecomponentoftheenzymecomplex.Itformsalongflexiblearm,allowingthelipoicacidprostheticgrouptorotatesequentiallybetweentheactivesitesofeachofthe+enzymesofthecomplex.(NAD,nicotinamideadeninedinucleotide;FAD,flavinadeninedinucleotide;TDP,thiamindiphosphate.)stratesarehandedonfromoneenzymetothenext,in-latedbyphosphorylationbyakinaseofthreeserinecreasesthereactionrateandeliminatessidereactions,residuesonthepyruvatedehydrogenasecomponentofincreasingoverallefficiency.themultienzymecomplex,resultingindecreasedactiv-ity,andbydephosphorylationbyaphosphatasethatPyruvateDehydrogenaseIsRegulatedcausesanincreaseinactivity.ThekinaseisactivatedbybyEnd-ProductInhibitionincreasesinthe[ATP]/[ADP],[acetyl-CoA]/[CoA],and[NADH]/[NAD+]ratios.Thus,pyruvatedehydro-&CovalentModificationgenase—andthereforeglycolysis—isinhibitednotonlyPyruvatedehydrogenaseisinhibitedbyitsproducts,byahigh-energypotentialbutalsowhenfattyacidsareacetyl-CoAandNADH(Figure17–6).Itisalsoregu-beingoxidized.Thus,instarvation,whenfreefattyacid
150142/CHAPTER17[Acetyl-CoA][NADH][ATP][CoA][NAD+][ADP]+++–DichloroacetateAcetyl-CoA2+––CaPDHKINASEPyruvate+NADH+HCO2+2MgATPADP–PDH–PDH-aPDH-b(ActiveDEPHOSPHO-ENZYME)(InactivePHOSPHO-ENZYME)PNAD+CoAPiH2OPyruvatePDHPHOSPHATASE+AB+Mg2+,Ca2+Insulin(inadiposetissue)Figure17–6.Regulationofpyruvatedehydrogenase(PDH).Arrowswithwavyshaftsindicateallostericef-fects.A:Regulationbyend-productinhibition.B:Regulationbyinterconversionofactiveandinactiveforms.concentrationsincrease,thereisadecreaseinthepro-ATPsynthasereactionhasbeencalculatedasapproxi-portionoftheenzymeintheactiveform,leadingtoamately51.6kJ.Itfollowsthatthetotalenergycapturedsparingofcarbohydrate.Inadiposetissue,whereglu-inATPpermoleofglucoseoxidizedis1961kJ,orap-coseprovidesacetylCoAforlipogenesis,theenzymeisproximately68%oftheenergyofcombustion.Mostofactivatedinresponsetoinsulin.theATPisformedbyoxidativephosphorylationresult-ingfromthereoxidationofreducedcoenzymesbytherespiratorychain.Theremainderisformedbysubstrate-OxidationofGlucoseYieldsUpto38Mollevelphosphorylation(Table17–1).ofATPUnderAerobicConditionsButOnly2MolWhenO2IsAbsentCLINICALASPECTSWhen1molofglucoseiscombustedinacalorimeterInhibitionofPyruvateMetabolismtoCO2andwater,approximately2870kJareliberatedLeadstoLacticAcidosisasheat.Whenoxidationoccursinthetissues,approxi-mately38molofATParegeneratedpermoleculeofArseniteandmercuricionsreactwiththe⎯SHgroupsglucoseoxidizedtoCO2andwater.Invivo,ΔGfortheoflipoicacidandinhibitpyruvatedehydrogenase,as
151GLYCOLYSIS&THEOXIDATIONOFPYRUVATE/143Table17–1.Generationofhigh-energyphosphateinthecatabolismofglucose.Numberof~PFormedperPathwayReactionCatalyzedbyMethodof~PProductionMoleofGlucoseGlycolysisGlyceraldehyde-3-phosphatedehydrogenaseRespiratorychainoxidationof2NADH6*PhosphoglyceratekinasePhosphorylationatsubstratelevel2PyruvatekinasePhosphorylationatsubstratelevel210AllowforconsumptionofATPbyreactionscatalyzedbyhexokinaseandphosphofructokinase−2Net8PyruvatedehydrogenaseRespiratorychainoxidationof2NADH6IsocitratedehydrogenaseRespiratorychainoxidationof2NADH6α-KetoglutaratedehydrogenaseRespiratorychainoxidationof2NADH6CitricacidcycleSuccinatethiokinasePhosphorylationatsubstratelevel2SuccinatedehydrogenaseRespiratorychainoxidationof2FADH24MalatedehydrogenaseRespiratorychainoxidationof2NADH6Net30Totalpermoleofglucoseunderaerobicconditions38Totalpermoleofglucoseunderanaerobicconditions2*ItisassumedthatNADHformedinglycolysisistransportedintomitochondriaviathemalateshuttle(seeFigure12–13).Iftheglyc-erophosphateshuttleisused,only2~PwouldbeformedpermoleofNADH,thetotalnetproductionbeing26insteadof38.The++calculationignoresthesmalllossofATPduetoatransportofHintothemitochondrionwithpyruvateandasimilartransportofHintheoperationofthemalateshuttle,totalingabout1molofATP.Notethatthereisasubstantialbenefitunderanaerobiccondi-tionsifglycogenisthestartingpoint,sincethenetproductionofhigh-energyphosphateinglycolysisisincreasedfrom2to3,asATPisnolongerrequiredbythehexokinasereaction.doesadietarydeficiencyofthiamin,allowingpyru-•Itcanfunctionanaerobicallybyregeneratingoxidized+vatetoaccumulate.NutritionallydeprivedalcoholicsNAD(requiredintheglyceraldehyde-3-phosphatede-arethiamin-deficientandmaydeveloppotentiallyfatalhydrogenasereaction)byreducingpyruvatetolactate.pyruvicandlacticacidosis.Patientswithinherited•Lactateistheendproductofglycolysisunderanaero-pyruvatedehydrogenasedeficiency,whichcanbeduebicconditions(eg,inexercisingmuscle)orwhenthetodefectsinoneormoreofthecomponentsoftheen-metabolicmachineryisabsentforthefurtheroxida-zymecomplex,alsopresentwithlacticacidosis,particu-tionofpyruvate(eg,inerythrocytes).larlyafteraglucoseload.Becauseofitsdependenceon•Glycolysisisregulatedbythreeenzymescatalyzingglucoseasafuel,brainisaprominenttissuewherethesenonequilibriumreactions:hexokinase,phosphofruc-metabolicdefectsmanifestthemselvesinneurologictokinase,andpyruvatekinase.disturbances.•Inerythrocytes,thefirstsiteinglycolysisforgenera-InheritedaldolaseAdeficiencyandpyruvatekinasetionofATPmaybebypassed,leadingtotheforma-deficiencyinerythrocytescausehemolyticanemia.tionof2,3-bisphosphoglycerate,whichisimportantTheexercisecapacityofpatientswithmusclephos-phofructokinasedeficiencyislow,particularlyonindecreasingtheaffinityofhemoglobinforO2.high-carbohydratediets.Byprovidinganalternative•Pyruvateisoxidizedtoacetyl-CoAbyamultienzymelipidfuel,eg,duringstarvation,whenbloodfreefattycomplex,pyruvatedehydrogenase,thatisdependentacidsandketonebodiesareincreased,workcapacityisonthevitamincofactorthiamindiphosphate.improved.•Conditionsthatinvolveaninabilitytometabolizepyruvatefrequentlyleadtolacticacidosis.SUMMARYREFERENCES•Glycolysisisthecytosolicpathwayofallmammaliancellsforthemetabolismofglucose(orglycogen)toBehalRHetal:Regulationofthepyruvatedehydrogenasemultien-pyruvateandlactate.zymecomplex.AnnuRevNutr1993;13:497.
152144/CHAPTER17BoiteuxA,HessB:Designofglycolysis.PhilTransRSocLondonSolsA:Multimodulationofenzymeactivity.CurrTopCellRegB1981;293:5.1981;19:77.Fothergill-GilmoreLA:Theevolutionoftheglycolyticpathway.SrerePA:Complexesofsequentialmetabolicenzymes.AnnuRevTrendsBiochemSci1986;11:47.Biochem1987;56:89.ScriverCRetal(editors):TheMetabolicandMolecularBasesofIn-heritedDisease,8thed.McGraw-Hill,2001.
153MetabolismofGlycogen18PeterA.Mayes,PhD,DSc,&DavidA.Bender,PhDBIOMEDICALIMPORTANCEphatasecatalyzeshydrolysisofpyrophosphateto2molofinorganicphosphate,shiftingtheequilibriumoftheGlycogenisthemajorstoragecarbohydrateinanimals,mainreactionbyremovingoneofitsproducts.correspondingtostarchinplants;itisabranchedpoly-Glycogensynthasecatalyzestheformationofagly-merofα-D-glucose.Itoccursmainlyinliver(upto6%)cosidebondbetweenC1oftheactivatedglucoseofandmuscle,whereitrarelyexceeds1%.However,be-UDPGlcandC4ofaterminalglucoseresidueofglyco-causeofitsgreatermass,musclecontainsaboutthreetogen,liberatinguridinediphosphate(UDP).Apreexist-fourtimesasmuchglycogenasdoesliver(Table18–1).ingglycogenmolecule,or“glycogenprimer,”mustbeMuscleglycogenisareadilyavailablesourceofglu-presenttoinitiatethisreaction.Theglycogenprimercoseforglycolysiswithinthemuscleitself.Liverglyco-mayinturnbeformedonaprimerknownasglyco-genfunctionstostoreandexportglucosetomaintaingenin,whichisa37-kDaproteinthatisglycosylatedbloodglucosebetweenmeals.After12–18hoursofonaspecifictyrosineresiduebyUDPGlc.Furtherglu-fasting,theliverglycogenisalmosttotallydepleted.coseresiduesareattachedinthe1→4positiontomakeGlycogenstoragediseasesareagroupofinheriteddis-ashortchainthatisasubstrateforglycogensynthase.orderscharacterizedbydeficientmobilizationofglyco-Inskeletalmuscle,glycogeninremainsattachedinthegenordepositionofabnormalformsofglycogen,lead-centeroftheglycogenmolecule(Figure13–15),ingtomuscularweaknessorevendeath.whereasinliverthenumberofglycogenmoleculesisgreaterthanthenumberofglycogeninmolecules.GLYCOGENESISOCCURSMAINLYINMUSCLE&LIVERBranchingInvolvesDetachmentofExistingGlycogenChainsThePathwayofGlycogenBiosynthesisInvolvesaSpecialNucleotideofGlucoseTheadditionofaglucoseresiduetoapreexistingglyco-(Figure18–1)genchain,or“primer,”occursatthenonreducing,outerendofthemoleculesothatthe“branches”oftheAsinglycolysis,glucoseisphosphorylatedtoglucoseglycogen“tree”becomeelongatedassuccessive1→46-phosphate,catalyzedbyhexokinaseinmuscleandlinkagesareformed(Figure18–3).Whenthechainhasglucokinaseinliver.Glucose6-phosphateisisomer-beenlengthenedtoatleast11glucoseresidues,branch-izedtoglucose1-phosphatebyphosphoglucomutase.ingenzymetransfersapartofthe1→4chain(atleastTheenzymeitselfisphosphorylated,andthephospho-sixglucoseresidues)toaneighboringchaintoformagrouptakespartinareversiblereactioninwhichglu-1→6linkage,establishingabranchpoint.Thecose1,6-bisphosphateisanintermediate.Next,glucosebranchesgrowbyfurtheradditionsof1→4-glucosyl1-phosphatereactswithuridinetriphosphate(UTP)tounitsandfurtherbranching.formtheactivenucleotideuridinediphosphateglu-cose(UDPGlc)*andpyrophosphate(Figure18–2),catalyzedbyUDPGlcpyrophosphorylase.Pyrophos-GLYCOGENOLYSISISNOTTHEREVERSEOFGLYCOGENESISBUTISASEPARATE*Othernucleosidediphosphatesugarcompoundsareknown,eg,PATHWAY(Figure18–1)UDPGal.Inaddition,thesamesugarmaybelinkedtodifferentnucleotides.Forexample,glucosemaybelinkedtouridine(asGlycogenphosphorylasecatalyzestherate-limitingshownabove)aswellastoguanosine,thymidine,adenosine,orcy-stepinglycogenolysisbypromotingthephosphorylytictidinenucleotides.cleavagebyinorganicphosphate(phosphorylysis;cfhy-145
154146/CHAPTER18Glycogen(1→4and1→6glucosylunits)xBRANCHINGENZYMEPi(1→4Glucosylunits)xInsulinUDPGLYCOGENGLYCOGENcAMPSYNTHASEPHOSPHORYLASEGlycogenprimerGlucagonEpinephrineGLUCAN*TRANSFERASEGlycogeninDEBRANCHINGUridineENZYMEdisphosphateglucose(UDPGlc)TouronicacidFreeglucosefrompathwaydebranchingUDPGlcPYROPHOSPHORYLASEenzymeINORGANICPYROPHOSPHATASEPPi2PiUridineUDPtriphosphate(UTP)Glucose1-phosphateMg2+PHOSPHOGLUCOMUTASEGlucose6-phosphateToglycolysisandpentosephosphatepathwayH2OADPNUCLEOSIDEDIPHOSPHO-ATPKINASEADPGLUCOSE-6-Mg2+GLUCOKINASEPHOSPHATASEPiATPGlucoseFigure18–1.Pathwayofglycogenesisandofglycogenolysisintheliver.Twohigh-energyphosphatesareusedintheincorporationof1molofglucoseintoglycogen.+,stimulation;−,inhibition.InsulindecreasesthelevelofcAMPonlyafterithasbeenraisedbyglucagonorepinephrine—ie,itantagonizestheiraction.Glucagonisactiveinheartmusclebutnotinskeletalmuscle.Atasterisk:Glucantransferaseanddebranchingenzymeap-peartobetwoseparateactivitiesofthesameenzyme.Table18–1.Storageofcarbohydrateinpostabsorptivenormaladulthumans(70kg).drolysis)ofthe1→4linkagesofglycogentoyieldglu-cose1-phosphate.Theterminalglucosylresiduesfrom1theoutermostchainsoftheglycogenmoleculearere-Liverglycogen4.0%=72g2movedsequentiallyuntilapproximatelyfourglucoseMuscleglycogen0.7%=245g3residuesremainoneithersideofa1→6branch(FigureExtracellularglucose0.1%=10g18–4).Anotherenzyme(-[1v4]v-[1v4]glucan327gtransferase)transfersatrisaccharideunitfromone1Liverweight1800g.branchtotheother,exposingthe1→6branchpoint.2Musclemass35kg.Hydrolysisofthe1→6linkagesrequiresthede-3Totalvolume10L.branchingenzyme.Furtherphosphorylaseactioncan
155METABOLISMOFGLYCOGEN/147Odephosphorylationofenzymeproteininresponseto6CH2OHhormoneaction(Chapter9).HNUracilHOCyclicAMP(cAMP)(Figure18–5)isformedfromHH1OATPbyadenylylcyclaseattheinnersurfaceofcellOHHNOOmembranesandactsasanintracellularsecondmessen-HOOPOPOCH2gerinresponsetohormonessuchasepinephrine,nor-HOHO–O–Oepinephrine,andglucagon.cAMPishydrolyzedbyphosphodiesterase,soterminatinghormoneaction.Inliver,insulinincreasestheactivityofphosphodiesterase.HHRiboseHHHOOHPhosphorylaseDiffersBetweenLiver&MuscleGlucoseDiphosphateUridineInliver,oneoftheserinehydroxylgroupsofactiveFigure18–2.Uridinediphosphateglucose(UDPGlc).phosphorylaseaisphosphorylated.Itisinactivatedbyhydrolyticremovalofthephosphatebyproteinphos-phatase-1toformphosphorylaseb.Reactivationre-thenproceed.Thecombinedactionofphosphorylasequiresrephosphorylationcatalyzedbyphosphorylaseandtheseotherenzymesleadstothecompletebreak-kinase.downofglycogen.Thereactioncatalyzedbyphospho-Musclephosphorylaseisdistinctfromthatofliver.Itglucomutaseisreversible,sothatglucose6-phosphateisadimer,eachmonomercontaining1molofpyridoxalcanbeformedfromglucose1-phosphate.Inliver(andphosphate(vitaminB6).Itispresentintwoforms:phos-kidney),butnotinmuscle,thereisaspecificenzyme,phorylasea,whichisphosphorylatedandactiveineitherglucose-6-phosphatase,thathydrolyzesglucosethepresenceorabsenceof5′-AMP(itsallostericmodi-6-phosphate,yieldingglucosethatisexported,leadingfier);andphosphorylaseb,whichisdephosphorylatedtoanincreaseinthebloodglucoseconcentration.andactiveonlyinthepresenceof5′-AMP.Thisoccursduringexercisewhenthelevelof5′-AMPrises,providing,CYCLICAMPINTEGRATESTHEbythismechanism,fuelforthemuscle.PhosphorylaseaisREGULATIONOFGLYCOGENOLYSISthenormalphysiologicallyactiveformoftheenzyme.&GLYCOGENESIScAMPActivatesMusclePhosphorylaseTheprincipalenzymescontrollingglycogenmetabo-lism—glycogenphosphorylaseandglycogensynthase—Phosphorylaseinmuscleisactivatedinresponsetoepi-areregulatedbyallostericmechanismsandcovalentnephrine(Figure18–6)actingviacAMP.IncreasingmodificationsduetoreversiblephosphorylationandtheconcentrationofcAMPactivatescAMP-dependent1→4-GlucosidicbondUnlabeledglucoseresidue1→6-Glucosidicbond14C-labeledglucoseresidue14C-GlucoseNew1→6-bondaddedGLYCOGENBRANCHINGSYNTHASEENZYMEFigure18–3.Thebiosynthesisofglycogen.Themechanismofbranchingasrevealed14byaddingC-labeledglucosetothedietinthelivinganimalandexaminingtheliverglycogenatfurtherintervals.
156148/CHAPTER18typesofsubunits—α,β,γ,andδ—inastructurerepre-sentedas(αβγδ)4.TheαandβsubunitscontainserineresiduesthatarephosphorylatedbycAMP-dependent2+proteinkinase.TheδsubunitbindsfourCaandis2+identicaltotheCa-bindingproteincalmodulin2+(Chapter43).ThebindingofCaactivatesthecat-alyticsiteoftheγsubunitwhilethemoleculeremainsinthedephosphorylatedbconfiguration.However,thephosphorylatedaformisonlyfullyactivatedinthe2+presenceofCa.Asecondmoleculeofcalmodulin,or2+TpC(thestructurallysimilarCa-bindingproteininmuscle),caninteractwithphosphorylasekinase,caus-ingfurtheractivation.Thus,activationofmusclecon-tractionandglycogenolysisarecarriedoutbythesamePHOSPHORYLASEGLUCANDEBRANCHING2+TRANSFERASEENZYMECa-bindingprotein,ensuringtheirsynchronization.GlucoseresiduesjoinedbyGlycogenolysisinLiverCan1→4-glucosidicbondsGlucoseresiduesjoinedbyBecAMP-Independent1→6-glucosidicbondsInadditiontotheactionofglucagonincausingforma-tionofcAMPandactivationofphosphorylaseinliver,Figure18–4.Stepsinglycogenolysis.1-adrenergicreceptorsmediatestimulationofglyco-genolysisbyepinephrineandnorepinephrine.Thisin-2+volvesacAMP-independentmobilizationofCaproteinkinase,whichcatalyzesthephosphorylationbyfrommitochondriaintothecytosol,followedbytheATPofinactivephosphorylasekinasebtoactivestimulationofaCa2+/calmodulin-sensitivephosphory-phosphorylasekinasea,whichinturn,bymeansofalasekinase.cAMP-independentglycogenolysisisalsofurtherphosphorylation,activatesphosphorylasebtocausedbyvasopressin,oxytocin,andangiotensinIIact-phosphorylasea.ingthroughcalciumorthephosphatidylinositolbis-phosphatepathway(Figure43–7).2+CaSynchronizestheActivationofPhosphorylaseWithMuscleContractionProteinPhosphatase-1Glycogenolysisincreasesinmuscleseveralhundred-foldInactivatesPhosphorylaseimmediatelyaftertheonsetofcontraction.Thisin-Bothphosphorylaseaandphosphorylasekinaseaarevolvestherapidactivationofphosphorylasebyactiva-2+dephosphorylatedandinactivatedbyproteinphos-tionofphosphorylasekinasebyCa,thesamesignalasphatase-1.Proteinphosphatase-1isinhibitedbyathatwhichinitiatescontractioninresponsetonerveprotein,inhibitor-1,whichisactiveonlyafterithasstimulation.MusclephosphorylasekinasehasfourbeenphosphorylatedbycAMP-dependentproteinki-nase.Thus,cAMPcontrolsboththeactivationandin-activationofphosphorylase(Figure18–6).Insulinre-NH2inforcesthiseffectbyinhibitingtheactivationofNNphosphorylaseb.Itdoesthisindirectlybyincreasinguptakeofglucose,leadingtoincreasedformationofNNglucose6-phosphate,whichisaninhibitorofphosphor-5′ylasekinase.OCH2GlycogenSynthase&PhosphorylaseO–OPOActivityAreReciprocallyRegulatedHH(Figure18–7)HHLikephosphorylase,glycogensynthaseexistsineitheraO3′OHphosphorylatedornonphosphorylatedstate.However,unlikephosphorylase,theactiveformisdephosphory-Figure18–5.3′,5′-Adenylicacid(cyclicAMP;cAMP).lated(glycogensynthasea)andmaybeinactivatedto
157–PROTEIN(n)Oi2PHOSPHATASE-1P+hormonalsignaleGlycogenGlucose1-phosphate+(active)(inactive)G6PInsulinPHOSPHORYLASEaPHOSPHORYLASEb–(n+1)allowsamplificationoftheiGlycogenPADPATPHdasacascade′-AMPO2ADP(active)HPHOSPHORYLASEKINASEaactionsarrang.)–ee++22ActiveCaofreKINASE–CaPROTEINCALMODULINnccAMP-DEPENDENTPROTEINKINASECOMPONENTOFPHOSPHORYLASEPHOSPHATASE-1ePHOSPHODIESTERASEqu6-phosphateesieP+.The(inactive)cAMP5ATPs;G6P,glucoseinmusclPHOSPHORYLASEKINASEbesidu+ereActiveadenylylcyclaseInactivePROTEINKINASErofglucosATPcAMP-DEPENDENTe+ControlofphosphorylasEpinephrine+Receptorp.(n=numbβ(active)eInhibitor-1(inactive)Inactiveadenylylcyclasere18–6.Inhibitor-1-phosphateuachsteATPADPFigat149
158150/CHAPTER18EpinephrineβReceptor+InactiveActiveadenylyladenylylcyclasecyclase+PHOSPHODIESTERASEATPcAMP5′-AMPPHOSPHORYLASE2+KINASECa++InactiveActivecAMP-DEPENDENTcAMP-DEPENDENTPROTEINKINASEPROTEINKINASEATPGlycogen(n+1)Inhibitor-1GSKADP(inactive)CALMODULIN-DEPENDENTPROTEINKINASEATPGLYCOGENSYNTHASEGLYCOGENSYNTHASE+ba(inactive)Ca2+(active)+InsulinG6P++ADPPROTEINPHOSPHATASEGlycogen(n)H2OPi+UDPGInhibitor-1-phosphatePROTEIN(active)PHOSPHATASE-1–Figure18–7.Controlofglycogensynthaseinmuscle(n=numberofglucoseresidues).Thesequenceofreac-tionsarrangedinacascadecausesamplificationateachstep,allowingonlynanomolequantitiesofhormonetocausemajorchangesinglycogenconcentration.(GSK,glycogensynthasekinase-3,-4,and-5;G6P,glucose6-phosphate.)glycogensynthasebbyphosphorylationonserineREGULATIONOFGLYCOGENresiduesbynofewerthansixdifferentproteinkinases.METABOLISMISEFFECTEDBY2+TwooftheproteinkinasesareCa/calmodulin-ABALANCEINACTIVITIESdependent(oneoftheseisphosphorylasekinase).An-otherkinaseiscAMP-dependentproteinkinase,whichBETWEENGLYCOGENallowscAMP-mediatedhormonalactiontoinhibitSYNTHASE&PHOSPHORYLASEglycogensynthesissynchronouslywiththeactivationof(Figure18–8)glycogenolysis.InsulinalsopromotesglycogenesisinmuscleatthesametimeasinhibitingglycogenolysisbyNotonlyisphosphorylaseactivatedbyariseinconcen-raisingglucose6-phosphateconcentrations,whichtrationofcAMP(viaphosphorylasekinase),butglyco-stimulatesthedephosphorylationandactivationofgensynthaseisatthesametimeconvertedtotheglycogensynthase.Dephosphorylationofglycogensyn-inactiveform;botheffectsaremediatedviacAMP-thasebiscarriedoutbyproteinphosphatase-1,whichdependentproteinkinase.Thus,inhibitionofgly-isunderthecontrolofcAMP-dependentproteinki-cogenolysisenhancesnetglycogenesis,andinhibitionofnase.glycogenesisenhancesnetglycogenolysis.Furthermore,
159METABOLISMOFGLYCOGEN/151EpinephrinePHOSPHODIESTERASE(liver,muscle)Inhibitor-1cAMP5′-AMPInhibitor-1Glucagonphosphate(liver)GLYCOGENPHOSPHORYLASESYNTHASEbKINASEbcAMP-PROTEINPROTEINDEPENDENTPHOSPHATASE-1PHOSPHATASE-1PROTEINKINASEGLYCOGENPHOSPHORYLASESYNTHASEaKINASEaGlycogenGlycogenPHOSPHORYLASEPHOSPHORYLASEUDPGIccycleabGlucose1-phosphatePROTEINPHOSPHATASE-1Glucose(liver)GlucoseLactate(muscle)Figure18–8.CoordinatedcontrolofglycogenolysisandglycogenesisbycAMP-dependentproteinki-nase.ThereactionsthatleadtoglycogenolysisasaresultofanincreaseincAMPconcentrationsareshownwithboldarrows,andthosethatareinhibitedbyactivationofproteinphosphatase-1areshownasbrokenarrows.ThereverseoccurswhencAMPconcentrationsdecreaseasaresultofphosphodiesteraseactivity,leadingtoglycogenesis.thedephosphorylationofphosphorylasea,phosphory-more,theyallowinsulin,viaglucose6-phosphateeleva-lasekinasea,andglycogensynthasebiscatalyzedbytion,tohaveeffectsthatactreciprocallytothoseofasingleenzymeofwidespecificity—proteinphos-cAMP(Figures18–6and18–7).phatase-1.Inturn,proteinphosphatase-1isinhibitedbycAMP-dependentproteinkinaseviainhibitor-1.Thus,glycogenolysiscanbeterminatedandglycogenesisCLINICALASPECTScanbestimulatedsynchronously,orviceversa,becauseGlycogenStorageDiseasesAreInheritedbothprocessesarekeyedtotheactivityofcAMP-depen-dentproteinkinase.Bothphosphorylasekinaseand“Glycogenstoragedisease”isagenerictermtodescribeglycogensynthasemaybereversiblyphosphorylatedinagroupofinheriteddisorderscharacterizedbydeposi-morethanonesitebyseparatekinasesandphosphatases.tionofanabnormaltypeorquantityofglycogenintheThesesecondaryphosphorylationsmodifythesensitivitytissues.Theprincipalglycogenosesaresummarizedinoftheprimarysitestophosphorylationanddephos-Table18–2.Deficienciesofadenylylkinaseandphorylation(multisitephosphorylation).WhatiscAMP-dependentproteinkinasehavealsobeenre-
160152/CHAPTER18Table18–2.Glycogenstoragediseases.GlycogenosisNameCauseofDisorderCharacteristicsTypeIVonGierke’sdiseaseDeficiencyofglucose-6-phosphataseLivercellsandrenaltubulecellsloadedwithglycogen.Hypoglycemia,lactic-acidemia,ketosis,hyperlipemia.TypeIIPompe’sdiseaseDeficiencyoflysosomalα-1→4-andFatal,accumulationofglycogeninlyso-1→6-glucosidase(acidmaltase)somes,heartfailure.TypeIIILimitdextrinosis,Forbes’orAbsenceofdebranchingenzymeAccumulationofacharacteristicCori’sdiseasebranchedpolysaccharide.TypeIVAmylopectinosis,Andersen’sAbsenceofbranchingenzymeAccumulationofapolysaccharidehav-diseaseingfewbranchpoints.Deathduetocardiacorliverfailureinfirstyearoflife.TypeVMyophosphorylasedeficiency,AbsenceofmusclephosphorylaseDiminishedexercisetolerance;musclesMcArdle’ssyndromehaveabnormallyhighglycogencon-tent(2.5–4.1%).Littleornolactateinbloodafterexercise.TypeVIHers’diseaseDeficiencyofliverphosphorylaseHighglycogencontentinliver,ten-dencytowardhypoglycemia.TypeVIITarui’sdiseaseDeficiencyofphosphofructokinaseAsfortypeVbutalsopossibilityofhe-inmuscleanderythrocytesmolyticanemia.TypeVIIIDeficiencyofliverphosphorylaseAsfortypeVI.kinaseported.Someoftheconditionsdescribedhavebene-REFERENCESfitedfromlivertransplantation.BollenM,KeppensS,StalmansW:Specificfeaturesofglycogenmetabolismintheliver.BiochemJ1998;336:19.SUMMARYCohenP:Theroleofproteinphosphorylationinthehormonal•Glycogenrepresentstheprincipalstorageformofcontrolofenzymeactivity.EurJBiochem1985;151:439.carbohydrateinthemammalianbody,mainlyintheErcanN,GannonMC,NuttallFQ:Incorporationofglycogeninintoahepaticproteoglycogenafteroralglucoseadministra-liverandmuscle.tion.JBiolChem1994;269:22328.•Intheliver,itsmajorfunctionistoprovideglucoseGeddesR:Glycogen:ametabolicviewpoint.BioscienceRepforextrahepatictissues.Inmuscle,itservesmainlyas1986;6:415.areadysourceofmetabolicfuelforuseinmuscle.McGarryJDetal:Fromdietaryglucosetoliverglycogen:thefull•Glycogenissynthesizedfromglucosebythepathwaycircleround.AnnuRevNutr1987;7:51.ofglycogenesis.Itisbrokendownbyaseparatepath-Meléndez-HeviaE,WaddellTG,SheltonED:Optimizationofwayknownasglycogenolysis.Glycogenolysisleadstomoleculardesignintheevolutionofmetabolism:theglyco-genmolecule.BiochemJ1993;295:477.glucoseformationinliverandlactateformationinmuscleowingtotherespectivepresenceorabsenceofRazI,KatzA,SpencerMK:Epinephrineinhibitsinsulin-mediatedglycogenesisbutenhancesglycolysisinhumanskeletalmus-glucose-6-phosphatase.cle.AmJPhysiol1991;260:E430.•CyclicAMPintegratestheregulationofglycogenoly-ScriverCRetal(editors):TheMetabolicandMolecularBasesofIn-sisandglycogenesisbypromotingthesimultaneousheritedDisease,8thed.McGraw-Hill,2001.activationofphosphorylaseandinhibitionofglyco-ShulmanGI,LandauBR:Pathwaysofglycogenrepletion.Physiolgensynthase.InsulinactsreciprocallybyinhibitingRev1992;72:1019.glycogenolysisandstimulatingglycogenesis.Villar-PalasiC:Onthemechanismofinactivationofmuscleglyco-•Inheriteddeficienciesinspecificenzymesofglycogengenphosphorylasebyinsulin.BiochimBiophysActa1994;1224:384.metabolisminbothliverandmusclearethecausesofglycogenstoragediseases.
161Gluconeogenesis&ControloftheBloodGlucose19PeterA.Mayes,PhD,DSc,&DavidA.Bender,PhDBIOMEDICALIMPORTANCEvate(Figure45–17).Asecondenzyme,phospho-enolpyruvatecarboxykinase,catalyzesthedecarboxy-Gluconeogenesisisthetermusedtoincludeallpath-lationandphosphorylationofoxaloacetatetophospho-waysresponsibleforconvertingnoncarbohydratepre-enolpyruvateusingGTP(orITP)asthephosphatecursorstoglucoseorglycogen.Themajorsubstratesaredonor.Thus,reversalofthereactioncatalyzedbypyru-theglucogenicaminoacidsandlactate,glycerol,andvatekinaseinglycolysisinvolvestwoendergonicreac-propionate.Liverandkidneyarethemajorgluco-tions.neogenictissues.GluconeogenesismeetstheneedsofInpigeon,chicken,andrabbitliver,phospho-thebodyforglucosewhencarbohydrateisnotavailableenolpyruvatecarboxykinaseisamitochondrialenzyme,insufficientamountsfromthedietorfromglycogenandphosphoenolpyruvateistransportedintothecy-reserves.Asupplyofglucoseisnecessaryespeciallyfortosolforgluconeogenesis.Intheratandthemouse,thethenervoussystemanderythrocytes.Failureofgluco-enzymeiscytosolic.Oxaloacetatedoesnotcrossthemi-neogenesisisusuallyfatal.Hypoglycemiacausesbraintochondrialinnermembrane;itisconvertedtomalate,dysfunction,whichcanleadtocomaanddeath.Glu-whichistransportedintothecytosol,andconvertedcoseisalsoimportantinmaintainingthelevelofinter-backtooxaloacetatebycytosolicmalatedehydrogenase.mediatesofthecitricacidcycleevenwhenfattyacidsInhumans,theguineapig,andthecow,theenzymeisarethemainsourceofacetyl-CoAinthetissues.Inad-equallydistributedbetweenmitochondriaandcytosol.dition,gluconeogenesisclearslactateproducedbymus-ThemainsourceofGTPforphosphoenolpyruvatecleanderythrocytesandglycerolproducedbyadiposecarboxykinaseinsidethemitochondrionisthereactiontissue.Propionate,theprincipalglucogenicfattyacidofsuccinyl-CoAsynthetase(Chapter16).Thisprovidesproducedinthedigestionofcarbohydratesbyrumi-alinkandlimitbetweencitricacidcycleactivityandnants,isamajorsubstrateforgluconeogenesisinthesetheextentofwithdrawalofoxaloacetateforgluconeo-species.genesis.GLUCONEOGENESISINVOLVESB.FRUCTOSE1,6-BISPHOSPHATEGLYCOLYSIS,THECITRICACIDCYCLE,&FRUCTOSE6-PHOSPHATE&SOMESPECIALREACTIONSTheconversionoffructose1,6-bisphosphatetofructose(Figure19–1)6-phosphate,toachieveareversalofglycolysis,iscat-ThermodynamicBarriersPreventalyzedbyfructose-1,6-bisphosphatase.ItspresenceaSimpleReversalofGlycolysisdetermineswhetherornotatissueiscapableofsynthe-sizingglycogennotonlyfrompyruvatebutalsofromThreenonequilibriumreactionscatalyzedbyhexoki-triosephosphates.Itispresentinliver,kidney,andnase,phosphofructokinase,andpyruvatekinasepreventskeletalmusclebutisprobablyabsentfromheartandsimplereversalofglycolysisforglucosesynthesissmoothmuscle.(Chapter17).Theyarecircumventedasfollows:A.PYRUVATE&PHOSPHOENOLPYRUVATEC.GLUCOSE6-PHOSPHATE&GLUCOSEMitochondrialpyruvatecarboxylasecatalyzesthecar-Theconversionofglucose6-phosphatetoglucoseisboxylationofpyruvatetooxaloacetate,anATP-requir-catalyzedbyglucose-6-phosphatase.Itispresentiningreactioninwhichthevitaminbiotinistheco-liverandkidneybutabsentfrommuscleandadiposeenzyme.BiotinbindsCO2frombicarbonateastissue,which,therefore,cannotexportglucoseintothecarboxybiotinpriortotheadditionoftheCO2topyru-bloodstream.153
162PiGlucoseATPGLUCOKINASEGLUCOSE-6-PHOSPHATASEHEXOKINASEGlucose6-HOADP2phosphateGlycogenAMPAMPPiFructose6-ATPphosphateFRUCTOSE-1,6-PHOSPHOFRUCTOKINASEBISPHOSPHATASEFructose1,6-HOADP2bisphosphateFructosecAMP2,6-bisphosphate(glucagon)Fructose2,6-bisphosphateGlyceraldehyde3-phosphateDihydroxyacetonephosphate+NADPi+NADH+H+GLYCEROL3-PHOSPHATENADH+HDEHYDROGENASEcAMP(glucagon)1,3-BisphosphoglycerateNAD+ADPGlycerol3-phosphateADPATPGLYCEROLKINASE3-PhosphoglycerateATPGlycerol2-PhosphoglyceratecAMP(glucagon)PhosphoenolpyruvateADPPYRUVATEKINASEAlanineGDP+CO2FattyATPacidsPHOSPHOENOLPYRUVATEPyruvateLactateCARBOXYKINASECitrate++GTPNADH+HNADPYRUVATEOxaloacetateCYTOSOLDEHYDROGENASEPyruvateAcetyl-CoANADH+H+CO+ATP2MITOCHONDRIONMg2+PYRUVATECARBOXYLASENAD+ADP+Pi+NADH+HOxaloacetateNAD+MalateMalateCitrateCitricacidcycleα-KetoglutarateFumarateSuccinyl-CoAPropionateFigure19–1.Majorpathwaysandregulationofgluconeogenesisandglycolysisintheliver.Entrypointsofglucogenicaminoacidsaftertransaminationareindicatedbyarrowsextendedfromcircles.(SeealsoFigure16–4.)Thekeygluconeogenicenzymesareenclosedindouble-borderedboxes.TheATPrequiredforgluconeogenesisissuppliedbytheoxidationoflong-chainfattyacids.Propionateisofquantitativeimportanceonlyinruminants.Arrowswithwavyshaftssignifyallostericeffects;dash-shaftedarrows,covalentmodificationbyreversiblephosphorylation.Highconcentrationsofalanineactasa“gluconeogenicsignal”byinhibitingglycolysisatthepyruvatekinasestep.154
163GLUCONEOGENESIS&CONTROLOFTHEBLOODGLUCOSE/155D.GLUCOSE1-PHOSPHATE&GLYCOGENphosphatebyNAD+catalyzedbyglycerol-3-phos-Thebreakdownofglycogentoglucose1-phosphateisphatedehydrogenase.catalyzedbyphosphorylase.Glycogensynthesisin-volvesadifferentpathwayviauridinediphosphateglu-SINCEGLYCOLYSIS&GLUCONEOGENESIScoseandglycogensynthase(Figure18–1).TherelationshipsbetweengluconeogenesisandtheSHARETHESAMEPATHWAYBUTINglycolyticpathwayareshowninFigure19–1.AfterOPPOSITEDIRECTIONS,THEYMUSTtransaminationordeamination,glucogenicaminoacidsBEREGULATEDRECIPROCALLYyieldeitherpyruvateorintermediatesofthecitricacidcycle.Therefore,thereactionsdescribedabovecanac-Changesintheavailabilityofsubstratesareresponsiblecountfortheconversionofbothglucogenicaminoformostchangesinmetabolismeitherdirectlyorindi-acidsandlactatetoglucoseorglycogen.Propionateisarectlyactingviachangesinhormonesecretion.Threemajorsourceofglucoseinruminantsandentersgluco-mechanismsareresponsibleforregulatingtheactivityneogenesisviathecitricacidcycle.Propionateisesteri-ofenzymesincarbohydratemetabolism:(1)changesinfiedwithCoA,thenpropionyl-CoA,iscarboxylatedtotherateofenzymesynthesis,(2)covalentmodificationbyreversiblephosphorylation,and(3)allostericeffects.D-methylmalonyl-CoA,catalyzedbypropionyl-CoAcarboxylase,abiotin-dependentenzyme(Figure19–2).Methylmalonyl-CoAracemasecatalyzestheconver-Induction&RepressionofKeyEnzymesionofD-methylmalonyl-CoAtoL-methylmalonyl-SynthesisRequiresSeveralHoursCoA,whichthenundergoesisomerizationtosuccinyl-CoAcatalyzedbymethylmalonyl-CoAisomerase.ThechangesinenzymeactivityintheliverthatoccurThisenzymerequiresvitaminB12asacoenzyme,andundervariousmetabolicconditionsarelistedinTabledeficiencyofthisvitaminresultsintheexcretionof19–1.Theenzymesinvolvedcatalyzenonequilibriummethylmalonate(methylmalonicaciduria).(physiologicallyirreversible)reactions.TheeffectsareC15andC17fattyacidsarefoundparticularlyinthegenerallyreinforcedbecausetheactivityoftheenzymeslipidsofruminants.Dietaryodd-carbonfattyacidscatalyzingthechangesintheoppositedirectionvariesuponoxidationyieldpropionate(Chapter22),whichisreciprocally(Figure19–1).Theenzymesinvolvedinasubstrateforgluconeogenesisinhumanliver.theutilizationofglucose(ie,thoseofglycolysisandli-Glycerolisreleasedfromadiposetissueasaresultofpogenesis)allbecomemoreactivewhenthereisasu-lipolysis,andonlytissuessuchasliverandkidneythatperfluityofglucose,andundertheseconditionstheen-possessglycerolkinase,whichcatalyzestheconversionzymesresponsibleforgluconeogenesisallhavelowofglyceroltoglycerol3-phosphate,canutilizeit.Glyc-activity.Thesecretionofinsulin,inresponsetoin-erol3-phosphatemaybeoxidizedtodihydroxyacetonecreasedbloodglucose,enhancesthesynthesisofthekeyCoASHACYL-CoACO2+H2OPROPIONYL-CoACH3SYNTHETASECH3CARBOXYLASECH3–CH2CH2HCCOO2+MgBiotinCOO–COCOSCoASCoAATPAMP+PPiATPADP+PiPropionatePropionyl-CoAD-Methyl-malonyl-CoAMETHYLMALONYL-CoARACEMASECOO–METHYLMALONYL-CoAISOMERASECH3IntermediatesCH2–OOCCHofcitricacidcycleCH2B12coenzymeCOSCoACOSCoAL-Methyl-Succinyl-CoAmalonyl-CoAFigure19–2.Metabolismofpropionate.
164156/CHAPTER19Table19–1.Regulatoryandadaptiveenzymesoftherat(mainlyliver).ActivityInCarbo-Starva-hydratetionandFeedingDiabetesInducerRepressorActivatorInhibitorEnzymesofglycogenesis,glycolysis,andpyruvateoxidationGlycogensynthase↑↓InsulinInsulinGlucagon(cAMP)phos-systemGlucose6-phorylase,glycogen1phosphate1HexokinaseGlucose6-phosphateGlucokinase↑↓InsulinGlucagon(cAMP)Phosphofructokinase-1↑↓InsulinGlucagonAMP,fructose6-Citrate(fattyacids,ketone11(cAMP)phosphate,Pi,fruc-bodies),ATP,glucagontose2,6-bisphos-(cAMP)1phatePyruvatekinase↑↓Insulin,fructoseGlucagonFructose1,6-ATP,alanine,glucagon1(cAMP)bisphosphate,in-(cAMP),epinephrinesulin+Pyruvatedehydro-↑↓CoA,NAD,insu-Acetyl-CoA,NADH,ATP2genaselin,ADP,pyruvate(fattyacids,ketonebodies)Enzymesofgluconeogenesis11Pyruvatecarboxylase↓↑Glucocorticoids,InsulinAcetyl-CoAADPglucagon,epi-nephrine(cAMP)Phosphoenolpyruvate↓↑Glucocorticoids,InsulinGlucagon?carboxykinaseglucagon,epi-nephrine(cAMP)Fructose-1,6-↓↑Glucocorticoids,InsulinGlucagon(cAMP)Fructose1,6-bisphosphate,bisphosphataseglucagon,epi-AMP,fructose2,6-bisphos-1nephrine(cAMP)phateGlucose-6-phosphatase↓↑Glucocorticoids,Insulinglucagon,epi-nephrine(cAMP)EnzymesofthepentosephosphatepathwayandlipogenesisGlucose-6-phosphate↑↓Insulindehydrogenase6-Phosphogluconate↑↓Insulindehydrogenase“Malicenzyme”↑↓InsulinATP-citratelyase↑↓Insulin1Acetyl-CoAcarboxylase↑↓Insulin?Citrate,insulinLong-chainacyl-CoA,cAMP,glucagonFattyacidsynthase↑↓Insulin?1Allosteric.2Inadiposetissuebutnotinliver.
165GLUCONEOGENESIS&CONTROLOFTHEBLOODGLUCOSE/157enzymesinglycolysis.Likewise,itantagonizestheeffectpresenceofadenylylkinaseinliverandmanyotheroftheglucocorticoidsandglucagon-stimulatedcAMP,tissuesallowsrapidequilibrationofthereaction:whichinducesynthesisofthekeyenzymesresponsibleforgluconeogenesis.BothdehydrogenasesofthepentosephosphateATPAMP+↔2ADPpathwaycanbeclassifiedasadaptiveenzymes,sincetheyincreaseinactivityinthewell-fedanimalandThus,whenATPisusedinenergy-requiringprocesseswheninsulinisgiventoadiabeticanimal.ActivityisresultinginformationofADP,[AMP]increases.Aslowindiabetesorstarvation.“Malicenzyme”and[ATP]maybe50times[AMP]atequilibrium,asmallATP-citratelyasebehavesimilarly,indicatingthatthesefractionaldecreasein[ATP]willcauseaseveralfoldin-twoenzymesareinvolvedinlipogenesisratherthancreasein[AMP].Thus,alargechangein[AMP]actsasgluconeogenesis(Chapter21).ametabolicamplifierofasmallchangein[ATP].Thismechanismallowstheactivityofphosphofructokinase-1tobehighlysensitivetoevensmallchangesinenergyCovalentModificationbyReversiblestatusofthecellandtocontrolthequantityofcarbohy-PhosphorylationIsRapiddrateundergoingglycolysispriortoitsentryintotheGlucagon,andtoalesserextentepinephrine,hor-citricacidcycle.Theincreasein[AMP]canalsoexplainmonesthatareresponsivetodecreasesinbloodglucose,whyglycolysisisincreasedduringhypoxiawhen[ATP]inhibitglycolysisandstimulategluconeogenesisinthedecreases.Simultaneously,AMPactivatesphosphory-liverbyincreasingtheconcentrationofcAMP.Thisinlase,increasingglycogenolysis.Theinhibitionofphos-turnactivatescAMP-dependentproteinkinase,leadingphofructokinase-1bycitrateandATPisanotherexpla-tothephosphorylationandinactivationofpyruvatenationofthesparingactionoffattyacidoxidationonkinase.TheyalsoaffecttheconcentrationoffructoseglucoseoxidationandalsoofthePasteureffect,2,6-bisphosphateandthereforeglycolysisandgluco-wherebyaerobicoxidation(viathecitricacidcycle)in-neogenesis,asexplainedbelow.hibitstheanaerobicdegradationofglucose.Aconse-quenceoftheinhibitionofphosphofructokinase-1isanaccumulationofglucose6-phosphatethat,inturn,in-AllostericModificationIsInstantaneoushibitsfurtheruptakeofglucoseinextrahepatictissuesbyallostericinhibitionofhexokinase.Ingluconeogenesis,pyruvatecarboxylase,whichcata-lyzesthesynthesisofoxaloacetatefrompyruvate,re-quiresacetyl-CoAasanallostericactivator.Thepres-Fructose2,6-BisphosphatePlaysaUniqueenceofacetyl-CoAresultsinachangeinthetertiaryRoleintheRegulationofGlycolysis&structureoftheprotein,loweringtheKmvalueforbi-GluconeogenesisinLivercarbonate.Thismeansthatasacetyl-CoAisformedfrompyruvate,itautomaticallyensurestheprovisionofThemostpotentpositiveallostericeffectorofphospho-oxaloacetateand,therefore,itsfurtheroxidationinthefructokinase-1andinhibitoroffructose-1,6-bisphos-citricacidcycle.Theactivationofpyruvatecarboxylasephataseinliverisfructose2,6-bisphosphate.Itre-andthereciprocalinhibitionofpyruvatedehydrogen-lievesinhibitionofphosphofructokinase-1byATPandasebyacetyl-CoAderivedfromtheoxidationoffattyincreasesaffinityforfructose6-phosphate.Itinhibitsacidsexplainstheactionoffattyacidoxidationinspar-fructose-1,6-bisphosphatasebyincreasingtheKmforingtheoxidationofpyruvateandinstimulatinggluco-fructose1,6-bisphosphate.Itsconcentrationisunderneogenesis.Thereciprocalrelationshipbetweenthesebothsubstrate(allosteric)andhormonalcontrol(cova-twoenzymesinbothliverandkidneyaltersthemeta-lentmodification)(Figure19–3).bolicfateofpyruvateasthetissuechangesfromcarbo-Fructose2,6-bisphosphateisformedbyphosphory-hydrateoxidation,viaglycolysis,togluconeogenesislationoffructose6-phosphatebyphosphofructoki-duringtransitionfromafedtoastarvedstate(Figurenase-2.Thesameenzymeproteinisalsoresponsiblefor19–1).Amajorroleoffattyacidoxidationinpromot-itsbreakdown,sinceithasfructose-2,6-bisphos-inggluconeogenesisistosupplytherequirementforphataseactivity.ThisbifunctionalenzymeisunderATP.Phosphofructokinase(phosphofructokinase-1)theallostericcontroloffructose6-phosphate,whichoccupiesakeypositioninregulatingglycolysisandisstimulatesthekinaseandinhibitsthephosphatase.alsosubjecttofeedbackcontrol.Itisinhibitedbycit-Hence,whenglucoseisabundant,theconcentrationofrateandbyATPandisactivatedby5′-AMP.5′-AMPfructose2,6-bisphosphateincreases,stimulatingglycol-actsasanindicatoroftheenergystatusofthecell.Theysisbyactivatingphosphofructokinase-1andinhibiting
166158/CHAPTER19GlycogenSubstrate(Futile)CyclesAllowFineTuningGlucoseItwillbeapparentthatthecontrolpointsinglycolysisandglycogenmetabolisminvolveacycleofphosphory-Fructose6-phosphatelationanddephosphorylationcatalyzedby:glucokinaseGlucagonandglucose-6-phosphatase;phosphofructokinase-1andfructose-1,6-bisphosphatase;pyruvatekinase,pyruvatecAMPcarboxylase,andphosphoenolypyruvatecarboxykinase;Piandglycogensynthaseandphosphorylase.IfthesewerecAMP-DEPENDENTallowedtocycleunchecked,theywouldamounttofu-PROTEINKINASEtilecycleswhosenetresultwashydrolysisofATP.Thisdoesnotoccurextensivelyduetothevariouscontrolmechanisms,whichensurethatonereactionisinhib-ADPATPitedastheotherisstimulated.However,thereisaphys-iologicadvantageinallowingsomecycling.TherateofActiveInactivenetglycolysismayincreaseseveralthousand-foldinre-F-2,6-PaseF-2,6-PasePsponsetostimulation,andthisismorereadilyachievedInactiveActivePFK-2PFK-2bybothincreasingtheactivityofphosphofructokinaseanddecreasingthatoffructosebisphosphataseifbothGLYCOLYSISareactive,thanbyswitchingoneenzyme“on”andtheH2OPiother“off”completely.This“finetuning”ofmetabolicGLUCONEOGENESIScontroloccursattheexpenseofsomelossofATP.PROTEINADPPHOSPHATASE-2CitrateTHECONCENTRATIONOFBLOODFructose2,6-bisphosphatePATPGLUCOSEISREGULATEDWITHINiNARROWLIMITSF-1,6-PasePFK-1Inthepostabsorptivestate,theconcentrationofbloodH2OADPglucoseinmostmammalsismaintainedbetween4.5and5.5mmol/L.Aftertheingestionofacarbohydratemeal,itmayriseto6.5–7.2mmol/L,andinstarvation,Fructose1,6-bisphosphateitmayfallto3.3–3.9mmol/L.Asuddendecreaseinbloodglucosewillcauseconvulsions,asininsulinover-Pyruvatedose,owingtotheimmediatedependenceofthebrainonasupplyofglucose.However,muchlowerconcen-Figure19–3.Controlofglycolysisandgluconeoge-trationscanbetolerated,providedprogressiveadapta-nesisintheliverbyfructose2,6-bisphosphateandthetionisallowed.Thebloodglucoselevelinbirdsiscon-bifunctionalenzymePFK-2/F-2,6-Pase(6-phospho-siderablyhigher(14.0mmol/L)andinruminantsfructo-2-kinase/fructose-2,6-bisphosphatase).(PFK-1,considerablylower(approximately2.2mmol/Linphosphofructokinase-1[6-phosphofructo-1-kinase];sheepand3.3mmol/Lincattle).TheselowernormalF-1,6-Pase,fructose-1,6-bisphosphatase.Arrowswithlevelsappeartobeassociatedwiththefactthatrumi-wavyshaftsindicateallostericeffects.)nantsfermentvirtuallyalldietarycarbohydratetolower(volatile)fattyacids,andtheselargelyreplaceglucoseasthemainmetabolicfuelofthetissuesinthefedcondi-fructose-1,6-bisphosphatase.Whenglucoseisshort,tion.glucagonstimulatestheproductionofcAMP,activat-ingcAMP-dependentproteinkinase,whichinturnin-activatesphosphofructokinase-2andactivatesfructoseBLOODGLUCOSEISDERIVEDFROM2,6-bisphosphatasebyphosphorylation.Therefore,glu-THEDIET,GLUCONEOGENESIS,coneogenesisisstimulatedbyadecreaseintheconcen-&GLYCOGENOLYSIStrationoffructose2,6-bisphosphate,whichdeactivatesphosphofructokinase-1anddeinhibitsfructose-1,6-bis-Thedigestibledietarycarbohydratesyieldglucose,phosphatase.Thismechanismalsoensuresthatglu-galactose,andfructosethataretransportedviathecagonstimulationofglycogenolysisinliverresultsinhepaticportalveintotheliverwheregalactoseandglucosereleaseratherthanglycolysis.fructosearereadilyconvertedtoglucose(Chapter20).
167GLUCONEOGENESIS&CONTROLOFTHEBLOODGLUCOSE/159GlucoseisformedfromtwogroupsofcompoundsMetabolic&HormonalMechanismsthatundergogluconeogenesis(Figures16–4and19–1):RegulatetheConcentration(1)thosewhichinvolveadirectnetconversiontoglu-oftheBloodGlucosecosewithoutsignificantrecycling,suchassomeaminoacidsandpropionate;and(2)thosewhicharetheThemaintenanceofstablelevelsofglucoseinthebloodproductsofthemetabolismofglucoseintissues.Thus,isoneofthemostfinelyregulatedofallhomeostaticlactate,formedbyglycolysisinskeletalmuscleandmechanisms,involvingtheliver,extrahepatictissues,erythrocytes,istransportedtotheliverandkidneyandseveralhormones.Livercellsarefreelypermeablewhereitre-formsglucose,whichagainbecomesavail-toglucose(viatheGLUT2transporter),whereascellsableviathecirculationforoxidationinthetissues.Thisofextrahepatictissues(apartfrompancreaticBislets)processisknownastheCoricycle,orlacticacidcyclearerelativelyimpermeable,andtheirglucosetrans-(Figure19–4).Triacylglycerolglycerolinadiposetissueportersareregulatedbyinsulin.Asaresult,uptakeisderivedfrombloodglucose.Thistriacylglycerolisfromthebloodstreamistherate-limitingstepinthecontinuouslyundergoinghydrolysistoformfreeglyc-utilizationofglucoseinextrahepatictissues.Theroleoferol,whichcannotbeutilizedbyadiposetissueandisvariousglucosetransporterproteinsfoundincellmem-convertedbacktoglucosebygluconeogenicmecha-branes,eachhaving12transmembranedomains,isnismsintheliverandkidney(Figure19–1).showninTable19–2.Oftheaminoacidstransportedfrommuscletotheliverduringstarvation,alaninepredominates.Theglu-GlucokinaseIsImportantinRegulatingcose-alaninecycle(Figure19–4)transportsglucoseBloodGlucoseAfteraMealfromlivertomusclewithformationofpyruvate,fol-lowedbytransaminationtoalanine,thentransportsHexokinasehasalowKmforglucoseandintheliverisalaninetotheliver,followedbygluconeogenesisbacksaturatedandactingataconstantrateunderallnormaltoglucose.Anettransferofaminonitrogenfrommus-conditions.GlucokinasehasaconsiderablyhigherKmcletoliverandoffreeenergyfromlivertomuscleisef-(loweraffinity)forglucose,sothatitsactivityincreasesfected.Theenergyrequiredforthehepaticsynthesisofoverthephysiologicrangeofglucoseconcentrationsglucosefrompyruvateisderivedfromtheoxidationof(Figure19–5).Itpromoteshepaticuptakeoflargefattyacids.amountsofglucoseatthehighconcentrationsfoundinGlucoseisalsoformedfromliverglycogenbythehepaticportalveinafteracarbohydratemeal.Itisglycogenolysis(Chapter18).absentfromtheliverofruminants,whichhavelittleBLOODGlucoseLIVERMUSCLEGlucose6-phosphateGlycogenGlycogenGlucose6-phosphateUreaPyruvateLactateLactatePyruvateTransaminationn–NH2–NH2natioLactateamisBLOODanrTPyruvateAlanineAlanineAlanineFigure19–4.Thelacticacid(Cori)cycleandglucose-alaninecycle.
168160/CHAPTER19Table19–2.Glucosetransporters.TissueLocationFunctionsFacilitativebidirectionaltransportersGLUT1Brain,kidney,colon,placenta,erythrocyteUptakeofglucoseGLUT2Liver,pancreaticBcell,smallintestine,kidneyRapiduptakeandreleaseofglucoseGLUT3Brain,kidney,placentaUptakeofglucoseGLUT4Heartandskeletalmuscle,adiposetissueInsulin-stimulateduptakeofglucoseGLUT5SmallintestineAbsorptionofglucoseSodium-dependentunidirectionaltransporterSGLT1SmallintestineandkidneyActiveuptakeofglucosefromlumenofintestineandreabsorptionofglucoseinproximaltubuleofkidneyagainstaconcentrationgradientglucoseenteringtheportalcirculationfromtheintes-coseviatheGLUT2transporter,andtheglucoseistines.phosphorylatedbyglucokinase.Therefore,increasingAtnormalsystemic-bloodglucoseconcentrationsbloodglucoseincreasesmetabolicfluxthroughglycoly-(4.5–5.5mmol/L),theliverisanetproducerofglu-sis,thecitricacidcycle,andthegenerationofATP.In-creasein[ATP]inhibitsATP-sensitiveK+channels,cose.However,astheglucoselevelrises,theoutputofglucoseceases,andthereisanetuptake.causingdepolarizationoftheBcellmembrane,which2+2+increasesCainfluxviavoltage-sensitiveCachannels,InsulinPlaysaCentralRoleinstimulatingexocytosisofinsulin.Thus,theconcentra-RegulatingBloodGlucosetionofinsulininthebloodparallelsthatofthebloodglucose.OthersubstancescausingreleaseofinsulinfromInadditiontothedirecteffectsofhyperglycemiainen-thepancreasincludeaminoacids,freefattyacids,ketonehancingtheuptakeofglucoseintotheliver,thehor-bodies,glucagon,secretin,andthesulfonylureadrugsmoneinsulinplaysacentralroleinregulatingbloodglu-tolbutamideandglyburide.Thesedrugsareusedtocose.ItisproducedbytheBcellsoftheisletsofstimulateinsulinsecretionintype2diabetesmellitusLangerhansinthepancreasinresponsetohyper-(NIDDM,non-insulin-dependentdiabetesmellitus);glycemia.TheBisletcellsarefreelypermeabletoglu-theyactbyinhibitingtheATP-sensitiveK+channels.Epinephrineandnorepinephrineblockthereleaseofin-sulin.Insulinlowersbloodglucoseimmediatelybyen-Vmax100hancingglucosetransportintoadiposetissueandmuscleHexokinasebyrecruitmentofglucosetransporters(GLUT4)fromtheinteriorofthecelltotheplasmamembrane.Al-thoughitdoesnotaffectglucoseuptakeintotheliverdirectly,insulindoesenhancelong-termuptakeasare-sultofitsactionsontheenzymescontrollingglycolysis,50Glucokinaseglycogenesis,andgluconeogenesis(Chapter18).ActivityGlucagonOpposestheActionsofInsulinGlucagonisthehormoneproducedbytheAcellsof0510152025thepancreaticislets.Itssecretionisstimulatedbyhypo-Bloodglucose(mmol/L)glycemia.Intheliver,itstimulatesglycogenolysisbyac-tivatingphosphorylase.Unlikeepinephrine,glucagonFigure19–5.Variationinglucosephosphorylatingdoesnothaveaneffectonmusclephosphorylase.activityofhexokinaseandglucokinasewithincreaseofGlucagonalsoenhancesgluconeogenesisfromaminobloodglucoseconcentration.TheKmforglucoseofacidsandlactate.Inalltheseactions,glucagonactsviahexokinaseis0.05mmol/Landofglucokinaseis10generationofcAMP(Table19–1).Bothhepaticmmol/L.glycogenolysisandgluconeogenesiscontributetothe
169GLUCONEOGENESIS&CONTROLOFTHEBLOODGLUCOSE/161hyperglycemiceffectofglucagon,whoseactionsop-mealsoratnight.Furthermore,prematureandlow-posethoseofinsulin.Mostoftheendogenousglucagonbirth-weightbabiesaremoresusceptibletohypo-(andinsulin)isclearedfromthecirculationbytheliver.glycemia,sincetheyhavelittleadiposetissuetogener-atealternativefuelssuchasfreefattyacidsorketonebodiesduringthetransitionfromfetaldependencytoOtherHormonesAffectBloodGlucosethefree-livingstate.TheenzymesofgluconeogenesisTheanteriorpituitaryglandsecreteshormonesthatmaynotbecompletelyfunctionalatthistime,andthetendtoelevatethebloodglucoseandthereforeantago-processisdependentonasupplyoffreefattyacidsfornizetheactionofinsulin.Thesearegrowthhormone,energy.Glycerol,whichwouldnormallybereleasedACTH(corticotropin),andpossiblyother“diabeto-fromadiposetissue,islessavailableforgluconeogenesis.genic”hormones.Growthhormonesecretionisstimu-latedbyhypoglycemia;itdecreasesglucoseuptakeinTheBody’sAbilitytoUtilizeGlucosemuscle.Someofthiseffectmaynotbedirect,sinceitMayBeAscertainedbyMeasuringItsstimulatesmobilizationoffreefattyacidsfromadiposeGlucoseTolerancetissue,whichthemselvesinhibitglucoseutilization.Theglucocorticoids(11-oxysteroids)aresecretedbytheGlucosetoleranceistheabilitytoregulatethebloodadrenalcortexandincreasegluconeogenesis.Thisisaglucoseconcentrationaftertheadministrationofatestresultofenhancedhepaticuptakeofaminoacidsanddoseofglucose(normally1g/kgbodyweight)(Figureincreasedactivityofaminotransferasesandkeyenzymes19–6).Diabetesmellitus(type1,orinsulin-dependentofgluconeogenesis.Inaddition,glucocorticoidsinhibitdiabetesmellitus;IDDM)ischaracterizedbydecreasedtheutilizationofglucoseinextrahepatictissues.Inallglucosetoleranceduetodecreasedsecretionofinsulintheseactions,glucocorticoidsactinamannerantago-inresponsetotheglucosechallenge.Glucosetolerancenistictoinsulin.isalsoimpairedintype2diabetesmellitus(NIDDM),Epinephrineissecretedbytheadrenalmedullaasawhichisoftenassociatedwithobesityandraisedlevelsresultofstressfulstimuli(fear,excitement,hemorrhage,ofplasmafreefattyacidsandinconditionswherethehypoxia,hypoglycemia,etc)andleadstoglycogenolysisliverisdamaged;insomeinfections;andinresponsetoinliverandmuscleowingtostimulationofphosphory-somedrugs.PoorglucosetolerancecanalsobeexpectedlaseviagenerationofcAMP.Inmuscle,glycogenolysisresultsinincreasedglycolysis,whereasinliverglucoseisthemainproductleadingtoincreaseinbloodglucose.15DiabeticFURTHERCLINICALASPECTSGlucosuriaOccursWhentheRenalThresholdforGlucoseIsExceeded10Whenthebloodglucoserisestorelativelyhighlevels,thekidneyalsoexertsaregulatoryeffect.Glucoseiscontinuouslyfilteredbytheglomerulibutisnormallycompletelyreabsorbedintherenaltubulesbyactivetransport.Thecapacityofthetubularsystemtoreab-Normal5sorbglucoseislimitedtoarateofabout350mg/min,Bloodglucose(mmol/L)andinhyperglycemia(asoccursinpoorlycontrolleddi-abetesmellitus)theglomerularfiltratemaycontainmoreglucosethancanbereabsorbed,resultinginglu-cosuria.Glucosuriaoccurswhenthevenousbloodglu-coseconcentrationexceeds9.5–10.0mmol/L;thisistermedtherenalthresholdforglucose.012Time(h)HypoglycemiaMayOccurDuringFigure19–6.Glucosetolerancetest.BloodglucosePregnancy&intheNeonatecurvesofanormalandadiabeticindividualafteroralDuringpregnancy,fetalglucoseconsumptionincreasesadministrationof50gofglucose.Notetheinitialraisedandthereisariskofmaternalandpossiblyfetalhypo-concentrationinthediabetic.Acriterionofnormalityisglycemia,particularlyiftherearelongintervalsbetweenthereturnofthecurvetotheinitialvaluewithin2hours.
170162/CHAPTER19duetohyperactivityofthepituitaryoradrenalcortex•Insulinissecretedasadirectresponsetohyper-becauseoftheantagonismofthehormonessecretedbyglycemia;itstimulatesthelivertostoreglucoseastheseglandstotheactionofinsulin.glycogenandfacilitatesuptakeofglucoseintoextra-Administrationofinsulin(asinthetreatmentofdi-hepatictissues.abetesmellitustype1)lowersthebloodglucoseandin-•Glucagonissecretedasaresponsetohypoglycemiacreasesitsutilizationandstorageintheliverandmuscleandactivatesbothglycogenolysisandgluconeogene-asglycogen.Anexcessofinsulinmaycausehypo-sisintheliver,causingreleaseofglucoseintotheglycemia,resultinginconvulsionsandevenindeathblood.unlessglucoseisadministeredpromptly.Increasedtol-erancetoglucoseisobservedinpituitaryoradrenocor-ticalinsufficiency—attributabletoadecreaseinthean-tagonismtoinsulinbythehormonesnormallysecretedREFERENCESbytheseglands.BurantCFetal:Mammalianglucosetransporters:structureandmolecularregulation.RecentProgHormRes1991;47:349.SUMMARYKrebsHA:Gluconeogenesis.ProcRSocLondon(Biol)1964;159:545.•Gluconeogenesisistheprocessofconvertingnoncar-LenzenS:HexoserecognitionmechanismsinpancreaticB-cells.bohydratestoglucoseorglycogen.ItisofparticularBiochemSocTrans1990;18:105.importancewhencarbohydrateisnotavailablefromNewgardCB,McGarryJD:Metaboliccouplingfactorsinpancre-thediet.Significantsubstratesareaminoacids,lac-aticbeta-cellsignaltransduction.AnnuRevBiochem1995;tate,glycerol,andpropionate.64:689.•Thepathwayofgluconeogenesisintheliverandkid-NewsholmeEA,StartC:RegulationinMetabolism.Wiley,1973.neyutilizesthosereactionsinglycolysiswhicharere-NordlieRC,FosterJD,LangeAJ:Regulationofglucoseproduc-versibleplusfouradditionalreactionsthatcircum-tionbytheliver.AnnuRevNutr1999;19:379.venttheirreversiblenonequilibriumreactions.PilkisSJ,El-MaghrabiMR,ClausTH:Hormonalregulationofhe-•Sinceglycolysisandgluconeogenesissharethesamepaticgluconeogenesisandglycolysis.AnnuRevBiochem1988;57:755.pathwaybutoperateinoppositedirections,theirac-PilkisSJ,GrannerDK:Molecularphysiologyoftheregulationoftivitiesareregulatedreciprocally.hepaticgluconeogenesisandglycolysis.AnnuRevPhysiol•Theliverregulatesthebloodglucoseafteramealbe-1992;54:885.causeitcontainsthehigh-Kmglucokinasethatpro-Yki-JarvinenH:Actionofinsulinonglucosemetabolisminvivo.motesincreasedhepaticutilizationofglucose.BaillieresClinEndocrinolMetab1993;7:903.
171ThePentosePhosphatePathway&OtherPathways20ofHexoseMetabolismPeterA.Mayes,PhD,DSc,&DavidA.Bender,PhDBIOMEDICALIMPORTANCEREACTIONSOFTHEPENTOSEThepentosephosphatepathwayisanalternativeroutePHOSPHATEPATHWAYOCCURforthemetabolismofglucose.ItdoesnotgenerateINTHECYTOSOLATPbuthastwomajorfunctions:(1)TheformationofTheenzymesofthepentosephosphatepathway,asofNADPHforsynthesisoffattyacidsandsteroidsandglycolysis,arecytosolic.Asinglycolysis,oxidation(2)thesynthesisofribosefornucleotideandnucleicisachievedbydehydrogenation;butNADP+andnotacidformation.Glucose,fructose,andgalactosearethe+NADisthehydrogenacceptor.Thesequenceofreac-mainhexosesabsorbedfromthegastrointestinaltract,tionsofthepathwaymaybedividedintotwophases:anderivedprincipallyfromdietarystarch,sucrose,andoxidativenonreversiblephaseandanonoxidativere-lactose,respectively.Fructoseandgalactosearecon-versiblephase.Inthefirstphase,glucose6-phosphatevertedtoglucose,mainlyintheliver.undergoesdehydrogenationanddecarboxylationtoyieldGeneticdeficiencyofglucose6-phosphatedehydro-apentose,ribulose5-phosphate.Inthesecondphase,genase,thefirstenzymeofthepentosephosphatepath-ribulose5-phosphateisconvertedbacktoglucose6-phos-way,isamajorcauseofhemolysisofredbloodcells,re-phatebyaseriesofreactionsinvolvingmainlytwoen-sultinginhemolyticanemiaandaffectingapproximatelyzymes:transketolaseandtransaldolase(Figure20–1).100millionpeopleworldwide.Glucuronicacidissynthe-sizedfromglucoseviatheuronicacidpathway,ofmajorTheOxidativePhaseGeneratesNADPHsignificancefortheexcretionofmetabolitesandforeignchemicals(xenobiotics)asglucuronides.Adeficiencyin(Figures20–1and20–2)thepathwayleadstoessentialpentosuria.ThelackofDehydrogenationofglucose6-phosphateto6-phos-oneenzymeofthepathway(gulonolactoneoxidase)inphogluconateoccursviatheformationof6-phospho-primatesandsomeotheranimalsexplainswhyascorbicgluconolactone,catalyzedbyglucose-6-phosphateacid(vitaminC)isadietaryrequirementforhumansbutdehydrogenase,anNADP-dependentenzyme.Thenotmostothermammals.Deficienciesintheenzymesofhydrolysisof6-phosphogluconolactoneisaccomplishedfructoseandgalactosemetabolismleadtoessentialfruc-bytheenzymegluconolactonehydrolase.Asecondtosuriaandthegalactosemias.oxidativestepiscatalyzedby6-phosphogluconatede-hydrogenase,whichalsorequiresNADP+ashydrogenTHEPENTOSEPHOSPHATEPATHWAYacceptorandinvolvesdecarboxylationfollowedbyfor-GENERATESNADPH&RIBOSEmationoftheketopentose,ribulose5-phosphate.PHOSPHATE(Figure20–1)TheNonoxidativePhaseGeneratesThepentosephosphatepathway(hexosemonophos-RibosePrecursorsphateshunt)isamorecomplexpathwaythanglycoly-sis.Threemoleculesofglucose6-phosphategiverisetoRibulose5-phosphateisthesubstratefortwoenzymes.threemoleculesofCO2andthreefive-carbonsugars.Ribulose5-phosphate3-epimerasealterstheconfigu-Thesearerearrangedtoregeneratetwomoleculesofrationaboutcarbon3,forminganotherketopentose,glucose6-phosphateandonemoleculeoftheglycolyticxylulose5-phosphate.Ribose5-phosphateketoisom-intermediate,glyceraldehyde3-phosphate.Sincetwoeraseconvertsribulose5-phosphatetothecorrespond-moleculesofglyceraldehyde3-phosphatecanregenerateingaldopentose,ribose5-phosphate,whichisthepre-glucose6-phosphate,thepathwaycanaccountforthecursoroftheriboserequiredfornucleotideandnucleiccompleteoxidationofglucose.acidsynthesis.Transketolasetransfersthetwo-carbon163
172164/CHAPTER20Glucose6-phosphateGlucose6-phosphateGlucose6-phosphateC6+C6+C6+NADP+H2ONADP+H2ONADP+H2OGLUCOSE-6-PHOSPHATEDEHYDROGENASE+++NADPH+HNADPH+HNADPH+H6-Phosphogluconate6-Phosphogluconate6-PhosphogluconateC6+C6+C6+NADPNADPNADP6-PHOSPHO-GLUCONATEDEHYDROGENASE+++NADPH+HNADPH+HNADPH+HCO2CO2CO2Ribulose5-phosphateRibulose5-phosphateRibulose5-phosphateC5C5C53-EPIMERASEKETO-ISOMERASE3-EPIMERASEXylulose5-phosphateRibose5-phosphateXylulose5-phosphateC5C5C5TRANSKETOLASESynthesisofnucleotides,RNA,DNAGlyceraldehyde3-phosphateSedoheptulose7-phosphateC3C7TRANSALDOLASEFructose6-phosphateErythrose4-phosphateC6C4TRANSKETOLASEFructose6-phosphateGlyceraldehyde3-phosphateC6C3PHOSPHOTRIOSEALDOLASEISOMERASEPHOSPHOHEXOSEPHOSPHOHEXOSE1/2Fructose1,6-bisphosphateISOMERASEISOMERASEC6FRUCTOSE-1,6-BISPHOSPHATASE1/2Fructose6-phosphateC6PHOSPHOHEXOSEISOMERASEGlucose6-phosphateGlucose6-phosphate1/2Glucose6-phosphateC6C6C6Figure20–1.Flowchartofpentosephosphatepathwayanditsconnectionswiththepathwayofglycolysis.Thefullpathway,asindicated,consistsofthreeinterconnectedcyclesinwhichglu-cose6-phosphateisbothsubstrateandendproduct.Thereactionsabovethebrokenlinearenonreversible,whereasallreactionsunderthatlinearefreelyreversibleapartfromthatcatalyzedbyfructose-1,6-bisphosphatase.
173THEPENTOSEPHOSPHATEPATHWAY&OTHERPATHWAYSOFHEXOSEMETABOLISM/165O–HOCHNADP+NADPH+H+CHOCOO2Mg2+Mg2+,Mn2+,HOCHHOCHHOCHorCa2+orCa2+HOCHHOCHHOCHOOHOCHGLUCOSE-6-PHOSPHATEHOCHGLUCONOLACTONEHOCHDEHYDROGENASEHYDROLASEHCHCHCOHCH2OPCH2OPCH2OPβ-D-Glucose6-phosphate6-Phosphogluconolactone6-PhosphogluconateNADP+6-PHOSPHOGLUCONATEMg2+,Mn2+,DEHYDROGENASEorCa2+NADP++H+–COOCHOHCHOHHOCH2RIBOSE5-PHOSPHATECOHKETOISOMERASECOCOHOCHHCOHHOCHHCOHHCOHHCOHCOCHOPCHOP2CHOP222EnediolformRibulose5-phosphate3-Keto6-phosphogluconateRIBULOSE5-PHOSPHATE3-EPIMERASECHOHCHOH22COCOHOCHHO*CHHOCHHOCHH*COHHOCHHOCHO*CHCOPHOCH2HCHCOHXylulose5-phosphateCH2OPCH2OPRibose5-phosphateSedoheptulose7-phosphateATPTRANSKETOLASEMg2+PRPPSYNTHETASEThiamin–PHO*C2Mg2+AMPHO*CHCHOH2HCOPP*CHC2OPCOHOCHGlyceraldehyde3-phosphateHOCHHOCHOTRANSALDOLASEHO*CHHCHOCHO*CHCH2OPHOCH*CHC2OPPRPPHOCHFructose6-phosphateCHOP2Erythrose4-phosphateCHOH2CHOHCO2COTRANSKETOLASEHOCHHOCHThiamin–P2HOCHOCHMg2+HOCHHOCHHOCHCH2OPCH2OPCH2OPXylulose5-phosphateGlyceraldehyde3-phosphateFructose6-phosphate2–Figure20–2.Thepentosephosphatepathway.(P,⎯PO3;PRPP,5-phosphoribosyl1-pyrophosphate.)
174166/CHAPTER20unitcomprisingcarbons1and2ofaketoseontotheRiboseCanBeSynthesizedinVirtuallyaldehydecarbonofanaldosesugar.ItthereforeeffectsAllTissuestheconversionofaketosesugarintoanaldosewithtwocarbonslessandsimultaneouslyconvertsanaldosesugarLittleornoribosecirculatesinthebloodstream,sotis-intoaketosewithtwocarbonsmore.Thereactionre-suesmustsynthesizetheriboserequiredfornucleotidequiresMg2+andthiamindiphosphate(vitaminB)asandnucleicacidsynthesis(Chapter34).Thesourceof1coenzyme.Thus,transketolasecatalyzesthetransferofribose5-phosphateisthepentosephosphatepathwaythetwo-carbonunitfromxylulose5-phosphatetoribose(Figure20–2).Musclehasonlylowactivityofglucose-5-phosphate,producingtheseven-carbonketosesedo-6-phosphatedehydrogenaseand6-phosphogluconateheptulose7-phosphateandthealdoseglyceraldehydedehydrogenase.Nevertheless,likemostothertissues,it3-phosphate.Transaldolaseallowsthetransferofaiscapableofsynthesizingribose5-phosphatebyreversalthree-carbondihydroxyacetonemoiety(carbons1–3)ofthenonoxidativephaseofthepentosephosphatefromtheketosesedoheptulose7-phosphateontotheal-pathwayutilizingfructose6-phosphate.Itisnotneces-doseglyceraldehyde3-phosphatetoformtheketosesarytohaveacompletelyfunctioningpentosephosphatefructose6-phosphateandthefour-carbonaldoseerythrosepathwayforatissuetosynthesizeribosephosphates.4-phosphate.Inafurtherreactioncatalyzedbytranske-tolase,xylulose5-phosphatedonatesatwo-carbonunitTHEPENTOSEPHOSPHATEPATHWAYtoerythrose4-phosphatetoformfructose6-phosphate&GLUTATHIONEPEROXIDASEPROTECTandglyceraldehyde3-phosphate.ERYTHROCYTESAGAINSTHEMOLYSISInordertooxidizeglucosecompletelytoCO2viathepentosephosphatepathway,theremustbeenzymesInerythrocytes,thepentosephosphatepathwaypro-presentinthetissuetoconvertglyceraldehyde3-phos-videsNADPHforthereductionofoxidizedglu-phatetoglucose6-phosphate.Thisinvolvesreversaloftathionecatalyzedbyglutathionereductase,aflavo-glycolysisandthegluconeogenicenzymefructose1,6-proteincontainingFAD.Reducedglutathioneremovesbisphosphatase.Intissuesthatlackthisenzyme,glyc-H2O2inareactioncatalyzedbyglutathioneperoxi-eraldehyde3-phosphatefollowsthenormalpathwayofdase,anenzymethatcontainstheseleniumanalogueglycolysistopyruvate.ofcysteine(selenocysteine)attheactivesite(Figure20–3).Thisreactionisimportant,sinceaccumulationTheTwoMajorPathwaysfortheofH2O2maydecreasethelifespanoftheerythrocytebycausingoxidativedamagetothecellmembrane,CatabolismofGlucoseHaveleadingtohemolysis.LittleinCommonAlthoughglucose6-phosphateiscommontobothGLUCURONATE,APRECURSOROFpathways,thepentosephosphatepathwayismarkedlyPROTEOGLYCANS&CONJUGATEDdifferentfromglycolysis.OxidationutilizesNADPGLUCURONIDES,ISAPRODUCTOFratherthanNAD,andCO2,whichisnotproducedinTHEURONICACIDPATHWAYglycolysis,isacharacteristicproduct.NoATPisgener-atedinthepentosephosphatepathway,whereasATPisInliver,theuronicacidpathwaycatalyzestheconver-amajorproductofglycolysis.sionofglucosetoglucuronicacid,ascorbicacid,andpentoses(Figure20–4).ItisalsoanalternativeoxidativeReducingEquivalentsAreGeneratedpathwayforglucose,but—likethepentosephosphateinThoseTissuesSpecializingpathway—itdoesnotleadtothegenerationofATP.Glucose6-phosphateisisomerizedtoglucose1-phos-inReductiveSynthesesphate,whichthenreactswithuridinetriphosphateThepentosephosphatepathwayisactiveinliver,adipose(UTP)toformuridinediphosphateglucose(UDPGlc)tissue,adrenalcortex,thyroid,erythrocytes,testis,andinareactioncatalyzedbyUDPGlcpyrophosphorylase,lactatingmammarygland.Itsactivityislowinnonlactat-asoccursinglycogensynthesis(Chapter18).UDPGlcisingmammaryglandandskeletalmuscle.Thosetissuesinoxidizedatcarbon6byNAD-dependentUDPGlcde-whichthepathwayisactiveuseNADPHinreductivehydrogenaseinatwo-stepreactiontoyieldUDP-glu-syntheses,eg,offattyacids,steroids,aminoacidsviaglu-curonate.UDP-glucuronateisthe“active”formofglu-tamatedehydrogenase,andreducedglutathione.Thecuronateforreactionsinvolvingincorporationofsynthesisofglucose-6-phosphatedehydrogenaseandglucuronicacidintoproteoglycansorforreactionsin6-phosphogluconatedehydrogenasemayalsobeinducedwhichsubstratessuchassteroidhormones,bilirubin,andbyinsulinduringconditionsassociatedwiththe“fedanumberofdrugsareconjugatedwithglucuronateforstate”(Table19–1),whenlipogenesisincreases.excretioninurineorbile(Figure32–14).
175THEPENTOSEPHOSPHATEPATHWAY&OTHERPATHWAYSOFHEXOSEMETABOLISM/167NADPH+H+2HOGGSS2PENTOSEGLUTATHIONEGLUTATHIONEPHOSPHATEFADSeREDUCTASEPEROXIDASEPATHWAYNADP+2GSHHO2H22Figure20–3.Roleofthepentosephosphatepathwayintheglutathioneperoxidasere-actionoferythrocytes.(G-S-S-G,oxidizedglutathione;G-SH,reducedglutathione;Se,sele-niumcofactor.)GlucuronateisreducedtoL-gulonateinanNADPH-tose.However,glucoseinhibitsthephosphorylationofdependentreaction;L-gulonateisthedirectprecursoroffructosesinceitisabettersubstrateforhexokinase.ascorbateinthoseanimalscapableofsynthesizingthisNevertheless,somefructosecanbemetabolizedinadi-vitamin.Inhumansandotherprimatesaswellasguineaposetissueandmuscle.Fructose,apotentialfuel,ispigs,ascorbicacidcannotbesynthesizedbecauseofthefoundinseminalplasmaandinthefetalcirculationofabsenceofL-gulonolactoneoxidase.L-Gulonateisme-ungulatesandwhales.AldosereductaseisfoundinthetabolizedultimatelytoD-xylulose5-phosphate,acon-placentaoftheeweandisresponsibleforthesecretionstituentofthepentosephosphatepathway.ofsorbitolintothefetalblood.Thepresenceofsor-bitoldehydrogenaseintheliver,includingthefetalINGESTIONOFLARGEQUANTITIESliver,isresponsiblefortheconversionofsorbitolintoOFFRUCTOSEHASPROFOUNDfructose.Thispathwayisalsoresponsiblefortheoccur-renceoffructoseinseminalfluid.METABOLICCONSEQUENCESDietshighinsucroseorinhigh-fructosesyrupsusedinmanufacturedfoodsandbeveragesleadtolargeamountsGALACTOSEISNEEDEDFORTHEoffructose(andglucose)enteringthehepaticportalvein.SYNTHESISOFLACTOSE,GLYCOLIPIDS,FructoseundergoesmorerapidglycolysisintheliverthanPROTEOGLYCANS,&GLYCOPROTEINSdoesglucosebecauseitbypassestheregulatorystepcat-alyzedbyphosphofructokinase(Figure20–5).ThisallowsGalactoseisderivedfromintestinalhydrolysisofthefructosetofloodthepathwaysintheliver,leadingtoen-disaccharidelactose,thesugarofmilk.Itisreadilycon-hancedfattyacidsynthesis,increasedesterificationoffattyvertedinthelivertoglucose.Galactokinasecatalyzesacids,andincreasedVLDLsecretion,whichmayraisethephosphorylationofgalactose,usingATPasphos-serumtriacylglycerolsandultimatelyraiseLDLcholes-phatedonor(Figure20–6A).Galactose1-phosphatere-terolconcentrations(Figure25–6).Aspecifickinase,actswithuridinediphosphateglucose(UDPGlc)tofructokinase,inliver(andkidneyandintestine)catalyzesformuridinediphosphategalactose(UDPGal)andglu-thephosphorylationoffructosetofructose1-phosphate.cose1-phosphate,inareactioncatalyzedbygalactoseThisenzymedoesnotactonglucose,and,unlikeglucoki-1-phosphateuridyltransferase.Theconversionofnase,itsactivityisnotaffectedbyfastingorbyinsulin,UDPGaltoUDPGlciscatalyzedbyUDPGal4-epim-whichmayexplainwhyfructoseisclearedfromtheblooderase.Epimerizationinvolvesanoxidationandreduc-ofdiabeticpatientsatanormalrate.Fructose1-phos-tionatcarbon4withNAD+ascoenzyme.Finally,glu-phateiscleavedtoD-glyceraldehydeanddihydroxyace-coseisliberatedfromUDPGlcafterconversiontotonephosphatebyaldolaseB,anenzymefoundintheglucose1-phosphate,probablyviaincorporationintoliver,whichalsofunctionsinglycolysisbycleavingfruc-glycogenfollowedbyphosphorolysis(Chapter18).tose1,6-bisphosphate.D-GlyceraldehydeentersglycolysisSincetheepimerasereactionisfreelyreversible,glu-viaphosphorylationtoglyceraldehyde3-phosphate,cat-cosecanbeconvertedtogalactose,sothatgalactoseisalyzedbytriokinase.Thetwotriosephosphates,dihy-notadietaryessential.Galactoseisrequiredinthebodydroxyacetonephosphateandglyceraldehyde3-phosphate,notonlyintheformationoflactosebutalsoasacon-maybedegradedbyglycolysisormaybesubstratesforal-stituentofglycolipids(cerebrosides),proteoglycans,dolaseandhencegluconeogenesis,whichisthefateofandglycoproteins.Inthesynthesisoflactoseinthemuchofthefructosemetabolizedintheliver.mammarygland,UDPGalcondenseswithglucosetoInextrahepatictissues,hexokinasecatalyzestheyieldlactose,catalyzedbylactosesynthase(Figurephosphorylationofmosthexosesugars,includingfruc-20–6B).
176168/CHAPTER20H*COHH*COPH*COUDPH*COUDPPHOSPHO-UDPGlcPYRO-UDPGlcHOCHGLUCOMUTASEHOCHPHOSPHORYLASEHOCHDEHYDROGENASEHOCHHOCHHOCHHOCHHOCHOOOOHOCHHOCHHOCHHOCHUTPPP2NAD+2NADHHCHCiHC+HC+HO+2H2CO–CH2OPCH2OHCH2OHOα-D-GlucoseGlucoseUridinediphosphateUridinediphosphate6-phosphate1-phosphateglucose(UDPGlc)glucuronateGlucuronidesH2OProteoglycansUDPOOO–O–CCH*COHNADHNADPHCH2OHCOHOCH+H+NAD+HOCHNADP++H+HOCH2COCOHOCHHOCHOHOCHHOCHHOCHHOCHHOCHHOCHHOCHHCO–*CH2OH*CH2OH*CH2OHCOL-Xylulose3-Keto-L-gulonateL-GulonateD-GlucuronateNADPH+H+OxalateH2OGlycolateL-GulonolactoneCO2O2NADP+BLOCKINPRIMATESANDGUINEAPIGSBLOCKINHUMANSGlycolaldehyde2-Keto-L-gulonolactoneBLOCKINPENTOSURIAD-Xylulose1-phosphate*CH2OH*CH2OHOONADHHOCHNAD++H+COC[2H]CHOCHHOCHD-XyluloseHOCOCOOHOCHHOCHHOCOCCH2OHD-XYLULOSECH2OHHCHCREDUCTASEOxalateXylitolATPHOCHHOCHDiet2+Mg*CH2OH*CH2OHADPL-AscorbateL-DehydroascorbateD-Xylulose5-phosphatePentosephosphatepathway2–Figure20–4.Uronicacidpathway.(Asteriskindicatesthefateofcarbon1ofglucose;P,⎯PO3.)
177THEPENTOSEPHOSPHATEPATHWAY&OTHERPATHWAYSOFHEXOSEMETABOLISM/169ATPGlycogenHEXOKINASEGLUCOKINASEALDOSE*REDUCTASEGlucose6-phosphateD-GlucoseD-Sorbitol+NADPHNADP+NADGLUCOSE-6-PHOSPHATASE+H+PHOSPHOHEXOSEISOMERASESORBITOLDEHYDROGENASENADH+H+HEXOKINASEFructose6-phosphateD-FructoseDietATPFRUCTOSE-1,6-ATPFRUCTOKINASEATPPHOSPHOFRUCTOKINASEBISPHOSPHATASEBLOCKINESSENTIALFRUCTOSURIAFructose1,6-bisphosphateFructose1-phosphateBLOCKINHEREDITARYFRUCTOSEINTOLERANCEALDOLASEBDihydroxyacetone-phosphateALDOLASEAPHOSPHO-FattyacidALDOLASEBTRIOSEesterificationISOMERASEATPGlyceraldehyde3-phosphateD-GlyceraldehydeTRIOKINASE2-PhosphoglyceratePyruvateFattyacidsynthesisFigure20–5.Metabolismoffructose.AldolaseAisfoundinalltissues,whereasaldolaseBisthepredominantforminliver.(*,notfoundinliver.)GlucoseIsthePrecursorofAllCLINICALASPECTSAminoSugars(Hexosamines)ImpairmentofthePentosePhosphateAminosugarsareimportantcomponentsofglycopro-PathwayLeadstoErythrocyteHemolysisteins(Chapter47),ofcertainglycosphingolipids(eg,gangliosides)(Chapter14),andofglycosaminoglycansGeneticdeficiencyofglucose-6-phosphatedehydrogen-(Chapter48).Themajoraminosugarsareglucosa-ase,withconsequentimpairmentofthegenerationofmine,galactosamine,andmannosamineandtheNADPH,iscommoninpopulationsofMediterraneannine-carboncompoundsialicacid.TheprincipalsialicandAfro-Caribbeanorigin.ThedefectismanifestedasacidfoundinhumantissuesisN-acetylneuraminicacidredcellhemolysis(hemolyticanemia)whensuscepti-(NeuAc).Asummaryofthemetabolicinterrelationshipsbleindividualsaresubjectedtooxidants,suchasthean-amongtheaminosugarsisshowninFigure20–7.timalarialprimaquine,aspirin,orsulfonamidesorwhen
178170/CHAPTER20AGalactoseGlycogenGLYCOGENSYNTHASEATPPiPHOSPHORYLASEMg2+GALACTOKINASEGlucose1-phosphateADPBLOCKINGalactoseGALACTOSEMIAPHOSPHOGLUCOMUTASE1-phosphateUDPGlcGALACTOSEURIDINE+1-PHOSPHATENADDIPHOSPHOGALACTOSEGLUCOSE-URIDYLTRANSFERASE4-EPIMERASE6-PHOSPHATASEGlucose1-phosphateUDPGalGlucose6-phosphateGlucoseBNAD+GlucoseUDPGlcUDPGalURIDINEATPDIPHOSPHOGALACTOSE4-EPIMERASE2+UDPGlcMgHEXOKINASELactosePYROPHOSPHORYLASELACTOSEPPiSYNTHASEADPPHOSPHOGLUCOMUTASEGlucose6-phosphateGlucose1-phosphateGlucoseFigure20–6.Pathwayofconversionof(A)galactosetoglucoseintheliverand(B)glucosetolactoseinthelactatingmammarygland.theyhaveeatenfavabeans(Viciafava—hencethetermuronicacidpathway.Forexample,administrationoffavism).Glutathioneperoxidaseisdependentuponabarbitalorofchlorobutanoltoratsresultsinasignifi-supplyofNADPH,whichinerythrocytescanbecantincreaseintheconversionofglucosetoglu-formedonlyviathepentosephosphatepathway.Itre-curonate,L-gulonate,andascorbate.ducesorganicperoxidesandH2O2aspartofthebody’sdefenseagainstlipidperoxidation(Figure14–21).Measurementoferythrocytetransketolaseanditsacti-LoadingoftheLiverWithFructosevationbythiamindiphosphateisusedtoassessthiaminMayPotentiateHyperlipidemianutritionalstatus(Chapter45).&HyperuricemiaDisruptionoftheUronicAcidPathwayIsIntheliver,fructoseincreasestriacylglycerolsynthesisCausedbyEnzymeDefects&SomeDrugsandVLDLsecretion,leadingtohypertriacylglyc-erolemia—andincreasedLDLcholesterol—whichcanIntherarehereditarydiseaseessentialpentosuria,con-beregardedaspotentiallyatherogenic(Chapter26).InsiderablequantitiesofL-xyluloseappearintheurineaddition,acuteloadingoftheliverwithfructose,ascanbecauseofabsenceoftheenzymenecessarytoreduceoccurwithintravenousinfusionorfollowingveryhighL-xylulosetoxylitol.Parenteraladministrationofxylitolfructoseintakes,causessequestrationofinorganicphos-mayleadtooxalosis,involvingcalciumoxalatedeposi-phateinfructose1-phosphateanddiminishedATPtioninbrainandkidneys(Figure20–4).Variousdrugssynthesis.AsaresultthereislessinhibitionofdenovomarkedlyincreasetherateatwhichglucoseentersthepurinesynthesisbyATPanduricacidformationisin-
179THEPENTOSEPHOSPHATEPATHWAY&OTHERPATHWAYSOFHEXOSEMETABOLISM/171GlycogenGlucose1-phosphateATPADPGlucoseGlucose6-phosphateFructose6-phosphateGlutamineAMIDOTRANSFERASEATPADPUTPGlutamateGlucosamineGlucosamineGlucosamineUDP-6-phosphatePHOSPHOGLUCO-1-phosphateglucosamine*MUTASEAcetyl-CoAPPi–Acetyl-CoAATPADPN-Acetyl-N-Acetyl-N-Acetyl-glucosamineglucosamineglucosamineGlycosaminoglycans6-phosphate1-phosphate(eg,heparin)UTPEPIMERASEPPiN-Acetyl-mannosamineUDP-Glycosaminoglycans6-phosphateN-acetylglucosamine*(hyaluronicacid),glycoproteinsPhosphoenolpyruvateNAD+EPIMERASEN-Acetyl-UDP-neuraminicacidN-acetylgalactosamine*9-phosphateInhibiting–allostericeffectSialicacid,Glycosaminoglycansgangliosides,(chondroitins),glycoproteinsglycoproteinsFigure20–7.Summaryoftheinterrelationshipsinmetabolismofaminosugars.(Atasterisk:Analo-goustoUDPGlc.)Otherpurineorpyrimidinenucleotidesmaybesimilarlylinkedtosugarsoraminosug-ars.Examplesarethymidinediphosphate(TDP)-glucosamineandTDP-N-acetylglucosamine.creased,causinghyperuricemia,whichisacauseofgoutfructose1-phosphate,leadstohereditaryfructosein-(Chapter34).tolerance.Dietslowinfructose,sorbitol,andsucrosearebeneficialforbothconditions.OneconsequenceofDefectsinFructoseMetabolismCausehereditaryfructoseintoleranceandofanothercondi-tionduetofructose-1,6-bisphosphatasedeficiencyisDisease(Figure20–5)fructose-inducedhypoglycemiadespitethepresenceofLackofhepaticfructokinasecausesessentialfructo-highglycogenreserves.Theaccumulationoffructosesuria,andabsenceofhepaticaldolaseB,whichcleaves1-phosphateandfructose1,6-bisphosphateallosterically
180172/CHAPTER20inhibitstheactivityofliverphosphorylase.Theseques-SUMMARYtrationofinorganicphosphatealsoleadstodepletionof•Thepentosephosphatepathway,presentinthecy-ATPandhyperuricemia.tosol,canaccountforthecompleteoxidationofglu-cose,producingNADPHandCO2butnotATP.Fructose&SorbitolintheLensAre•Thepathwayhasanoxidativephase,whichisirre-AssociatedWithDiabeticCataractversibleandgeneratesNADPH;andanonoxidativeBothfructoseandsorbitolarefoundinthelensofthephase,whichisreversibleandprovidesriboseprecur-eyeinincreasedconcentrationsindiabetesmellitusandsorsfornucleotidesynthesis.Thecompletepathwaymaybeinvolvedinthepathogenesisofdiabeticispresentonlyinthosetissueshavingarequirementcataract.Thesorbitol(polyol)pathway(notfoundinforNADPHforreductivesyntheses,eg,lipogenesisliver)isresponsibleforfructoseformationfromglucoseorsteroidogenesis,whereasthenonoxidativephaseis(Figure20–5)andincreasesinactivityastheglucosepresentinallcellsrequiringribose.concentrationrisesindiabetesinthosetissuesthatare•Inerythrocytes,thepathwayhasamajorfunctioninnotinsulin-sensitive,ie,thelens,peripheralnerves,andpreventinghemolysisbyprovidingNADPHtorenalglomeruli.Glucoseisreducedtosorbitolbyal-maintainglutathioneinthereducedstateasthesub-dosereductase,followedbyoxidationofsorbitoltostrateforglutathioneperoxidase.fructoseinthepresenceofNAD+andsorbitoldehydro-•Theuronicacidpathwayisthesourceofglucuronicgenase(polyoldehydrogenase).Sorbitoldoesnotdif-acidforconjugationofmanyendogenousandexoge-fusethroughcellmembraneseasilyandaccumulates,noussubstancesbeforeexcretionasglucuronidesincausingosmoticdamage.Simultaneously,myoinositolurineandbile.levelsfall.Sorbitolaccumulation,myoinositoldeple-•Fructosebypassesthemainregulatorystepinglycol-tion,anddiabeticcataractcanbepreventedbyaldoseysis,catalyzedbyphosphofructokinase,andstimu-reductaseinhibitorsindiabeticrats,andpromisingre-latesfattyacidsynthesisandhepatictriacylglycerolsultshavebeenobtainedinclinicaltrials.secretion.Whensorbitolisadministeredintravenously,itisconvertedtofructoseratherthantoglucose.Itispoorly•Galactoseissynthesizedfromglucoseinthelactatingabsorbedinthesmallintestine,andmuchisfermentedmammaryglandandinothertissueswhereitisre-bycolonicbacteriatoshort-chainfattyacids,CO,andquiredforthesynthesisofglycolipids,proteoglycans,2H2,leadingtoabdominalpainanddiarrhea(sorbitolandglycoproteins.intolerance).EnzymeDeficienciesintheGalactoseREFERENCESPathwayCauseGalactosemiaCouetC,JanP,DebryG:Lactoseandcataractinhumans:are-Inabilitytometabolizegalactoseoccursinthegalac-view.JAmCollNutr1991;10:79.tosemias,whichmaybecausedbyinheriteddefectsinCoxTM:AldolaseBandfructoseintolerance.FASEBJ1994;8:62.galactokinase,uridyltransferase,or4-epimerase(FigureCrossNCP,CoxTM:Hereditaryfructoseintolerance.IntJBiochem1990;22:685.20–6A),thoughadeficiencyinuridyltransferaseisthebestknowncause.ThegalactoseconcentrationinKadorPF:Theroleofaldosereductaseinthedevelopmentofdia-beticcomplications.MedResRev1988;8:325.thebloodandintheeyeisreducedbyaldosereductaseKaufmanFR,DevganS:Classicalgalactosemia:areview.Endocri-togalactitol,whichaccumulates,causingcataract.Innologist1995;5:189.uridyltransferasedeficiency,galactose1-phosphateac-MacdonaldI,VranaA(editors):MetabolicEffectsofDietaryCarbo-cumulatesanddepletestheliverofinorganicphos-hydrates.Karger,1986.phate.Ultimately,liverfailureandmentaldeteriorationMayesPA:Intermediarymetabolismoffructose.AmJClinNutrresult.Astheepimeraseispresentinadequateamounts,1993(5Suppl);58:754S.thegalactosemicindividualcanstillformUDPGalVandenBergheG:Inbornerrorsoffructosemetabolism.Annufromglucose,andnormalgrowthanddevelopmentcanRevNutr1994;14:41.occurregardlessofthegalactose-freedietsusedtocon-WoodT:Physiologicalfunctionsofthepentosephosphatepath-trolthesymptomsofthedisease.way.CellBiolFunct1986;4:241.
181BiosynthesisofFattyAcids21PeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DScBIOMEDICALIMPORTANCETheFattyAcidSynthaseComplexIsaPolypeptideContainingFattyacidsaresynthesizedbyanextramitochondrialSevenEnzymeActivitiessystem,whichisresponsibleforthecompletesynthesisofpalmitatefromacetyl-CoAinthecytosol.Intherat,Inbacteriaandplants,theindividualenzymesofthethepathwayiswellrepresentedinadiposetissueandfattyacidsynthasesystemareseparate,andtheacylliver,whereasinhumansadiposetissuemaynotbeanradicalsarefoundincombinationwithaproteincalledimportantsite,andliverhasonlylowactivity.Inbirds,theacylcarrierprotein(ACP).However,inyeast,lipogenesisisconfinedtotheliver,whereitisparticu-mammals,andbirds,thesynthasesystemisamultien-larlyimportantinprovidinglipidsforeggformation.InzymepolypeptidecomplexthatincorporatesACP,mostmammals,glucoseistheprimarysubstrateforwhichtakesovertheroleofCoA.Itcontainsthevita-lipogenesis,butinruminantsitisacetate,themainfuelminpantothenicacidintheformof4′-phosphopan-moleculeproducedbythediet.Criticaldiseasesofthetetheine(Figure45–18).Theuseofonemultienzymepathwayhavenotbeenreportedinhumans.However,functionalunithastheadvantagesofachievingtheef-inhibitionoflipogenesisoccursintype1(insulin-de-fectofcompartmentalizationoftheprocesswithinthependent)diabetesmellitus,andvariationsinitsactiv-cellwithouttheerectionofpermeabilitybarriers,anditymayaffectthenatureandextentofobesity.synthesisofallenzymesinthecomplexiscoordinatedsinceitisencodedbyasinglegene.THEMAINPATHWAYFORDENOVOInmammals,thefattyacidsynthasecomplexisaSYNTHESISOFFATTYACIDSdimercomprisingtwoidenticalmonomers,eachcon-(LIPOGENESIS)OCCURStainingallsevenenzymeactivitiesoffattyacidsynthaseononepolypeptidechain(Figure21–2).Initially,aINTHECYTOSOLprimingmoleculeofacetyl-CoAcombineswithacys-Thissystemispresentinmanytissues,includingliver,teine⎯SHgroupcatalyzedbyacetyltransacylasekidney,brain,lung,mammarygland,andadiposetis-(Figure21–3,reaction1a).Malonyl-CoAcombinessue.ItscofactorrequirementsincludeNADPH,ATP,withtheadjacent⎯SHonthe4′-phosphopantetheineMn2+,biotin,andHCO−(asasourceofCO).Acetyl-ofACPoftheothermonomer,catalyzedbymalonyl32CoAistheimmediatesubstrate,andfreepalmitateistransacylase(reaction1b),toformacetyl(acyl)-mal-theendproduct.onylenzyme.Theacetylgroupattacksthemethylenegroupofthemalonylresidue,catalyzedby3-ketoacylProductionofMalonyl-CoAIssynthase,andliberatesCO2,forming3-ketoacylen-theInitial&ControllingStepzyme(acetoacetylenzyme)(reaction2),freeingthecys-teine⎯SHgroup.DecarboxylationallowsthereactioninFattyAcidSynthesistogotocompletion,pullingthewholesequenceofre-BicarbonateasasourceofCO2isrequiredintheinitialactionsintheforwarddirection.The3-ketoacylgroupreactionforthecarboxylationofacetyl-CoAtomal-isreduced,dehydrated,andreducedagain(reactions3,onyl-CoAinthepresenceofATPandacetyl-CoAcar-4,5)toformthecorrespondingsaturatedacyl-S-boxylase.Acetyl-CoAcarboxylasehasarequirementenzyme.Anewmalonyl-CoAmoleculecombineswithforthevitaminbiotin(Figure21–1).Theenzymeisathe⎯SHof4′-phosphopantetheine,displacingthesat-multienzymeproteincontainingavariablenumberofuratedacylresidueontothefreecysteine⎯SHgroup.identicalsubunits,eachcontainingbiotin,biotincar-Thesequenceofreactionsisrepeatedsixmoretimesboxylase,biotincarboxylcarrierprotein,andtranscar-untilasaturated16-carbonacylradical(palmityl)hasboxylase,aswellasaregulatoryallostericsite.Thereac-beenassembled.Itisliberatedfromtheenzymecom-tiontakesplaceintwosteps:(1)carboxylationofbiotinplexbytheactivityofaseventhenzymeinthecomplex,involvingATPand(2)transferofthecarboxyltothioesterase(deacylase).Thefreepalmitatemustbeac-acetyl-CoAtoformmalonyl-CoA.tivatedtoacyl-CoAbeforeitcanproceedviaanyother173
182174/CHAPTER21CHCOSCoA–OOCCHCOSCoA32Acetyl-CoAMalonyl-CoAEnzbiotinCOO–Enzbiotin–ADP+PiATP+HCO3Figure21–1.Biosynthesisofmalonyl-CoA.(Enz,acetyl-CoAcarboxylase.)metabolicpathway.Itsusualfateisesterificationinto+CHCOSCoA22⋅⋅+71HOOCCHCOSCoA⋅⋅⋅+4NADPH+14Hacylglycerols,chainelongationordesaturation,orester-+ificationtocholesterylester.Inmammarygland,there→+CHCH32()14COOH768CO22+HO+CoASH⋅+14NADPisaseparatethioesterasespecificforacylresiduesofC8,C10,orC12,whicharesubsequentlyfoundinmilkTheacetyl-CoAusedasaprimerformscarbonlipids.atoms15and16ofpalmitate.TheadditionofalltheTheequationfortheoverallsynthesisofpalmitatesubsequentC2unitsisviamalonyl-CoA.Propionyl-fromacetyl-CoAandmalonyl-CoAis:CoAactsasprimerforthesynthesisoflong-chainfattyHydrataseMalonylEnoyltransacylasereductaseAcetylKetoacyltransacylasereductaseKetoacyl1.FunctionalACPThioesterasesynthasedivision4′-Phospho-CyspantetheineSHSubunitSHSHdivisionSH4′-Phospho-Cys2.pantetheineKetoacylThioesteraseACPsynthaseKetoacylAcetylreductasetransacylaseEnoylMalonylreductasetransacylaseHydrataseFigure21–2.Fattyacidsynthasemultienzymecomplex.Thecomplexisadimeroftwoidenticalpolypeptidemonomers,1and2,eachconsistingofsevenenzymeactivitiesandtheacylcarrierprotein(ACP).(Cys⎯SH,cys-teinethiol.)The⎯SHofthe4′-phosphopantetheineofonemonomerisincloseproximitytothe⎯SHofthecys-teineresidueoftheketoacylsynthaseoftheothermonomer,suggestinga“head-to-tail”arrangementofthetwomonomers.Thougheachmonomercontainsallthepartialactivitiesofthereactionsequence,theactualfunc-tionalunitconsistsofone-halfofonemonomerinteractingwiththecomplementaryhalfoftheother.Thus,twoacylchainsareproducedsimultaneously.ThesequenceoftheenzymesineachmonomerisbasedonWakil.
183*CO2Acetyl-CoA*Malonyl-CoAC2ACETYL-CoAC3CARBOXYLASE1aHSPan1CysSH1bCoAACETYLCtransferfromTRANSACYLASEMALONYLnTRANSACYLASECoA21toHSCys2PanSHC2FattyacidsynthaseOmultienzymecomplex1CysSCCH3O–2PanSCCH2*COO(C3)Acyl(acetyl)-malonylenzyme3-KETOACYL2SYNTHASE*CO21CysSHOO2PanSCCH2CCH33-Ketoacylenzyme(acetoacetylenzyme)NADPH+H+3-KETOACYL3REDUCTASENADP+1CysSHOOH2PanSCCH2CHCH3NADPHGENERATORSD(–)-3-HydroxyacylenzymePentosephosphatepathwayHYDRATASE4IsocitrateH2OdehydrogenaseMalicenzyme1CysSHO2PanSCCHCHCH32,3-UnsaturatedacylenzymeNADPH+H+ENOYLREDUCTASE5NADP+H2O1CysSHTHIOESTERASEOAftercyclingthroughsteps2–5seventimes2PanSCCH2CH2CH3(Cn)AcylenzymePalmitateKEY:12,,individualmonomersoffattyacidsynthaseFigure21–3.Biosynthesisoflong-chainfattyacids.Detailsofhowadditionofamalonylresiduecausestheacylchaintogrowbytwocarbonatoms.(Cys,cysteineresidue;Pan,4′-phosphopante-theine.)TheblocksshownindarkbluecontaininitiallyaC2unitderivedfromacetyl-CoA(asillustrated)andsubsequentlytheCnunitformedinreaction5.175
184176/CHAPTER21acidshavinganoddnumberofcarbonatoms,foundphatepathway.Moreover,bothmetabolicpathwaysareparticularlyinruminantfatandmilk.foundinthecytosolofthecell,sotherearenomem-branesorpermeabilitybarriersagainstthetransferofTheMainSourceofNADPHNADPH.OthersourcesofNADPHincludethereac-forLipogenesisIsthePentosetionthatconvertsmalatetopyruvatecatalyzedbythePhosphatePathway“malicenzyme”(NADPmalatedehydrogenase)(Fig-ure21–4)andtheextramitochondrialisocitratedehy-NADPHisinvolvedasdonorofreducingequivalentsdrogenasereaction(probablynotasubstantialsource,inboththereductionofthe3-ketoacylandofthe2,3-exceptinruminants).unsaturatedacylderivatives(Figure21–3,reactions3and5).Theoxidativereactionsofthepentosephos-Acetyl-CoAIsthePrincipalBuildingphatepathway(seeChapter20)arethechiefsourceofBlockofFattyAcidsthehydrogenrequiredforthereductivesynthesisoffattyacids.Significantly,tissuesspecializinginactiveAcetyl-CoAisformedfromglucoseviatheoxidationoflipogenesis—ie,liver,adiposetissue,andthelactatingpyruvatewithinthemitochondria.However,itdoesmammarygland—alsopossessanactivepentosephos-notdiffusereadilyintotheextramitochondrialcytosol,GlucosePalmitateGlucose6-phosphateNADP+NADP+PPPFructose6-phosphateNADPH+Malic+H+NADPH+HenzymeGlyceraldehydeMALATEDEHYDROGENASEMalonyl-CoA3-phosphateNAD+MalateCOGLYCERALDEHYDE-2ATPACETYL-3-PHOSPHATECoADEHYDROGENASECARBOXY-NADH+H+OxaloacetateCO2LASEPyruvateAcetyl-CoACoACYTOSOLATP-CITRATECoAATPAcetateLYASEATPCitrateH+CitrateIsocitrateISOCITRATEOutsideDEHYDROGENASETPINNERMITOCHONDRIALMEMBRANETInsidePYRUVATEDEHYDROGENASEMalatePyruvateAcetyl-CoAMITOCHONDRIONα-KetoglutarateNADH+H+OxaloacetateCitrateCitricacidcycleNAD+Malateα-KetoglutarateKFigure21–4.Theprovisionofacetyl-CoAandNADPHforlipogenesis.(PPP,pentosephosphatepath-way;T,tricarboxylatetransporter;K,α-ketoglutaratetransporter;P,pyruvatetransporter.)
185BIOSYNTHESISOFFATTYACIDS/177theprincipalsiteoffattyacidsynthesis.Citrate,formedOOaftercondensationofacetyl-CoAwithoxaloacetateinthecitricacidcyclewithinmitochondria,istranslo-RCH2CSCoA+CH2CSCoAcatedintotheextramitochondrialcompartmentviatheCOOHtricarboxylatetransporter,whereinthepresenceofAcyl-CoAMalonyl-CoACoAandATPitundergoescleavagetoacetyl-CoAandoxaloacetatecatalyzedbyATP-citratelyase,whichin-creasesinactivityinthewell-fedstate.Theacetyl-CoA3-KETOACYL-CoAisthenavailableformalonyl-CoAformationandsyn-SYNTHASECoASH+CO2thesistopalmitate(Figure21–4).Theresultingox-aloacetatecanformmalateviaNADH-linkedmalatedehydrogenase,followedbythegenerationofNADPHOOviathemalicenzyme.TheNADPHbecomesavailableRCH2CCH2CSCoAforlipogenesis,andthepyruvatecanbeusedtoregen-erateacetyl-CoAaftertransportintothemitochon-3-Ketoacyl-CoAdrion.ThispathwayisameansoftransferringreducingNADPH+H+equivalentsfromextramitochondrialNADHtoNADP.Alternatively,malateitselfcanbetransportedintothe3-KETOACYL-CoAmitochondrion,whereitisabletore-formoxaloacetate.REDUCTASENotethatthecitrate(tricarboxylate)transporterinthe+NADPmitochondrialmembranerequiresmalatetoexchangeOHOwithcitrate(seeFigure12-10).ThereislittleATP-citratelyaseormalicenzymeinruminants,probablyRCH2CHCH2CSCoAbecauseinthesespeciesacetate(derivedfromthe3-Hydroxyacyl-CoArumenandactivatedtoacetylCoAextramitochondri-ally)isthemainsourceofacetyl-CoA.3-HYDROXYACYL-CoAElongationofFattyAcidChainsOccursDEHYDRASEHO2intheEndoplasmicReticulumThispathway(the“microsomalsystem”)elongatessat-Ouratedandunsaturatedfattyacyl-CoAs(fromC10up-RCH2CHCHCSCoAward)bytwocarbons,usingmalonyl-CoAasacetyldonorandNADPHasreductant,andiscatalyzedby2-trans-Enoyl-CoAthemicrosomalfattyacidelongasesystemofenzymes+NADPH+H(Figure21–5).Elongationofstearyl-CoAinbrainin-2-trans-ENOYL-CoAcreasesrapidlyduringmyelinationinordertoprovideREDUCTASEC22andC24fattyacidsforsphingolipids.NADP+OTHENUTRITIONALSTATEREGULATESLIPOGENESISRCH2CH2CH2CSCoAExcesscarbohydrateisstoredasfatinmanyanimalsinAcyl-CoAanticipationofperiodsofcaloricdeficiencysuchasstar-Figure21–5.Microsomalelongasesystemforfattyvation,hibernation,etc,andtoprovideenergyforuseacidchainelongation.NADHisalsousedbythereduc-betweenmealsinanimals,includinghumans,thattaketases,butNADPHispreferred.theirfoodatspacedintervals.Lipogenesisconvertssur-plusglucoseandintermediatessuchaspyruvate,lactate,andacetyl-CoAtofat,assistingtheanabolicphaseofafatdiet,orwhenthereisadeficiencyofinsulin,asinthisfeedingcycle.Thenutritionalstateoftheorganismdiabetesmellitus.Theselatterconditionsareassociatedisthemainfactorregulatingtherateoflipogenesis.withincreasedconcentrationsofplasmafreefattyacids,Thus,therateishighinthewell-fedanimalwhosedietandaninverserelationshiphasbeendemonstratedbe-containsahighproportionofcarbohydrate.Itisde-tweenhepaticlipogenesisandtheconcentrationofpressedunderconditionsofrestrictedcaloricintake,onserum-freefattyacids.Lipogenesisisincreasedwhensu-
186178/CHAPTER21croseisfedinsteadofglucosebecausefructosebypassesPROTEINthephosphofructokinasecontrolpointinglycolysisandPiPHOSPHATASEfloodsthelipogenicpathway(Figure20–5).H2OSHORT-&LONG-TERMMECHANISMSACETYL-CoAACETYL-CoAREGULATELIPOGENESISCARBOXYLASEPCARBOXYLASE(active)(inactive)Long-chainfattyacidsynthesisiscontrolledintheshorttermbyallostericandcovalentmodificationofAcetyl-enzymesandinthelongtermbychangesingeneex-CoApressiongoverningratesofsynthesisofenzymes.ATPAMPKADPAcetyl-CoACarboxylaseIstheMostH2O(active)ImportantEnzymeintheRegulationMalonyl-CoAofLipogenesisPAMPKKAcetyl-CoAcarboxylaseisanallostericenzymeandisactivatedbycitrate,whichincreasesinconcentrationinAMPK++thewell-fedstateandisanindicatorofaplentifulsup-(inactive)PiATPplyofacetyl-CoA.Citrateconvertstheenzymefromaninactivedimertoanactivepolymericform,havingaAcyl-CoAmolecularmassofseveralmillion.Inactivationispro-cAMP-DEPENDENTmotedbyphosphorylationoftheenzymeandbylong-GlucagoncAMP++PROTEINKINASEchainacyl-CoAmolecules,anexampleofnegativefeed-backinhibitionbyaproductofareaction.Thus,ifFigure21–6.Regulationofacetyl-CoAcarboxylaseacyl-CoAaccumulatesbecauseitisnotesterifiedbyphosphorylation/dephosphorylation.TheenzymeisquicklyenoughorbecauseofincreasedlipolysisoraninactivatedbyphosphorylationbyAMP-activatedpro-influxoffreefattyacidsintothetissue,itwillautomati-callyreducethesynthesisofnewfattyacid.Acyl-CoAteinkinase(AMPK),whichinturnisphosphorylatedmayalsoinhibitthemitochondrialtricarboxylateandactivatedbyAMP-activatedproteinkinasekinasetransporter,thuspreventingactivationoftheenzyme(AMPKK).Glucagon(andepinephrine),afterincreasingbyegressofcitratefromthemitochondriaintothecy-cAMP,activatethislatterenzymeviacAMP-dependenttosol.proteinkinase.Thekinasekinaseenzymeisalsobe-Acetyl-CoAcarboxylaseisalsoregulatedbyhor-lievedtobeactivatedbyacyl-CoA.Insulinactivatesmonessuchasglucagon,epinephrine,andinsulinviaacetyl-CoAcarboxylase,probablythroughan“activa-changesinitsphosphorylationstate(detailsinFiguretor”proteinandaninsulin-stimulatedproteinkinase.21–6).PyruvateDehydrogenaseIsAlsoInsulinAlsoRegulatesLipogenesisRegulatedbyAcyl-CoAbyOtherMechanismsAcyl-CoAcausesaninhibitionofpyruvatedehydrogen-Insulinstimulateslipogenesisbyseveralothermecha-asebyinhibitingtheATP-ADPexchangetransporterofnismsaswellasbyincreasingacetyl-CoAcarboxylasetheinnermitochondrialmembrane,whichleadstoin-activity.Itincreasesthetransportofglucoseintothecreasedintramitochondrial[ATP]/[ADP]ratiosandcell(eg,inadiposetissue),increasingtheavailabilityofthereforetoconversionofactivetoinactivepyruvatebothpyruvateforfattyacidsynthesisandglyceroldehydrogenase(seeFigure17–6),thusregulatingthe3-phosphateforesterificationofthenewlyformedfattyavailabilityofacetyl-CoAforlipogenesis.Furthermore,acids,andalsoconvertstheinactiveformofpyruvateoxidationofacyl-CoAduetoincreasedlevelsoffreedehydrogenasetotheactiveforminadiposetissuebutfattyacidsmayincreasetheratiosof[acetyl-CoA]/notinliver.Insulinalso—byitsabilitytodepressthe[CoA]and[NADH]/[NAD+]inmitochondria,inhibit-levelofintracellularcAMP—inhibitslipolysisinadi-ingpyruvatedehydrogenase.posetissueandtherebyreducestheconcentrationof
187BIOSYNTHESISOFFATTYACIDS/179plasmafreefattyacidsandthereforelong-chainacyl-•Acetyl-CoAcarboxylaseisrequiredtoconvertacetyl-CoA,aninhibitoroflipogenesis.CoAtomalonyl-CoA.Inturn,fattyacidsynthase,amultienzymecomplexofonepolypeptidechainwithTheFattyAcidSynthaseComplexsevenseparateenzymaticactivities,catalyzestheas-&Acetyl-CoACarboxylaseAresemblyofpalmitatefromoneacetyl-CoAandsevenAdaptiveEnzymesmalonyl-CoAmolecules.•Lipogenesisisregulatedattheacetyl-CoAcarboxy-Theseenzymesadapttothebody’sphysiologicneedslasestepbyallostericmodifiers,phosphorylation/de-byincreasingintotalamountinthefedstateandbyphosphorylation,andinductionandrepressionofen-decreasinginstarvation,feedingoffat,andindiabetes.zymesynthesis.Citrateactivatestheenzyme,andInsulinisanimportanthormonecausinggeneexpres-long-chainacyl-CoAinhibitsitsactivity.Insulinacti-sionandinductionofenzymebiosynthesis,andvatesacetyl-CoAcarboxylasewhereasglucagonandglucagon(viacAMP)antagonizesthiseffect.Feedingepinephrinehaveoppositeactions.fatscontainingpolyunsaturatedfattyacidscoordinatelyregulatestheinhibitionofexpressionofkeyenzymesofglycolysisandlipogenesis.ThesemechanismsforREFERENCESlonger-termregulationoflipogenesistakeseveraldaystobecomefullymanifestedandaugmentthedirectandHudginsLCetal:Humanfattyacidsynthesisisstimulatedbyaimmediateeffectoffreefattyacidsandhormonessucheucaloriclowfat,highcarbohydratediet.JClinInvestasinsulinandglucagon.1996;97:2081.JumpDBetal:Coordinateregulationofglycolyticandlipogenicgeneexpressionbypolyunsaturatedfattyacids.JLipidResSUMMARY1994;35:1076.•Thesynthesisoflong-chainfattyacids(lipogenesis)isKimKH:Regulationofmammalianacetyl-coenzymeAcarboxy-lase.AnnuRevNutr1997;17:77.carriedoutbytwoenzymesystems:acetyl-CoAcar-SalatiLM,GoodridgeAG:Fattyacidsynthesisineukaryotes.In:boxylaseandfattyacidsynthase.BiochemistryofLipids,LipoproteinsandMembranes.Vance•Thepathwayconvertsacetyl-CoAtopalmitateandDE,VanceJE(editors).Elsevier,1996.2+requiresNADPH,ATP,Mn,biotin,pantothenicWakilSJ:Fattyacidsynthase,aproficientmultifunctionalenzyme.−Biochemistry1989;28:4523.acid,andHCO3ascofactors.
188MetabolismofUnsaturatedFattyAcids&Eicosanoids23PeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DScBIOMEDICALIMPORTANCE4569ducedattheΔ,Δ,Δ,andΔpositions(seeChapter914)inmostanimals,butneverbeyondtheΔposition.UnsaturatedfattyacidsinphospholipidsofthecellIncontrast,plantsareabletosynthesizethenutrition-membraneareimportantinmaintainingmembraneallyessentialfattyacidsbyintroducingdoublebondsatfluidity.Ahighratioofpolyunsaturatedfattyacidsto1215theΔandΔpositions.saturatedfattyacids(P:Sratio)inthedietisamajorfactorinloweringplasmacholesterolconcentrationsandisconsideredtobebeneficialinpreventingcoro-naryheartdisease.Animaltissueshavelimitedcapacity169COOHfordesaturatingfattyacids,andthatprocessrequires9)Palmitoleicacid(ω7,16:1,Δcertaindietarypolyunsaturatedfattyacidsderivedfromplants.Theseessentialfattyacidsareusedtoformeicosanoic(C20)fattyacids,whichinturngive189COOHrisetotheprostaglandinsandthromboxanesandtoOleicacid(ω9,18:1,Δ9)leukotrienesandlipoxins—knowncollectivelyaseicosanoids.Theprostaglandinsandthromboxanesare129COOHlocalhormonesthataresynthesizedrapidlywhenre-quired.Prostaglandinsmediateinflammation,produce189,12)pain,andinducesleepaswellasbeinginvolvedinthe*Linoleicacid(ω6,18:2,Δregulationofbloodcoagulationandreproduction.Nonsteroidalanti-inflammatorydrugssuchasaspirin1815129COOHactbyinhibitingprostaglandinsynthesis.Leukotrienes9,12,15)havemusclecontractantandchemotacticproperties*α-Linolenicacid(ω3,18:3,Δandareimportantinallergicreactionsandinflamma-141185tion.COOH20*Arachidonicacid(ω6,20:4,Δ5,8,11,14)SOMEPOLYUNSATURATEDFATTYACIDSCANNOTBESYNTHESIZEDBYMAMMALS&ARE2017141185COOHNUTRITIONALLYESSENTIAL5,8,11,14,17Eicosapentaenoicacid(ω3,20:5,Δ)Certainlong-chainunsaturatedfattyacidsofmetabolicFigure23–1.StructureofsomeunsaturatedfattysignificanceinmammalsareshowninFigure23–1.acids.AlthoughthecarbonatomsinthemoleculesareOtherC20,C22,andC24polyenoicfattyacidsmaybeconventionallynumbered—ie,numberedfromthecar-derivedfromoleic,linoleic,andα-linolenicacidsbychainelongation.Palmitoleicandoleicacidsarenotes-boxylterminal—theωnumbers(eg,ω7inpalmitoleicsentialinthedietbecausethetissuescanintroduceaacid)arecalculatedfromthereverseend(themethyldoublebondattheΔ9positionofasaturatedfattyacid.terminal)ofthemolecules.Theinformationinparen-Linoleicand-linolenicacidsaretheonlyfattyacidsthesesshows,forinstance,thatα-linolenicacidcon-knowntobeessentialforthecompletenutritionoftainsdoublebondsstartingatthethirdcarbonfrommanyspeciesofanimals,includinghumans,andarethemethylterminal,has18carbonsand3doubleknownasthenutritionallyessentialfattyacids.Inbonds,andhasthesedoublebondsatthe9th,12th,mostmammals,arachidonicacidcanbeformedfromand15thcarbonsfromthecarboxylterminal.(Asterisks:linoleicacid(Figure23–4).Doublebondscanbeintro-Classifiedas“essentialfattyacids.”)190
189METABOLISMOFUNSATURATEDFATTYACIDS&EICOSANOIDS/191StearoylCoAareabletosynthesizetheω9(oleicacid)familyofunsat-uratedfattyacidscompletelybyacombinationofchainO+NADH+H+2elongationanddesaturation(Figure23–3).However,asindicatedabove,linoleic(ω6)orα-linolenic(ω3)acids9ΔDESATURASECytb5requiredforthesynthesisoftheothermembersofthe+ω6orω3familiesmustbesuppliedinthediet.NAD+2H2OLinoleatemaybeconvertedtoarachidonatevia-OleoylCoAlinolenatebythepathwayshowninFigure23–4.Thenutritionalrequirementforarachidonatemaythusbe9Figure23–2.MicrosomalΔdesaturase.dispensedwithifthereisadequatelinoleateinthediet.Thedesaturationandchainelongationsystemisgreatlydiminishedinthestarvingstate,inresponsetoglucagonMONOUNSATURATEDFATTYandepinephrineadministration,andintheabsenceofACIDSARESYNTHESIZEDBYinsulinasintype1diabetesmellitus.9ADESATURASESYSTEMSeveraltissuesincludingtheliverareconsideredtobere-DEFICIENCYSYMPTOMSAREPRODUCEDsponsiblefortheformationofnonessentialmonounsatu-WHENTHEESSENTIALFATTYACIDSratedfattyacidsfromsaturatedfattyacids.Thefirstdou-(EFA)AREABSENTFROMTHEDIETblebondintroducedintoasaturatedfattyacidisnearly99alwaysintheΔposition.Anenzymesystem—desat-RatsfedapurifiednonlipiddietcontainingvitaminsAurase(Figure23–2)—intheendoplasmicreticulumwillandDexhibitareducedgrowthrateandreproductivecatalyzetheconversionofpalmitoyl-CoAorstearoyl-CoAdeficiencywhichmaybecuredbytheadditionoftopalmitoleoyl-CoAoroleoyl-CoA,respectively.Oxygenlinoleic,-linolenic,andarachidonicacidstothediet.andeitherNADHorNADPHarenecessaryforthereac-Thesefattyacidsarefoundinhighconcentrationsintion.Theenzymesappeartobesimilartoamonooxyge-vegetableoils(Table14–2)andinsmallamountsinani-nasesysteminvolvingcytochromeb5(Chapter11).malcarcasses.Theseessentialfattyacidsarerequiredforprostaglandin,thromboxane,leukotriene,andlipoxinSYNTHESISOFPOLYUNSATURATEDformation(seebelow),andtheyalsohavevariousotherFATTYACIDSINVOLVESDESATURASEfunctionswhicharelesswelldefined.Essentialfattyacidsarefoundinthestructurallipidsofthecell,ofteninthe&ELONGASEENZYMESYSTEMS2positionofphospholipids,andareconcernedwiththeAdditionaldoublebondsintroducedintoexistingmo-structuralintegrityofthemitochondrialmembrane.nounsaturatedfattyacidsarealwaysseparatedfromeachArachidonicacidispresentinmembranesandac-otherbyamethylenegroup(methyleneinterrupted)ex-countsfor5–15%ofthefattyacidsinphospholipids.9Docosahexaenoicacid(DHA;ω3,22:6),whichissyn-ceptinbacteria.SinceanimalshaveaΔdesaturase,theyω921314Oleicacid18:220:220:322:322:4Family18:11Accumulatesinessential11—fattyaciddeficiency24:122:120:121314ω6Linoleicacid18:320:320:422:422:5Family18:21—20:2ω3α-Linolenic21314Family18:320:320:422:422:5acid18:3Figure23–3.Biosynthesisoftheω9,ω6,andω3familiesofpolyunsaturatedfattyacids.Eachstepiscatalyzedbythemicrosomalchainelongationordesaturasesys-tem:1,elongase;2,Δ6desaturase;3,Δ5desaturase;4,Δ4desaturase.(—,Inhibition.)
190192/CHAPTER23Ofattyacidsinphospholipids,othercomplexlipids,and5,8,11129membranes,particularlywithΔ-eicosatrienoicacidCSCoA(ω920:3)(Figure23–3).Thetriene:tetraeneratioin18plasmalipidscanbeusedtodiagnosetheextentofes-Linoleoyl-CoA(Δ9,12-octadecadienoyl-CoA)sentialfattyaciddeficiency.O+NADH+H+2TransFattyAcidsAreImplicatedΔ6inVariousDisordersDESATURASESmallamountsoftrans-unsaturatedfattyacidsarefound2HO+NAD+2inruminantfat(eg,butterfathas2–7%),wheretheyarisefromtheactionofmicroorganismsintherumen,1296butthemainsourceinthehumandietisfrompartiallyhydrogenatedvegetableoils(eg,margarine).Transfatty18CSCoAacidscompetewithessentialfattyacidsandmayexacer-Obateessentialfattyaciddeficiency.Moreover,theyareγ-Linolenoyl-CoA(Δ6,9,12-octadecatrienoyl-CoA)structurallysimilartosaturatedfattyacids(Chapter14)andhavecomparableeffectsinthepromotionofhyper-C2cholesterolemiaandatherosclerosis(Chapter26).(Malonyl-CoA,MICROSOMALCHAINNADPH)ELONGATIONSYSTEM(ELONGASE)EICOSANOIDSAREFORMEDFROMC20POLYUNSATURATEDFATTYACIDSArachidonateandsomeotherC20polyunsaturatedfatty14118acidsgiverisetoeicosanoids,physiologicallyandphar-macologicallyactivecompoundsknownasprosta-20CSCoAglandins(PG),thromboxanes(TX),leukotrienesO(LT),andlipoxins(LX)(Chapter14).Physiologically,Dihomo-γ-linolenoyl-CoA(Δ8,11,14-eicosatrienoyl-CoA)theyareconsideredtoactaslocalhormonesfunction-ingthroughG-protein-linkedreceptorstoelicittheirO+NADH+H+2biochemicaleffects.5Therearethreegroupsofeicosanoidsthataresyn-ΔDESATURASEthesizedfromC20eicosanoicacidsderivedfromthees-sentialfattyacidslinoleateand-linolenate,ordi-2HO+NAD+2rectlyfromdietaryarachidonateandeicosapentaenoateO(Figure23–5).Arachidonate,usuallyderivedfromthe1411852positionofphospholipidsintheplasmamembranebyCSCoAtheactionofphospholipaseA2(Figure24–6)—butalso20fromthediet—isthesubstrateforthesynthesisoftheArachidonoyl-CoA(Δ5,8,11,14-eicosatetraenoyl-CoA)PG2,TX2series(prostanoids)bythecyclooxygenasepathway,ortheLT4andLX4seriesbythelipoxyge-Figure23–4.Conversionoflinoleatetoarachido-nasepathway,withthetwopathwayscompetingfornate.Catscannotcarryoutthisconversionowingtoab-thearachidonatesubstrate(Figure23–5).6senceofΔdesaturaseandmustobtainarachidonateintheirdiet.THECYCLOOXYGENASEPATHWAYISRESPONSIBLEFORPROSTANOIDSYNTHESISthesizedfromα-linolenicacidorobtaineddirectlyfromfishoils,ispresentinhighconcentrationsinretina,Prostanoidsynthesis(Figure23–6)involvesthecon-cerebralcortex,testis,andsperm.DHAisparticularlysumptionoftwomoleculesofO2catalyzedbyneededfordevelopmentofthebrainandretinaandisprostaglandinHsynthase(PGHS),whichconsistsofsuppliedviatheplacentaandmilk.Patientswithretini-twoenzymes,cyclooxygenaseandperoxidase.PGHStispigmentosaarereportedtohavelowbloodlevelsofispresentastwoisoenzymes,PGHS-1andPGHS-2.DHA.Inessentialfattyaciddeficiency,nonessentialTheproduct,anendoperoxide(PGH),isconvertedtopolyenoicacidsoftheω9familyreplacetheessentialprostaglandinsD,E,andFaswellastoathromboxane
191METABOLISMOFUNSATURATEDFATTYACIDS&EICOSANOIDS/193DietMembranephospholipidPHOSPHOLIPASEAngiotensinIILinoleateA2+BradykininEpinephrineThrombin–2Hγ-LinolenateGROUP1DietGROUP2ProstanoidsProstanoids+2CPGE1PGD21PGF11PGE2TXA1PGF2COOHCOOH–2HPGI2TXA2LeukotrienesLeukotrienesLipoxinsLTA38,11,14-Eicosatrienoate2LTC5,8,11,14-LTA4LXA4(dihomoγ-linolenate)3Eicosatetraenoate2LTBLXBLTD443LTC4LXC4ArachidonateLTD4LXD4LTE4LXE4GROUP3ProstanoidsPGD31PGE3PGF3COOH–2HPGI3EicosatetraenoateTXA3Leukotrienes+2C5,8,11,14,17-LTA5Eicosapentaenoate2LTB5LTC5Octadecatetraenoate–2HDietα-LinolenateDietFigure23–5.Thethreegroupsofeicosanoidsandtheirbiosyntheticorigins.(PG,prostaglandin;PGI,prosta-cyclin;TX,thromboxane;LT,leukotriene;LX,lipoxin;1,cyclooxygenasepathway;2,lipoxygenasepathway.)Thesubscriptdenotesthetotalnumberofdoublebondsinthemoleculeandtheseriestowhichthecompoundbelongs.(TXA2)andprostacyclin(PGI2).Eachcelltypepro-EssentialFattyAcidsDoNotExertducesonlyonetypeofprostanoid.Aspirin,anons-AllTheirPhysiologicEffectsViateroidalanti-inflammatorydrug(NSAID),inhibitscy-ProstaglandinSynthesisclooxygenaseofbothPGHS-1andPGHS-2byacetylation.MostotherNSAIDs,suchasindomethacinTheroleofessentialfattyacidsinmembraneformationandibuprofen,inhibitcyclooxygenasesbycompetingisunrelatedtoprostaglandinformation.Prostaglandinswitharachidonate.TranscriptionofPGHS-2—butnotdonotrelievesymptomsofessentialfattyaciddefi-ofPGHS-1—iscompletelyinhibitedbyanti-inflam-ciency,andanessentialfattyaciddeficiencyisnotmatorycorticosteroids.causedbyinhibitionofprostaglandinsynthesis.
192194/CHAPTER23COOHArachidonate*2O2CYCLOOXYGENASEAspirin–IndomethacinOIbuprofenCOOHCOOHOPGI2OOHPROSTACYCLINPGG*2PEROXIDASEOOSYNTHASEOCHCOOHCOOH+CHOOHOHOHOOHPGHMalondialdehyde+HHT2OCOOHISOMERASETHROMBOXANEImidazoleOOSYNTHASE–COOHCOOHOOOHOHOHOHOHTXA26-KetoPGF1αPGE2ISOMERASEREDUCTASEOHOHOHCOOHCOOHCOOHHOOOHOHOOHOHPGF2αPGD2TXB2Figure23–6.Conversionofarachidonicacidtoprostaglandinsandthromboxanesofseries2.(PG,prostaglandin;TX,thromboxane;PGI,prostacyclin;HHT,hydroxyheptadecatrienoate.)(Asterisk:Bothofthesestarredactivitiesareattributedtooneenzyme:prostaglandinHsynthase.Similarconversionsoccurinprostaglandinsandthromboxanesofseries1and3.)CyclooxygenaseIsa“SuicideEnzyme”nasepathwayinresponsetobothimmunologicandnonimmunologicstimuli.Threedifferentlipoxygenases“Switchingoff”ofprostaglandinactivityispartlyachieved(dioxygenases)insertoxygenintothe5,12,and15po-byaremarkablepropertyofcyclooxygenase—thatofsitionsofarachidonicacid,givingrisetohydroperox-self-catalyzeddestruction;ie,itisa“suicideenzyme.”ides(HPETE).Only5-lipoxygenaseformsleuko-Furthermore,theinactivationofprostaglandinsby15-trienes(detailsinFigure23–7).Lipoxinsareafamilyofhydroxyprostaglandindehydrogenaseisrapid.Block-conjugatedtetraenesalsoarisinginleukocytes.Theyareingtheactionofthisenzymewithsulfasalazineorin-formedbythecombinedactionofmorethanonedomethacincanprolongthehalf-lifeofprostaglandinslipoxygenase(Figure23–7).inthebody.CLINICALASPECTSLEUKOTRIENES&LIPOXINSAREFORMEDBYTHESymptomsofEssentialFattyAcidLIPOXYGENASEPATHWAYDeficiencyinHumansIncludeSkinLesions&ImpairmentofLipidTransportTheleukotrienesareafamilyofconjugatedtrienesformedfromeicosanoicacidsinleukocytes,mastocy-Inadultssubsistingonordinarydiets,nosignsofes-tomacells,platelets,andmacrophagesbythelipoxyge-sentialfattyaciddeficiencieshavebeenreported.How-
193METABOLISMOFUNSATURATEDFATTYACIDS&EICOSANOIDS/195COOH15-LIPOXYGENASE12-LIPOXYGENASEArachidonateCOOHCOOHO2HOOOOH112-HPETE15-HPETE5-LIPOXYGENASECOOH1COOHOHHO15-HETEOOHOH12-HETECOOHCOOH5-LIPOXYGENASE15-HPETE5-HETEOHH2OOHOHCOOHCOOHH2OOCOOHOH15-LIPOXYGENASEOH2LeukotrieneB4LeukotrieneA4Lipoxins,eg,LXA4Glutathione3GlutamicacidONH2GlycineGlycineOHONHOONH2NH2NHNHHOHOHOCysteineGlutamicacidCysteineGlycineCysteineOSOSOSCOOH4COOH5COOHOHOHOHLeukotrieneC4LeukotrieneD4LeukotrieneE4Figure23–7.Conversionofarachidonicacidtoleukotrienesandlipoxinsofseries4viathelipoxygenasepath-way.Somesimilarconversionsoccurinseries3and5leukotrienes.(HPETE,hydroperoxyeicosatetraenoate;HETE,hydroxyeicosatetraenoate;1,peroxidase;2,leukotrieneA4epoxidehydrolase;3,glutathioneS-transferase;4,γ-glutamyltranspeptidase;5,cysteinyl-glycinedipeptidase.)ever,infantsreceivingformuladietslowinfatandpa-AbnormalMetabolismofEssentialFattytientsmaintainedforlongperiodsexclusivelybyintra-AcidsOccursinSeveralDiseasesvenousnutritionlowinessentialfattyacidsshowdefi-ciencysymptomsthatcanbepreventedbyanessentialAbnormalmetabolismofessentialfattyacids,whichfattyacidintakeof1–2%ofthetotalcaloricrequire-maybeconnectedwithdietaryinsufficiency,hasbeenment.notedincysticfibrosis,acrodermatitisenteropathica,
194196/CHAPTER23hepatorenalsyndrome,Sjögren-Larssonsyndrome,immediatehypersensitivityreactions,suchasasthma.multisystemneuronaldegeneration,Crohn’sdisease,Leukotrienesarevasoactive,and5-lipoxygenasehascirrhosisandalcoholism,andReye’ssyndrome.Ele-beenfoundinarterialwalls.Evidencesupportsarolevatedlevelsofverylongchainpolyenoicacidshaveforlipoxinsinvasoactiveandimmunoregulatoryfunc-beenfoundinthebrainsofpatientswithZellweger’stion,eg,ascounterregulatorycompounds(chalones)ofsyndrome(Chapter22).DietswithahighP:S(polyun-theimmuneresponse.saturated:saturatedfattyacid)ratioreduceserumcho-lesterollevelsandareconsideredtobebeneficialintermsoftheriskofdevelopmentofcoronaryheartdis-SUMMARYease.•Biosynthesisofunsaturatedlong-chainfattyacidsisachievedbydesaturaseandelongaseenzymes,whichProstanoidsArePotentBiologicallyintroducedoublebondsandlengthenexistingacylActiveSubstanceschains,respectively.4569Thromboxanesaresynthesizedinplateletsandupon•HigheranimalshaveΔ,Δ,Δ,andΔdesaturasesreleasecausevasoconstrictionandplateletaggregation.butcannotinsertnewdoublebondsbeyondthe9Theirsynthesisisspecificallyinhibitedbylow-doseas-positionoffattyacids.Thus,theessentialfattyacidspirin.Prostacyclins(PGI2)areproducedbybloodves-linoleic(ω6)andα-linolenic(ω3)mustbeobtainedselwallsandarepotentinhibitorsofplateletaggrega-fromthediet.tion.Thus,thromboxanesandprostacyclinsare•EicosanoidsarederivedfromC20(eicosanoic)fattyantagonistic.PG3andTX3,formedfromeicosapen-acidssynthesizedfromtheessentialfattyacidsandtaenoicacid(EPA)infishoils,inhibitthereleaseofcompriseimportantgroupsofphysiologicallyandarachidonatefromphospholipidsandtheformationpharmacologicallyactivecompounds,includingtheofPG2andTX2.PGI3isaspotentanantiaggregatorofprostaglandins,thromboxanes,leukotrienes,andplateletsasPGI2,butTXA3isaweakeraggregatorthanlipoxins.TXA2,changingthebalanceofactivityandfavoringlongerclottingtimes.Aslittleas1ng/mLofplasmaprostaglandinscausescontractionofsmoothmuscleinREFERENCESanimals.PotentialtherapeuticusesincludepreventionConnorWE:Thebeneficialeffectsofomega-3fattyacids:cardio-ofconception,inductionoflaboratterm,terminationvasculardiseaseandneurodevelopment.CurrOpinLipidolofpregnancy,preventionoralleviationofgastriculcers,1997;8:1.controlofinflammationandofbloodpressure,andre-FischerS:Dietarypolyunsaturatedfattyacidsandeicosanoidfor-liefofasthmaandnasalcongestion.Inaddition,PGD2mationinhumans.AdvLipidRes1989;23:169.isapotentsleep-promotingsubstance.ProstaglandinsLagardeM,GualdeN,RigaudM:MetabolicinteractionsbetweenincreasecAMPinplatelets,thyroid,corpusluteum,eicosanoidsinbloodandvascularcells.BiochemJ1989;fetalbone,adenohypophysis,andlungbutreduce257:313.cAMPinrenaltubulecellsandadiposetissue(Chap-NeuringerM,AndersonGJ,ConnorWE:Theessentialityofn-3ter25).fattyacidsforthedevelopmentandfunctionoftheretinaandbrain.AnnuRevNutr1988;8:517.SerhanCN:LipoxinbiosynthesisanditsimpactininflammatoryLeukotrienes&LipoxinsArePotentandvascularevents.BiochimBiophysActa1994;1212:1.RegulatorsofManyDiseaseProcessesSmithWL,FitzpatrickFA:Theeicosanoids:Cyclooxygenase,Slow-reactingsubstanceofanaphylaxis(SRS-A)isalipoxygenase,andepoxygenasepathways.In:BiochemistryofLipids,LipoproteinsandMembranes.VanceDE,VanceJEmixtureofleukotrienesC4,D4,andE4.Thismixtureof(editors).Elsevier,1996.leukotrienesisapotentconstrictorofthebronchialair-TocherDR,LeaverMJ,HodgsonPA:Recentadvancesinthebio-waymusculature.Theseleukotrienestogetherwithchemistryandmolecularbiologyoffattyacyldesaturases.leukotrieneB4alsocausevascularpermeabilityandat-ProgLipidRes1998;37:73.tractionandactivationofleukocytesandareimportantValenzuelaA,MorgadoN:Transfattyacidisomersinhumanregulatorsinmanydiseasesinvolvinginflammatoryorhealthandthefoodindustry.BiolRes1999;32:273.
195MetabolismofAcylglycerols&Sphingolipids24PeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DScBIOMEDICALIMPORTANCEpossessglycerolkinase,foundinsignificantamountsinliver,kidney,intestine,brownadiposetissue,andAcylglycerolsconstitutethemajorityoflipidsinthelactatingmammarygland.body.Triacylglycerolsarethemajorlipidsinfatde-positsandinfood,andtheirrolesinlipidtransportandstorageandinvariousdiseasessuchasobesity,diabetes,TRIACYLGLYCEROLS&andhyperlipoproteinemiawillbedescribedinsubse-PHOSPHOGLYCEROLSAREFORMEDBYquentchapters.Theamphipathicnatureofphospho-lipidsandsphingolipidsmakesthemideallysuitableasACYLATIONOFTRIOSEPHOSPHATESthemainlipidcomponentofcellmembranes.Phos-Themajorpathwaysoftriacylglycerolandphosphoglyc-pholipidsalsotakepartinthemetabolismofmanyerolbiosynthesisareoutlinedinFigure24–1.Impor-otherlipids.Somephospholipidshavespecializedfunc-tantsubstancessuchastriacylglycerols,phosphatidyl-tions;eg,dipalmitoyllecithinisamajorcomponentofcholine,phosphatidylethanolamine,phosphatidylinositol,lungsurfactant,whichislackinginrespiratorydistressandcardiolipin,aconstituentofmitochondrialmem-syndromeofthenewborn.Inositolphospholipidsinthebranes,areformedfromglycerol-3-phosphate.Significantcellmembraneactasprecursorsofhormonesecondbranchpointsinthepathwayoccuratthephosphati-messengers,andplatelet-activatingfactorisanalkyl-dateanddiacylglycerolsteps.Fromdihydroxyacetonephospholipid.Glycosphingolipids,containingsphingo-phosphatearederivedphosphoglycerolscontainingansineandsugarresiduesaswellasfattyacidandfoundinetherlink(⎯C⎯O⎯C⎯),thebest-knownofwhichtheouterleafletoftheplasmamembranewiththeirareplasmalogensandplatelet-activatingfactor(PAF).oligosaccharidechainsfacingoutward,formpartoftheGlycerol3-phosphateanddihydroxyacetonephosphateglycocalyxofthecellsurfaceandareimportant(1)inareintermediatesinglycolysis,makingaveryimportantcelladhesionandcellrecognition;(2)asreceptorsforconnectionbetweencarbohydrateandlipidmetabo-bacterialtoxins(eg,thetoxinthatcausescholera);andlism.(3)asABObloodgroupsubstances.Adozenorsogly-colipidstoragediseaseshavebeendescribed(eg,Gaucher’sdisease,Tay-Sachsdisease),eachduetoage-neticdefectinthepathwayforglycolipiddegradationinthelysosomes.Glycerol3-phosphateDihydroxyacetonephosphateHYDROLYSISINITIATESCATABOLISMOFTRIACYLGLYCEROLSPhosphatidatePlasmalogensPAFTriacylglycerolsmustbehydrolyzedbyalipasetotheirconstituentfattyacidsandglycerolbeforefurthercatab-olismcanproceed.Muchofthishydrolysis(lipolysis)DiacylglycerolCardiolipinPhosphatidylinositoloccursinadiposetissuewithreleaseoffreefattyacidsintotheplasma,wheretheyarefoundcombinedwithserumalbumin.Thisisfollowedbyfreefattyacidup-PhosphatidylcholineTriacylglycerolPhosphatidylinositoltakeintotissues(includingliver,heart,kidney,muscle,Phosphatidylethanolamine4,5-bisphosphatelung,testis,andadiposetissue,butnotreadilybybrain),wheretheyareoxidizedorreesterified.Theuti-Figure24–1.Overviewofacylglycerolbiosynthesis.lizationofglyceroldependsuponwhethersuchtissues(PAF,platelet-activatingfactor.)197
196ATPADPNAD+NADH+H+H2COHH2COHH2COHHOCHHOCHCOGlycolysisH2COHGLYCEROLKINASEH2COPGLYCEROL-H2COP3-PHOSPHATEGlycerols-GlycerolnDihydroxyacetoneDEHYDROGENASE3-phosphatephosphateAcyl-CoA(mainlysaturated)GLYCEROL-23-PHOSPHATEACYLTRANSFERASECoAOH2COCR1HOCHH2COHH2COPR2COCH1-Acylglycerol-3-phosphateOH2COH(lysophosphatidate)2-MonoacylglycerolAcyl-CoA(usuallyunsaturated)1-ACYLGLYCEROL-3-PHOSPHATEACYLTRANSFERASEAcyl-CoA1CoAMONOACYLGLYCEROLACYLTRANSFERASEO(INTESTINE)H2COCR1CoAR2COCHOH2COP1,2-Diacylglycerolphosphate(phosphatidate)CholineH2OCTPATPPHOSPHATIDATECDP-DGPHOSPHOHYDROLASESYNTHASECHOLINEKINASEP1PP1ADPOOPhosphocholineH2COCR1H2COCR1CTPR2COCHR2COCHCTP:PHOSPHOCHOLINEOH2COHOH2COPPCYTIDYL1,2-DiacylglycerolTRANSFERASECytidineCDP-diacylglycerolCardiolipinPP1CDP-cholineAcyl-CoAInositolCDP-CHOLINE:DIACYLGLYCEROLDIACYLGLYCEROLPHOSPHATIDYL-PHOSPHOCHOLINEACYLTRANSFERASEINOSITOLSYNTHASETRANSFERASECMPCoACMPATPADPOOOKINASEOH2COCR1H2COCR1H2COCR1H2COCR1R2COCHR2COCHOR2COCHR2COCHOHC2OPOHC2OCR3OHC2OPOHC2OPInositolPTriacyglycerolCholineInositolPhosphatidylinositol4-phosphatePhosphatidylinositolPhosphatidylcholineATPPHOSPHATIDYLETHANOLAMINEN-METHYLTRANSFERASE(–CH3)3KINASEPhosphatidylethanolamineSerineCO2OADPH2COCR1PhosphatidylserineEthanolamineR2COCHFigure24–2.Biosynthesisoftriacylglycerolandphospholipids.OHC2OPInositolP(1,Monoacylglycerolpathway;2,glycerolphosphatepathway.)PPhosphatidylinositol4,5-bisphosphatePhosphatidylethanolaminemaybeformedfromethanolaminebyapathwaysimilartothatshownfortheformationofphosphatidyl-cholinefromcholine.
197METABOLISMOFACYLGLYCEROLS&SPHINGOLIPIDS/199PhosphatidateIstheCommonPrecursorfromphosphatidylglycerol,whichinturnissynthesizedintheBiosynthesisofTriacylglycerols,fromCDP-diacylglycerol(Figure24–2)andglycerolManyPhosphoglycerols,&Cardiolipin3-phosphateaccordingtotheschemeshowninFigure24–3.Cardiolipin,foundintheinnermembraneofBothglycerolandfattyacidsmustbeactivatedbyATPmitochondria,isspecificallyrequiredforthefunction-beforetheycanbeincorporatedintoacylglycerols.ingofthephosphatetransporterandforcytochromeGlycerolkinasecatalyzestheactivationofglyceroltooxidaseactivity.sn-glycerol3-phosphate.Iftheactivityofthisenzymeisabsentorlow,asinmuscleoradiposetissue,mostoftheglycerol3-phosphateisformedfromdihydroxyace-B.BIOSYNTHESISOFGLYCEROLETHERPHOSPHOLIPIDStonephosphatebyglycerol-3-phosphatedehydrogen-Thispathwayislocatedinperoxisomes.Dihydroxyace-ase(Figure24–2).tonephosphateistheprecursoroftheglycerolmoietyofglyceroletherphospholipids(Figure24–4).ThisA.BIOSYNTHESISOFTRIACYLGLYCEROLScompoundcombineswithacyl-CoAtogive1-acyldihy-Twomoleculesofacyl-CoA,formedbytheactivationdroxyacetonephosphate.Theetherlinkisformedinoffattyacidsbyacyl-CoAsynthetase(Chapter22),thenextreaction,producing1-alkyldihydroxyacetonecombinewithglycerol3-phosphatetoformphosphati-phosphate,whichisthenconvertedto1-alkylglyceroldate(1,2-diacylglycerolphosphate).Thistakesplacein3-phosphate.Afterfurtheracylationinthe2position,twostages,catalyzedbyglycerol-3-phosphateacyl-theresulting1-alkyl-2-acylglycerol3-phosphate(analo-transferaseand1-acylglycerol-3-phosphateacyltrans-goustophosphatidateinFigure24–2)ishydrolyzedtoferase.Phosphatidateisconvertedbyphosphatidategivethefreeglycerolderivative.Plasmalogens,whichphosphohydrolaseanddiacylglycerolacyltransferasecomprisemuchofthephospholipidinmitochondria,to1,2-diacylglycerolandthentriacylglycerol.Inintesti-areformedbydesaturationoftheanalogous3-phos-nalmucosa,monoacylglycerolacyltransferasecon-phoethanolaminederivative(Figure24–4).Platelet-vertsmonoacylglycerolto1,2-diacylglycerolintheactivatingfactor(PAF)(1-alkyl-2-acetyl-sn-glycerol-3-monoacylglycerolpathway.Mostoftheactivityofphosphocholine)issynthesizedfromthecorrespondingtheseenzymesresidesintheendoplasmicreticulumof3-phosphocholinederivative.Itisformedbymanythecell,butsomeisfoundinmitochondria.Phosphati-bloodcellsandothertissuesandaggregatesplateletsatdatephosphohydrolaseisfoundmainlyinthecytosol,concentrationsaslowas10−11mol/L.Italsohashy-buttheactiveformoftheenzymeismembrane-bound.potensiveandulcerogenicpropertiesandisinvolvedinInthebiosynthesisofphosphatidylcholineandavarietyofbiologicresponses,includinginflammation,phosphatidylethanolamine(Figure24–2),cholineorchemotaxis,andproteinphosphorylation.ethanolaminemustfirstbeactivatedbyphosphoryla-tionbyATPfollowedbylinkagetoCTP.TheresultingCDP-cholineorCDP-ethanolaminereactswith1,2-di-acylglyceroltoformeitherphosphatidylcholineorCDP-Diacyl-sn-Glycerolphosphatidylethanolamine,respectively.Phosphatidyl-glycerol3-phosphateserineisformedfromphosphatidylethanolaminedi-rectlybyreactionwithserine(Figure24–2).Phos-CMPphatidylserinemayre-formphosphatidylethanolaminebydecarboxylation.Analternativepathwayinliveren-PhosphatidylglycerolphosphateablesphosphatidylethanolaminetogiverisedirectlytoH2Ophosphatidylcholinebyprogressivemethylationoftheethanolamineresidue.Inspiteofthesesourcesofcholine,itisconsideredtobeanessentialnutrientinPimanymammalianspecies,butthishasnotbeenestab-Phosphatidylglycerollishedinhumans.Theregulationoftriacylglycerol,phosphatidyl-choline,andphosphatidylethanolaminebiosynthesisisdrivenbytheavailabilityoffreefattyacids.Thosethatescapeoxidationarepreferentiallyconvertedtophos-CMPpholipids,andwhenthisrequirementissatisfiedtheyCardiolipinareusedfortriacylglycerolsynthesis.(diphosphatidylglycerol)Aphospholipidpresentinmitochondriaiscardio-lipin(diphosphatidylglycerol;Figure14–8).ItisformedFigure24–3.Biosynthesisofcardiolipin.
198200/CHAPTER24NADPHOR2(CH2)2OH+H+NADP+Acyl-CoAH2COHH2COCR1H2CO(CH2)2R2H2CO(CH2)2R2OCOCOCHOCHH2COPACYL-H2COPSYNTHASEH2COPREDUCTASEH2COPTRANSFERASEHOOCR1Dihydroxyacetone1-Acyldihydroxyacetone1-Alkyldihydroxyacetone1-Alkylglycerol3-phosphatephosphatephosphatephosphateAcyl-CoAACYL-*TRANSFERASECDP-CMPEthanolaminePiH2OOH2CO(CH2)2R2OH2CO(CH2)2R2OH2CO(CH2)2R2R3COCHR3COCHR3COCHH2COPCH2CH2NH2CDP-ETHANOLAMINE:H2COHPHOSPHOHYDROLASEH2COPALKYLACYLGLYCEROLPHOSPHOETHANOLAMINE1-Alkyl-2-acylglycerolTRANSFERASE3-phosphoethanolamine1-Alkyl-2-acylglycerol3-phosphate1-Alkyl-2-acylglycerolCDP-cholineNADPH,O2,CDP-CHOLINE:DESATURASECytb5ALKYLACYLGLYCEROLAlkyl,diacylglycerolsPHOSPHOCHOLINETRANSFERASECMPOH2COCHCHR2OH2CO(CH2)2R2R3COCHH2OR3COOHR3COCHH2CO(CH2)2R2H2COP(CH2)2NH2H2COPHOCH1-Alkenyl-2-acylglycerolPHOSPHOLIPASEA2H2COPCholine3-phosphoethanolamineplasmalogen1-Alkyl-2-acylglycerolCholine3-phosphocholine1-Alkyl-2-lysoglycerolAcetyl-CoA3-phosphocholineACETYLTRANSFERASEOH2CO(CH2)2R2H3CCOCHH2COPCholine1-Alkyl-2-acetylglycerol3-phosphocholinePAFFigure24–4.Biosynthesisofetherlipids,includingplasmalogens,andplatelet-activatingfactor(PAF).InthedenovopathwayforPAFsynthesis,acetyl-CoAisincorporatedatstage*,avoidingthelasttwostepsinthepath-wayshownhere.PhospholipasesAllowDegradationsolecithin)isattackedbylysophospholipase,forming&RemodelingofPhosphoglycerolsthecorrespondingglycerylphosphorylbase,whichinturnmaybesplitbyahydrolaseliberatingglycerolAlthoughphospholipidsareactivelydegraded,each3-phosphateplusbase.PhospholipasesA1,A2,B,C,portionofthemoleculeturnsoveratadifferentrate—andDattackthebondsindicatedinFigure24–6.eg,theturnovertimeofthephosphategroupisdiffer-PhospholipaseA2isfoundinpancreaticfluidandentfromthatofthe1-acylgroup.Thisisduetothesnakevenomaswellasinmanytypesofcells;phos-presenceofenzymesthatallowpartialdegradationfol-pholipaseCisoneofthemajortoxinssecretedbybac-lowedbyresynthesis(Figure24–5).PhospholipaseA2teria;andphospholipaseDisknowntobeinvolvedincatalyzesthehydrolysisofglycerophospholipidstoformmammaliansignaltransduction.afreefattyacidandlysophospholipid,whichinturnLysolecithin(lysophosphatidylcholine)maybemaybereacylatedbyacyl-CoAinthepresenceofanformedbyanalternativeroutethatinvolveslecithin:acyltransferase.Alternatively,lysophospholipid(eg,ly-cholesterolacyltransferase(LCAT).Thisenzyme,
199METABOLISMOFACYLGLYCEROLS&SPHINGOLIPIDS/201Ofoundinplasma,catalyzesthetransferofafattyacidresiduefromthe2positionoflecithintocholesteroltoOH2COCR1formcholesterylesterandlysolecithinandisconsideredR2COCHtoberesponsibleformuchofthecholesterylesterinH2COPCholineplasmalipoproteins.Long-chainsaturatedfattyacidsarefoundpredominantlyinthe1positionofphospho-Phosphatidylcholinelipids,whereasthepolyunsaturatedacids(eg,thepre-H2Ocursorsofprostaglandins)areincorporatedmoreintothe2position.TheincorporationoffattyacidsintoACYLTRANSFERASEPHOSPHOLIPASEA2lecithinoccursbycompletesynthesisofthephospho-R2COOHlipid,bytransacylationbetweencholesterylesterandOlysolecithin,andbydirectacylationoflysolecithinbyacyl-CoA.Thus,acontinuousexchangeofthefattyH2COCR1acidsispossible,particularlywithregardtointroducingHOCHessentialfattyacidsintophospholipidmolecules.H2COPCholineAcyl-CoALysophosphatidylcholine(lysolecithin)ALLSPHINGOLIPIDSAREFORMEDHOFROMCERAMIDE2LYSOPHOSPHOLIPASECeramideissynthesizedintheendoplasmicreticulumfromtheaminoacidserineaccordingtoFigure24–7.R1COOHCeramideisanimportantsignalingmolecule(secondmessenger)regulatingpathwaysincludingapoptosisH2COH(processesleadingtocelldeath),cellsenescence,andHOCHdifferentiation,andopposessomeoftheactionsofdi-H2COPCholineacylglycerol.GlycerylphosphocholineSphingomyelins(Figure14–11)arephospholipidsandareformedwhenceramidereactswithphos-H2Ophatidylcholinetoformsphingomyelinplusdiacylglyc-GLYCERYLPHOSPHO-erol(Figure24–8A).ThisoccursmainlyintheGolgiCHOLINEHYDROLASEapparatusandtoalesserextentintheplasmamem-brane.H2COHGlycosphingolipidsAreaCombinationHOCH+CholineofCeramideWithOneorMoreH2COPSugarResiduessn-Glycerol3-phosphateThesimplestglycosphingolipids(cerebrosides)areFigure24–5.Metabolismofphosphatidylcholinegalactosylceramide(GalCer)andglucosylceramide(lecithin).(GlcCer).GalCerisamajorlipidofmyelin,whereasGlcCeristhemajorglycosphingolipidofextraneuraltissuesandaprecursorofmostofthemorecomplexPHOSPHOLIPASEBPHOSPHOLIPASEA1glycosphingolipids.Galactosylceramide(Figure24–8B)OisformedinareactionbetweenceramideandUDPGal(formedbyepimerizationfromUDPGlc—FigureH2COCR1O20–6).SulfogalactosylceramideandothersulfolipidsPHOSPHOLIPASEDsuchasthesulfo(galacto)-glycerolipidsandtheR2COCHOsteroidsulfatesareformedafterfurtherreactionsin-volving3′-phosphoadenosine-5′-phosphosulfate(PAPS;H2COPON-BASE“activesulfate”).GangliosidesaresynthesizedfromPHOSPHOLIPASEA–2Oceramidebythestepwiseadditionofactivatedsugars(eg,PHOSPHOLIPASECUDPGlcandUDPGal)andasialicacid,usuallyN-acetylneuraminicacid(Figure24–9).AlargenumberFigure24–6.Sitesofthehydrolyticactivityofphos-ofgangliosidesofincreasingmolecularweightmaybepholipasesonaphospholipidsubstrate.formed.Mostoftheenzymestransferringsugarsfrom
200202/CHAPTER24+nucleotidesugars(glycosyltransferases)arefoundinONH3theGolgiapparatus.CH(CH)CSCoA−OOCCHCHOH32142GlycosphingolipidsareconstituentsoftheouterPalmitoyl-CoASerineleafletofplasmamembranesandareimportantincelladhesionandcellrecognition.Someareantigens,eg,ABObloodgroupsubstances.Certaingangliosides2+Pyridoxalphosphate,Mnfunctionasreceptorsforbacterialtoxins(eg,forcholeratoxin,whichsubsequentlyactivatesadenylylcyclase).SERINEPALMITOYLTRANSFERASECLINICALASPECTSCoASHCO2ODeficiencyofLungSurfactantCausesRespiratoryDistressSyndromeCH3(CH2)12CH2CH2CCHCH2OH+LungsurfactantiscomposedmainlyoflipidwithNH33-Ketosphinganinesomeproteinsandcarbohydrateandpreventsthealve-NADPH+H+olifromcollapsing.Surfactantactivityislargelyattrib-utedtodipalmitoylphosphatidylcholine,whichis3-KETOSPHINGANINEREDUCTASEsynthesizedshortlybeforeparturitioninfull-termin-+fants.DeficiencyoflungsurfactantinthelungsofNADPmanypretermnewbornsgivesrisetorespiratorydis-CH3(CH2)12CH2CH2CHCHCH2OHtresssyndrome.Administrationofeithernaturalorar-OHNH+tificialsurfactanthasbeenoftherapeuticbenefit.3Dihydrosphingosine(sphinganine)Phospholipids&SphingolipidsRCOSCoAAreInvolvedinMultipleSclerosisAcyl-CoADIHYDROSPHINGOSINEN-ACYLTRANSFERASEandLipidosesCoASHCertaindiseasesarecharacterizedbyabnormalquanti-CH3(CH2)12CH2CH2CHCHCH2OHtiesoftheselipidsinthetissues,ofteninthenervoussystem.Theymaybeclassifiedintotwogroups:(1)trueOHNHCORdemyelinatingdiseasesand(2)sphingolipidoses.DihydroceramideInmultiplesclerosis,whichisademyelinatingdis-DIHYDROCERAMIDEease,thereislossofbothphospholipids(particularly2HDESATURASEethanolamineplasmalogen)andofsphingolipidsfromwhitematter.Thus,thelipidcompositionofwhiteCH3(CH2)12CHCHCHCHCH2OHmatterresemblesthatofgraymatter.Thecerebrospinalfluidshowsraisedphospholipidlevels.OHNHCORThesphingolipidoses(lipidstoragediseases)areaCeramidegroupofinheriteddiseasesthatareoftenmanifestedinFigure24–7.Biosynthesisofceramide.childhood.Thesediseasesarepartofalargergroupoflysosomaldisordersandexhibitseveralconstantfea-tures:(1)Complexlipidscontainingceramideaccumu-lateincells,particularlyneurons,causingneurodegen-ACeramideSphingomyelinPhosphatidylcholineDiacylglycerolFigure24–8.Biosynthesisofsphingomyelin(A),UDPGalUDPPAPSgalactosylceramideanditssulfoderivative(B).(PAPS,Sulfogalactosyl-Galactosylceramideceramide“activesulfate,”adenosine3′-phosphate-5′-phospho-BCeramide(cerebroside)(sulfatide)sulfate.)
201METABOLISMOFACYLGLYCEROLS&SPHINGOLIPIDS/203UDPGlcUDPUDPGalUDPCMP-NeuAcCMPGlucosylCeramideceramideCer-Glc-GalCer-Glc-Gal(Cer-Glc)NeuAc(GM3)UDP-N-acetylgalactosamineUDPUDPGalUDPHighergangliosidesCer-Glc-Gal-GalNAc-GalCer-Glc-Gal-GalNAc(disialo-andtrisialo-gangliosides)NeuAcNeuAc(GM1)(GM2)Figure24–9.Biosynthesisofgangliosides.(NeuAc,N-acetylneuraminicacid.)erationandshorteningthelifespan.(2)Therateofdase)inthetreatmentofGaucher’sdisease.Arecentsynthesisofthestoredlipidisnormal.(3)Theenzy-promisingapproachissubstratereductiontherapytomaticdefectisinthelysosomaldegradationpathwayinhibitthesynthesisofsphingolipids,andgenetherapyofsphingolipids.(4)Theextenttowhichtheactivityofforlysosomaldisordersiscurrentlyunderinvestigation.theaffectedenzymeisdecreasedissimilarinalltissues.Someexamplesofthemoreimportantlipidstoragedis-Thereisnoeffectivetreatmentformanyofthediseases,easesareshowninTable24–1.thoughsomesuccesshasbeenachievedwithenzymesMultiplesulfatasedeficiencyresultsinaccumula-thathavebeenchemicallymodifiedtoensurebindingtionofsulfogalactosylceramide,steroidsulfates,andtoreceptorsoftargetcells,eg,tomacrophagesintheproteoglycansowingtoacombineddeficiencyofaryl-liverinordertodeliverβ-glucosidase(glucocerebrosi-sulfatasesA,B,andCandsteroidsulfatase.Table24–1.Examplesofsphingolipidoses.1DiseaseEnzymeDeficiencyLipidAccumulatingClinicalSymptoms:Tay-SachsdiseaseHexosaminidaseACer—Glc—Gal(NeuAc)—GalNAcMentalretardation,blindness,muscularweakness.:GM2Ganglioside:Fabry’sdiseaseα-GalactosidaseCer—Glc—Gal—GalSkinrash,kidneyfailure(fullsymptomsonlyin:Globotriaosylceramidemales;X-linkedrecessive).:MetachromaticArylsulfataseACer—Gal—:OSO3Mentalretardationandpsychologicdisturbancesinleukodystrophy3-Sulfogalactosylceramideadults;demyelination.:Krabbe’sdiseaseβ-GalactosidaseCer—GalMentalretardation;myelinalmostabsent.:Galactosylceramide:Gaucher’sdiseaseβ-GlucosidaseCer—GlcEnlargedliverandspleen,erosionoflongbones,:Glucosylceramidementalretardationininfants.:Niemann-PickSphingomyelinaseCer—P—cholineEnlargedliverandspleen,mentalretardation;fatalin:diseaseSphingomyelinearlylife.:Farber’sdiseaseCeramidaseAcyl—SphingosineHoarseness,dermatitis,skeletaldeformation,mental:Ceramideretardation;fatalinearlylife.1NeuAc,N-acetylneuraminicacid;Cer,ceramide;Glc,glucose;Gal,galactose.—:,siteofdeficientenzymereaction.:
202204/CHAPTER24SUMMARY(demyelination),andsphingolipidoses(inabilitytobreakdownsphingolipidsinlysosomesduetoinher-•Triacylglycerolsarethemajorenergy-storinglipids,iteddefectsinhydrolaseenzymes).whereasphosphoglycerols,sphingomyelin,andgly-cosphingolipidsareamphipathicandhavestructuralfunctionsincellmembranesaswellasotherspecial-izedroles.REFERENCES•Triacylglycerolsandsomephosphoglycerolsaresyn-GrieseM:Pulmonarysurfactantinhealthandhumanlungdis-thesizedbyprogressiveacylationofglycerol3-phos-eases:stateoftheart.EurRespirJ1999;13:1455.phate.Thepathwaybifurcatesatphosphatidate,MerrillAH,SweeleyCC:Sphingolipids:metabolismandcellsig-forminginositolphospholipidsandcardiolipinonnaling.In:BiochemistryofLipids,LipoproteinsandMem-theonehandandtriacylglycerolandcholineandbranes.VanceDE,VanceJE(editors).Elsevier,1996.ethanolaminephospholipidsontheother.PrescottSMetal:Platelet-activatingfactorandrelatedlipidmedia-•Plasmalogensandplatelet-activatingfactor(PAF)aretors.AnnuRevBiochem2000;69:419.etherphospholipidsformedfromdihydroxyacetoneRuvoloPP:Ceramideregulatescellularhomeostasisviadiversestresssignalingpathways.Leukemia2001;15:1153.phosphate.SchuetteCGetal:Theglycosphingolipidoses—fromdiseaseto•Sphingolipidsareformedfromceramide(N-acyl-basicprinciplesofmetabolism.BiolChem1999;380:759.sphingosine).Sphingomyelinispresentinmem-ScriverCRetal(editors):TheMetabolicandMolecularBasesofIn-branesoforganellesinvolvedinsecretoryprocessesheritedDisease,8thed.McGraw-Hill,2001.(eg,Golgiapparatus).Thesimplestglycosphin-TijburgLBM,GeelenMJH,vanGoldeLMG:Regulationofthegolipidsareacombinationofceramideplusasugarbiosynthesisoftriacylglycerol,phosphatidylcholineandphos-residue(eg,GalCerinmyelin).Gangliosidesarephatidylethanolamineintheliver.BiochimBiophysActamorecomplexglycosphingolipidscontainingmore1989;1004:1.sugarresiduesplussialicacid.TheyarepresentintheVanceDE:Glycerolipidbiosynthesisineukaryotes.In:Biochem-outerlayeroftheplasmamembrane,wheretheycon-istryofLipids,LipoproteinsandMembranes.VanceDE,VanceJE(editors).Elsevier,1996.tributetotheglycocalyxandareimportantasanti-vanEchtenG,SandhoffK:Gangliosidemetabolism.Enzymology,gensandcellreceptors.topology,andregulation.JBiolChem1993;268:5341.•Phospholipidsandsphingolipidsareinvolvedinsev-WaiteM:Phospholipases.In:BiochemistryofLipids,Lipoproteinseraldiseaseprocesses,includingrespiratorydistressandMembranes.VanceDE,VanceJE(editors).Elsevier,syndrome(lackoflungsurfactant),multiplesclerosis1996.
203LipidTransport&Storage25PeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DScBIOMEDICALIMPORTANCEFourMajorGroupsofPlasmaLipoproteinsHaveBeenIdentifiedFatabsorbedfromthedietandlipidssynthesizedbytheliverandadiposetissuemustbetransportedbetweenBecausefatislessdensethanwater,thedensityofathevarioustissuesandorgansforutilizationandstor-lipoproteindecreasesastheproportionoflipidtopro-age.Sincelipidsareinsolubleinwater,theproblemofteinincreases(Table25–1).InadditiontoFFA,fourhowtotransportthemintheaqueousbloodplasmaismajorgroupsoflipoproteinshavebeenidentifiedthatsolvedbyassociatingnonpolarlipids(triacylglycerolareimportantphysiologicallyandinclinicaldiagnosis.andcholesterylesters)withamphipathiclipids(phos-Theseare(1)chylomicrons,derivedfromintestinalpholipidsandcholesterol)andproteinstomakewater-absorptionoftriacylglycerolandotherlipids;(2)verymisciblelipoproteins.lowdensitylipoproteins(VLDL,orpre-β-lipopro-Inameal-eatingomnivoresuchasthehuman,ex-teins),derivedfromtheliverfortheexportoftriacyl-cesscaloriesareingestedintheanabolicphaseoftheglycerol;(3)low-densitylipoproteins(LDL,orβ-feedingcycle,followedbyaperiodofnegativecaloriclipoproteins),representingafinalstageinthecatabolismbalancewhentheorganismdrawsuponitscarbohy-ofVLDL;and(4)high-densitylipoproteins(HDL,ordrateandfatstores.Lipoproteinsmediatethiscyclebyα-lipoproteins),involvedinVLDLandchylomicrontransportinglipidsfromtheintestinesaschylomi-metabolismandalsoincholesteroltransport.Triacyl-crons—andfromtheliverasverylowdensitylipopro-glycerolisthepredominantlipidinchylomicronsandteins(VLDL)—tomosttissuesforoxidationandtoVLDL,whereascholesterolandphospholipidaretheadiposetissueforstorage.Lipidismobilizedfromadi-predominantlipidsinLDLandHDL,respectivelyposetissueasfreefattyacids(FFA)attachedtoserum(Table25–1).Lipoproteinsmaybeseparatedaccordingalbumin.Abnormalitiesoflipoproteinmetabolismtotheirelectrophoreticpropertiesinto-,-,andpre-causevarioushypo-orhyperlipoproteinemias.The-lipoproteins.mostcommonoftheseisdiabetesmellitus,wherein-sulindeficiencycausesexcessivemobilizationofFFAandunderutilizationofchylomicronsandVLDL,lead-LipoproteinsConsistofaNonpolaringtohypertriacylglycerolemia.Mostotherpatho-Core&aSingleSurfaceLayeroflogicconditionsaffectinglipidtransportareduepri-AmphipathicLipidsmarilytoinheriteddefects,someofwhichcauseThenonpolarlipidcoreconsistsofmainlytriacylglyc-hypercholesterolemia,andprematureatherosclerosis.erolandcholesterylesterandissurroundedbyasin-Obesity—particularlyabdominalobesity—isariskfac-glesurfacelayerofamphipathicphospholipidandtorforincreasedmortality,hypertension,type2dia-cholesterolmolecules(Figure25–1).Theseareorientedbetesmellitus,hyperlipidemia,hyperglycemia,andvari-sothattheirpolargroupsfaceoutwardtotheaqueousousendocrinedysfunctions.medium,asinthecellmembrane(Chapter14).Theproteinmoietyofalipoproteinisknownasanapo-LIPIDSARETRANSPORTEDINTHElipoproteinorapoprotein,constitutingnearly70%ofPLASMAASLIPOPROTEINSsomeHDLandaslittleas1%ofchylomicrons.Someapolipoproteinsareintegralandcannotberemoved,FourMajorLipidClassesArePresentwhereasothersarefreetotransfertootherlipoproteins.inLipoproteinsPlasmalipidsconsistoftriacylglycerols(16%),phos-TheDistributionofApolipoproteinspholipids(30%),cholesterol(14%),andcholesterylCharacterizestheLipoproteinesters(36%)andamuchsmallerfractionofunesteri-fiedlong-chainfattyacids(freefattyacids)(4%).ThisOneormoreapolipoproteins(proteinsorpolypeptides)latterfraction,thefreefattyacids(FFA),ismetaboli-arepresentineachlipoprotein.Themajorapolipopro-callythemostactiveoftheplasmalipids.teinsofHDL(α-lipoprotein)aredesignatedA(Table205
204206/CHAPTER25Table25–1.Compositionofthelipoproteinsinplasmaofhumans.CompositionDiameterDensityProteinLipidMainLipidLipoproteinSource(nm)(g/mL)(%)(%)ComponentsApolipoproteins1ChylomicronsIntestine90–1000<0.951–298–99TriacylglycerolA-I,A-II,A-IV,B-48,C-I,C-II,C-III,EChylomicronChylomicrons45–150<1.0066–892–94Triacylglycerol,B-48,Eremnantsphospholipids,cholesterolVLDLLiver(intestine)30–900.95–1.0067–1090–93TriacylglycerolB-100,C-I,C-II,C-IIIIDLVLDL25–351.006–1.0191189Triacylglycerol,B-100,EcholesterolLDLVLDL20–251.019–1.0632179CholesterolB-1002HDLLiver,intestine,Phospholipids,A-I,A-II,A-IV,C-I,C-II,C-III,D,EHDL1VLDL,chylo-20–251.019–1.0633268cholesterolmicronsHDL210–201.063–1.1253367HDL35–101.125–1.21057433Preβ-HDL<5>1.210A-IAlbumin/freeAdipose>1.281991FreefattyacidsfattyacidstissueAbbreviations:HDL,high-densitylipoproteins;IDL,intermediate-densitylipoproteins;LDL,low-densitylipoproteins;VLDL,verylowdensitylipoproteins.1SecretedwithchylomicronsbuttransferstoHDL.2AssociatedwithHDL2andHDL3subfractions.3Partofaminorfractionknownasveryhighdensitylipoproteins(VHDL).25–1).ThemainapolipoproteinofLDL(β-lipopro-teinreceptorsintissues,eg,apoB-100andapoEfortein)isapolipoproteinB(B-100)andisfoundalsointheLDLreceptor,apoEfortheLDLreceptor-relatedVLDL.Chylomicronscontainatruncatedformofapoprotein(LRP),whichhasbeenidentifiedastherem-B(B-48)thatissynthesizedintheintestine,whilenantreceptor,andapoA-IfortheHDLreceptor.TheB-100issynthesizedintheliver.ApoB-100isoneoffunctionsofapoA-IVandapoD,however,arenotyetthelongestsinglepolypeptidechainsknown,havingclearlydefined.4536aminoacidsandamolecularmassof550,000Da.ApoB-48(48%ofB-100)isformedfromthesamemRNAasapoB-100aftertheintroductionofastopsig-FREEFATTYACIDSAREnalbyanRNAeditingenzyme.ApoC-I,C-II,andRAPIDLYMETABOLIZEDC-IIIaresmallerpolypeptides(molecularmass7000–9000Da)freelytransferablebetweenseveraldifferentThefreefattyacids(FFA,nonesterifiedfattyacids,un-lipoproteins.ApoEisfoundinVLDL,HDL,chylomi-esterifiedfattyacids)ariseintheplasmafromlipolysiscrons,andchylomicronremnants;itaccountsfor5–oftriacylglycerolinadiposetissueorasaresultofthe10%oftotalVLDLapolipoproteinsinnormalsubjects.actionoflipoproteinlipaseduringuptakeofplasmatri-Apolipoproteinscarryoutseveralroles:(1)theycanacylglycerolsintotissues.Theyarefoundincombina-formpartofthestructureofthelipoprotein,eg,apoB;tionwithalbumin,averyeffectivesolubilizer,incon-(2)theyareenzymecofactors,eg,C-IIforlipoproteincentrationsvaryingbetween0.1and2.0μeq/mLoflipase,A-Iforlecithin:cholesterolacyltransferase,oren-plasma.Levelsarelowinthefullyfedconditionandzymeinhibitors,eg,apoA-IIandapoC-IIIforlipopro-riseto0.7–0.8μeq/mLinthestarvedstate.Inuncon-teinlipase,apoC-Iforcholesterylestertransferprotein;trolleddiabetesmellitus,thelevelmayrisetoasmuchand(3)theyactasligandsforinteractionwithlipopro-as2μeq/mL.
205LIPIDTRANSPORT&STORAGE/207Peripheralapoproteinarealsotobefoundinchyle;however,mostofthe(eg,apoC)plasmaVLDLareofhepaticorigin.Theyarethevehi-clesoftransportoftriacylglycerolfromthelivertoFreePhospholipidtheextrahepatictissues.cholesterolTherearestrikingsimilaritiesinthemechanismsofCholesterylformationofchylomicronsbyintestinalcellsandofesterVLDLbyhepaticparenchymalcells(Figure25–2),per-Triacylglycerolhapsbecause—apartfromthemammarygland—theintestineandliveraretheonlytissuesfromwhichpar-ticulatelipidissecreted.Newlysecretedor“nascent”chylomicronsandVLDLcontainonlyasmallamountofapolipoproteinsCandE,andthefullcomplementisacquiredfromHDLinthecirculation(Figures25–3Coreofmainlyand25–4).ApoBisessentialforchylomicronandnonpolarlipidsVLDLformation.Inabetalipoproteinemia(araredis-ease),lipoproteinscontainingapoBarenotformedandIntegralapoproteinMonolayerofmainlylipiddropletsaccumulateintheintestineandliver.(eg,apoB)amphipathiclipidsAmoredetailedaccountofthefactorscontrollinghepaticVLDLsecretionisgivenbelow.Figure25–1.Generalizedstructureofaplasmalipoprotein.Thesimilaritieswiththestructureoftheplasmamembranearetobenoted.SmallamountsofCHYLOMICRONS&VERYLOWcholesterylesterandtriacylglycerolaretobefoundinDENSITYLIPOPROTEINSAREthesurfacelayerandalittlefreecholesterolinthecore.RAPIDLYCATABOLIZEDTheclearanceoflabeledchylomicronsfromthebloodFreefattyacidsareremovedfromthebloodex-israpid,thehalf-timeofdisappearancebeingunder1tremelyrapidlyandoxidized(fulfilling25–50%ofen-hourinhumans.Largerparticlesarecatabolizedmoreergyrequirementsinstarvation)oresterifiedtoformquicklythansmallerones.Fattyacidsoriginatingfromtriacylglycerolinthetissues.Instarvation,esterifiedchylomicrontriacylglycerolaredeliveredmainlytoadi-lipidsfromthecirculationorinthetissuesareoxidizedposetissue,heart,andmuscle(80%),whileabout20%aswell,particularlyinheartandskeletalmusclecells,goestotheliver.However,theliverdoesnotmetabo-whereconsiderablestoresoflipidaretobefound.lizenativechylomicronsorVLDLsignificantly;thus,Thefreefattyaciduptakebytissuesisrelateddi-thefattyacidsinthelivermustbesecondarytotheirrectlytotheplasmafreefattyacidconcentration,whichmetabolisminextrahepatictissues.inturnisdeterminedbytherateoflipolysisinadiposetissue.Afterdissociationofthefattyacid-albumincom-plexattheplasmamembrane,fattyacidsbindtoaTriacylglycerolsofChylomicrons&VLDLmembranefattyacidtransportproteinthatactsasaAreHydrolyzedbyLipoproteinLipasetransmembranecotransporterwithNa+.OnenteringLipoproteinlipaseislocatedonthewallsofbloodcap-thecytosol,freefattyacidsareboundbyintracellularillaries,anchoredtotheendotheliumbynegativelyfattyacid-bindingproteins.Theroleoftheseproteinschargedproteoglycanchainsofheparansulfate.Ithasinintracellulartransportisthoughttobesimilartothatbeenfoundinheart,adiposetissue,spleen,lung,renalofserumalbumininextracellulartransportoflong-medulla,aorta,diaphragm,andlactatingmammarychainfattyacids.gland,thoughitisnotactiveinadultliver.Itisnotnormallyfoundinblood;however,followinginjectionTRIACYLGLYCEROLISTRANSPORTEDofheparin,lipoproteinlipaseisreleasedfromitshep-FROMTHEINTESTINESINaransulfatebindingintothecirculation.Hepaticli-CHYLOMICRONS&FROMTHELIVERINpaseisboundtothesinusoidalsurfaceoflivercellsandVERYLOWDENSITYLIPOPROTEINSisreleasedbyheparin.Thisenzyme,however,doesnotreactreadilywithchylomicronsorVLDLbutiscon-Bydefinition,chylomicronsarefoundinchyleformedcernedwithchylomicronremnantandHDLmetabo-onlybythelymphaticsystemdrainingtheintestine.lism.TheyareresponsibleforthetransportofalldietaryBothphospholipidsandapoC-IIarerequiredaslipidsintothecirculation.SmallquantitiesofVLDLcofactorsforlipoproteinlipaseactivity,whileapoA-II
206208/CHAPTER25AIntestinallumenB•••••••••RER••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••G•••••••••••RERSER•••••••••••••••••••••••••••••••••••••••••••••N••••••SERBileGcanaliculusNCVLDLFenestraSDEndothelialcellBloodLymphvesselleadingEcapillarytothoracicductLumenofbloodsinusoidFigure25–2.Theformationandsecretionof(A)chylomicronsbyanintestinalcelland(B)verylowdensitylipoproteinsbyahepaticcell.(RER,roughendoplasmicreticulum;SER,smoothendoplasmicreticulum;G,Golgiapparatus;N,nucleus;C,chylomicrons;VLDL,verylowdensitylipoproteins;E,endothelium;SD,spaceofDisse,containingbloodplasma.)ApolipoproteinB,synthesizedintheRER,isincorporatedintolipoproteinsintheSER,themainsiteofsynthesisoftriacylglycerol.AfteradditionofcarbohydrateresiduesinG,theyarereleasedfromthecellbyreversepinocytosis.Chylomicronspassintothelymphaticsystem.VLDLaresecretedintothespaceofDisseandthenintothehepaticsinusoidsthroughfenestraeintheendotheliallining.andapoC-IIIactasinhibitors.HydrolysistakesplaceTheActionofLipoproteinLipaseFormswhilethelipoproteinsareattachedtotheenzymeonRemnantLipoproteinstheendothelium.Triacylglycerolishydrolyzedprogres-sivelythroughadiacylglyceroltoamonoacylglycerolReactionwithlipoproteinlipaseresultsinthelossofthatisfinallyhydrolyzedtofreefattyacidplusglycerol.approximately90%ofthetriacylglycerolofchylomi-Someofthereleasedfreefattyacidsreturntothecircu-cronsandinthelossofapoC(whichreturnstoHDL)lation,attachedtoalbumin,butthebulkistransportedbutnotapoE,whichisretained.Theresultingchy-intothetissue(Figures25–3and25–4).Heartlipopro-lomicronremnantisabouthalfthediameteroftheteinlipasehasalowKmfortriacylglycerol,aboutone-parentchylomicronandisrelativelyenrichedincholes-tenthofthatfortheenzymeinadiposetissue.Thisen-terolandcholesterylestersbecauseofthelossoftriacyl-ablesthedeliveryoffattyacidsfromtriacylglyceroltoglycerol(Figure25–3).SimilarchangesoccurtoberedirectedfromadiposetissuetotheheartintheVLDL,withtheformationofVLDLremnantsorIDLstarvedstatewhentheplasmatriacylglyceroldecreases.(intermediate-densitylipoprotein)(Figure25–4).Asimilarredirectiontothemammaryglandoccursduringlactation,allowinguptakeoflipoproteintriacyl-TheLiverIsResponsiblefortheUptakeglycerolfattyacidformilkfatsynthesis.TheVLDLre-ofRemnantLipoproteinsceptorplaysanimportantpartinthedeliveryoffattyacidsfromVLDLtriacylglyceroltoadipocytesbybind-Chylomicronremnantsaretakenupbytheliverbyre-ingVLDLandbringingitintoclosecontactwithceptor-mediatedendocytosis,andthecholesterylesterslipoproteinlipase.Inadiposetissue,insulinenhancesandtriacylglycerolsarehydrolyzedandmetabolized.lipoproteinlipasesynthesisinadipocytesanditsUptakeismediatedbyareceptorspecificforapoEtranslocationtotheluminalsurfaceofthecapillaryen-(Figure25–3),andboththeLDL(apoB-100,E)re-dothelium.ceptorandtheLRP(LDLreceptor-relatedprotein)
207LIPIDTRANSPORT&STORAGE/209DietaryTGNascentchylomicronB-48SMALLLymphaticsINTESTINETGChylomicronCApoEB-48,ACpoEXTRAHEPATICATGTISSUESELDLAC(apoB-100,E)CEPCCreceptorAHDLACholesterolpoLIPOPROTEINLIPASEA,ApoCFattyacidsB-48HLTGCFattyacidsLIVEREChylomicronGlycerolLRPreceptorremnantFigure25–3.Metabolicfateofchylomicrons.(A,apolipoproteinA;B-48,apolipoproteinB-48;C,apolipoproteinC;E,apolipoproteinE;HDL,high-densitylipoprotein;TG,triacylglycerol;C,cholesterolandcholesterylester;P,phospholipid;HL,hepaticlipase;LRP,LDLreceptor-relatedprotein.)Onlythepredominantlipidsareshown.arebelievedtotakepart.Hepaticlipasehasadualrole:gradedinextrahepatictissuesand70%intheliver.A(1)inactingasaligandtothelipoproteinand(2)inpositivecorrelationexistsbetweentheincidenceofhydrolyzingitstriacylglycerolandphospholipid.coronaryatherosclerosisandtheplasmaconcentra-VLDListheprecursorofIDL,whichisthencon-tionofLDLcholesterol.ForfurtherdiscussionofthevertedtoLDL.OnlyonemoleculeofapoB-100isregulationoftheLDLreceptor,seeChapter26.presentineachoftheselipoproteinparticles,andthisisconservedduringthetransformations.Thus,eachLDLHDLTAKESPARTINBOTHparticleisderivedfromonlyoneVLDLparticle(FigureLIPOPROTEINTRIACYLGLYCEROL25–4).TwopossiblefatesawaitIDL.Itcanbetakenup&CHOLESTEROLMETABOLISMbytheliverdirectlyviatheLDL(apoB-100,E)recep-tor,oritisconvertedtoLDL.Inhumans,arelativelyHDLissynthesizedandsecretedfrombothliverandlargeproportionformsLDL,accountingforthein-intestine(Figure25–5).However,apoCandapoEarecreasedconcentrationsofLDLinhumanscomparedsynthesizedintheliverandtransferredfromliverHDLwithmanyothermammals.tointestinalHDLwhenthelatterenterstheplasma.AmajorfunctionofHDListoactasarepositoryfortheLDLISMETABOLIZEDVIAapoCandapoErequiredinthemetabolismofchy-THELDLRECEPTORlomicronsandVLDL.NascentHDLconsistsofdiscoidphospholipidbilayerscontainingapoAandfreecholes-Theliverandmanyextrahepatictissuesexpresstheterol.TheselipoproteinsaresimilartotheparticlesLDL(B-100,E)receptor.ItissodesignatedbecauseitfoundintheplasmaofpatientswithadeficiencyoftheisspecificforapoB-100butnotB-48,whichlackstheplasmaenzymelecithin:cholesterolacyltransferasecarboxylterminaldomainofB-100containingthe(LCAT)andintheplasmaofpatientswithobstructiveLDLreceptorligand,anditalsotakesuplipoproteinsjaundice.LCAT—andtheLCATactivatorapoA-I—richinapoE.Thisreceptorisdefectiveinfamilialhy-bindtothedisk,andthesurfacephospholipidandfreepercholesterolemia.Approximately30%ofLDLisde-cholesterolareconvertedintocholesterylestersand
208210/CHAPTER25NascentVLDLB-100TGCVLDLEEApo,B-100CCoEXTRAHEPATICpATGTISSUESELDLAC(apoB-100,E)CreceptorEPCCHDLApoCFattyacidsLIPOPROTEINLIPASEB-100TGCholesterol?B-100CECFattyacidsIDLLIVER(VLDLremnant)LDLLDL(apoB-100,E)GlycerolreceptorFinaldestructioninliver,extrahepatictissues(eg,lympho-EXTRAHEPATICcytes,fibroblasts)TISSUESviaendocytosisFigure25–4.Metabolicfateofverylowdensitylipoproteins(VLDL)andproductionoflow-densitylipoproteins(LDL).(A,apolipoproteinA;B-100,apolipoproteinB-100;C,apolipoproteinC;E,apolipoproteinE;HDL,high-densitylipoprotein;TG,triacylglycerol;IDL,intermediate-densitylipoprotein;C,cholesterolandcholesterylester;P,phospholipid.)Onlythepredominantlipidsareshown.ItispossiblethatsomeIDLisalsometabolizedviatheLRP.lysolecithin(Chapter24).Thenonpolarcholesteryles-thenesterifiedbyLCAT,increasingthesizeofthepar-tersmoveintothehydrophobicinteriorofthebilayer,ticlestoformthelessdenseHDL2.Thecycleiscom-whereaslysolecithinistransferredtoplasmaalbumin.pletedbythere-formationofHDL3,eitherafterselec-Thus,anonpolarcoreisgenerated,formingaspherical,tivedeliveryofcholesterylestertotheliverviathepseudomicellarHDLcoveredbyasurfacefilmofpolarSR-B1orbyhydrolysisofHDL2phospholipidandtri-lipidsandapolipoproteins.Inthisway,theLCATsys-acylglycerolbyhepaticlipase.Inaddition,freeapoA-Itemisinvolvedintheremovalofexcessunesterifiedisreleasedbytheseprocessesandformspre-HDLcholesterolfromlipoproteinsandtissues.TheclassBafterassociatingwithaminimumamountofphospho-scavengerreceptorB1(SR-B1)hasrecentlybeenlipidandcholesterol.Preβ-HDListhemostpotentidentifiedasanHDLreceptorintheliverandinformofHDLininducingcholesteroleffluxfromthesteroidogenictissues.HDLbindstothereceptorviatissuestoformdiscoidalHDL.SurplusapoA-Iisde-apoA-Iandcholesterylesterisselectivelydeliveredtostroyedinthekidney.thecells,buttheparticleitself,includingapoA-I,isnotHDLconcentrationsvaryreciprocallywithplasmatakenup.Thetransportofcholesterolfromthetissuestriacylglycerolconcentrationsanddirectlywiththeac-totheliverisknownasreversecholesteroltransporttivityoflipoproteinlipase.ThismaybeduetosurplusandismediatedbyanHDLcycle(Figure25–5).Thesurfaceconstituents,eg,phospholipidandapoA-IsmallerHDL3acceptscholesterolfromthetissuesviabeingreleasedduringhydrolysisofchylomicronsandtheATP-bindingcassettetransporter-1(ABC-1).VLDLandcontributingtowardtheformationofpreβ-ABC-1isamemberofafamilyoftransporterproteinsHDLanddiscoidalHDL.HDL2concentrationsarein-thatcouplethehydrolysisofATPtothebindingofaverselyrelatedtotheincidenceofcoronaryathero-substrate,enablingittobetransportedacrossthemem-sclerosis,possiblybecausetheyreflecttheefficiencyofbrane.AfterbeingacceptedbyHDL3,thecholesterolisreversecholesteroltransport.HDLc(HDL1)isfoundin
209LIPIDTRANSPORT&STORAGE/211BileCandbileacidsPLA-1SMALLINTESTINELIVERCSynthesisLCATSynthesisCKidneyCCEPLDiscoidalHDLSR-B1HEPATICA-1LIPASEPLCA-1Preβ-HDLPhospholipidbilayerA-1CA-1TISSUESCECPLCELCATABC-1CPLHDL2HDL3Figure25–5.Metabolismofhigh-densitylipoprotein(HDL)inreversecholesteroltransport.(LCAT,lecithin:cholesterolacyltransferase;C,cholesterol;CE,cholesterylester;PL,phospholipid;A-I,apolipoproteinA-I;SR-B1,scavengerreceptorB1;ABC-1,ATPbindingcassettetransporter1.)Preβ-HDL,HDL2,HDL3—seeTable25–1.Surplussurfaceconstituentsfromtheactionoflipopro-teinlipaseonchylomicronsandVLDLareanothersourceofpreβ-HDL.Hepaticlipaseactivityisincreasedbyandrogensanddecreasedbyestrogens,whichmayaccountforhigherconcentra-tionsofplasmaHDL2inwomen.thebloodofdiet-inducedhypercholesterolemicani-HepaticVLDLSecretionIsRelatedmals.Itisrichincholesterol,anditssoleapolipopro-toDietary&HormonalStatusteinisapoE.Itappearsthatallplasmalipoproteinsareinterrelatedcomponentsofoneormoremetaboliccy-ThecellulareventsinvolvedinVLDLformationandclesthattogetherareresponsibleforthecomplexsecretionhavebeendescribedabove.Hepatictriacyl-processofplasmalipidtransport.glycerolsynthesisprovidestheimmediatestimulusfortheformationandsecretionofVLDL.Thefattyacidsusedarederivedfromtwopossiblesources:(1)synthe-THELIVERPLAYSACENTRALROLEINsiswithintheliverfromacetyl-CoAderivedmainlyfromcarbohydrate(perhapsnotsoimportantinhu-LIPIDTRANSPORT&METABOLISMmans)and(2)uptakeoffreefattyacidsfromthecircu-Thelivercarriesoutthefollowingmajorfunctionsinlation.Thefirstsourceispredominantinthewell-fedlipidmetabolism:(1)Itfacilitatesthedigestionandab-condition,whenfattyacidsynthesisishighandthesorptionoflipidsbytheproductionofbile,whichcon-levelofcirculatingfreefattyacidsislow.Astriacylglyc-tainscholesterolandbilesaltssynthesizedwithintheeroldoesnotnormallyaccumulateintheliverunderliverdenovoorfromuptakeoflipoproteincholesterolthiscondition,itmustbeinferredthatitistransported(Chapter26).(2)TheliverhasactiveenzymesystemsfromtheliverinVLDLasrapidlyasitissynthesizedforsynthesizingandoxidizingfattyacids(Chapters21andthatthesynthesisofapoB-100isnotrate-limiting.and22)andforsynthesizingtriacylglycerolsandphos-Freefattyacidsfromthecirculationarethemainsourcepholipids(Chapter24).(3)Itconvertsfattyacidstoke-duringstarvation,thefeedingofhigh-fatdiets,orindi-tonebodies(ketogenesis)(Chapter22).(4)Itplaysanabetesmellitus,whenhepaticlipogenesisisinhibited.integralpartinthesynthesisandmetabolismofplasmaFactorsthatenhanceboththesynthesisoftriacylglyc-lipoproteins(thischapter).erolandthesecretionofVLDLbytheliverinclude(1)
210212/CHAPTER25thefedstateratherthanthestarvedstate;(2)thefeed-causinglipidperoxidation.Someprotectionagainstthisingofdietshighincarbohydrate(particularlyiftheyisprovidedbytheantioxidantactionofvitaminE-sup-containsucroseorfructose),leadingtohighratesofli-plementeddiets.Theactionofethionineisthoughttopogenesisandesterificationoffattyacids;(3)highlev-beduetoareductioninavailabilityofATPduetoitselsofcirculatingfreefattyacids;(4)ingestionofreplacingmethionineinS-adenosylmethionine,trap-ethanol;and(5)thepresenceofhighconcentrationsofpingavailableadenineandpreventingsynthesisofinsulinandlowconcentrationsofglucagon,whichen-ATP.Oroticacidalsocausesfattyliver;itisbelievedtohancefattyacidsynthesisandesterificationandinhibitinterferewithglycosylationofthelipoprotein,thusin-theiroxidation(Figure25–6).hibitingrelease,andmayalsoimpairtherecruitmentoftriacylglyceroltotheparticles.AdeficiencyofvitaminEenhancesthehepaticnecrosisofthecholinedefi-CLINICALASPECTSciencytypeoffattyliver.AddedvitaminEorasourceofseleniumhasaprotectiveeffectbycombatinglipidImbalanceintheRateofTriacylglycerolperoxidation.Inadditiontoproteindeficiency,essen-Formation&ExportCausesFattyLivertialfattyacidandvitamindeficiencies(eg,linoleicacid,Foravarietyofreasons,lipid—mainlyastriacylglyc-pyridoxine,andpantothenicacid)cancausefattyinfil-erol—canaccumulateintheliver(Figure25–6).Exten-trationoftheliver.siveaccumulationisregardedasapathologiccondition.WhenaccumulationoflipidintheliverbecomesEthanolAlsoCausesFattyLiverchronic,fibroticchangesoccurinthecellsthatprogresstocirrhosisandimpairedliverfunction.Alcoholismleadstofataccumulationintheliver,hy-Fattyliversfallintotwomaincategories.Thefirstperlipidemia,andultimatelycirrhosis.Theexacttypeisassociatedwithraisedlevelsofplasmafreemechanismofactionofethanolinthelongtermisstillfattyacidsresultingfrommobilizationoffatfromadi-uncertain.Ethanolconsumptionoveralongperiodposetissueorfromthehydrolysisoflipoproteintriacyl-leadstotheaccumulationoffattyacidsintheliverthatglycerolbylipoproteinlipaseinextrahepatictissues.arederivedfromendogenoussynthesisratherthanfromTheproductionofVLDLdoesnotkeeppacewiththeincreasedmobilizationfromadiposetissue.Thereisnoincreasinginfluxandesterificationoffreefattyacids,al-impairmentofhepaticsynthesisofproteinafterethanollowingtriacylglyceroltoaccumulate,causingafattyingestion.Oxidationofethanolbyalcoholdehydrogen-liver.ThisoccursduringstarvationandthefeedingofaseleadstoexcessproductionofNADH.high-fatdiets.TheabilitytosecreteVLDLmayalsobeimpaired(eg,instarvation).InuncontrolleddiabetesALCOHOLmellitus,twinlambdisease,andketosisincattle,DEHYDROGENASEfattyinfiltrationissufficientlyseveretocausevisibleCH3CH2OHCH3CHOpallor(fattyappearance)andenlargementoftheliverNAD+NADH+H+withpossibleliverdysfunction.EthanolAcetaldehydeThesecondtypeoffattyliverisusuallyduetoametabolicblockintheproductionofplasmalipo-TheNADHgeneratedcompeteswithreducingproteins,thusallowingtriacylglyceroltoaccumulate.equivalentsfromothersubstrates,includingfattyacids,Theoretically,thelesionmaybedueto(1)ablockinfortherespiratorychain,inhibitingtheiroxidation,andapolipoproteinsynthesis,(2)ablockinthesynthesisofdecreasingactivityofthecitricacidcycle.Theneteffectthelipoproteinfromlipidandapolipoprotein,(3)aofinhibitingfattyacidoxidationistocauseincreasedfailureinprovisionofphospholipidsthatarefoundinesterificationoffattyacidsintriacylglycerol,resultinglipoproteins,or(4)afailureinthesecretorymechanisminthefattyliver.Oxidationofethanolleadstothefor-itself.mationofacetaldehyde,whichisoxidizedbyaldehydeOnetypeoffattyliverthathasbeenstudiedexten-dehydrogenase,producingacetate.Othereffectsofsivelyinratsisduetoadeficiencyofcholine,whichethanolmayincludeincreasedlipogenesisandcholes-hasthereforebeencalledalipotropicfactor.Thean-terolsynthesisfromacetyl-CoA,andlipidperoxidation.tibioticpuromycin,ethionine(α-amino-γ-mercaptobu-Theincreased[NADH]/[NAD+]ratioalsocausesin-tyricacid),carbontetrachloride,chloroform,phospho-creased[lactate]/[pyruvate],resultinginhyperlactic-rus,lead,andarsenicallcausefattyliverandamarkedacidemia,whichdecreasesexcretionofuricacid,aggra-reductioninconcentrationofVLDLinrats.Cholinevatinggout.SomemetabolismofethanoltakesplacewillnotprotecttheorganismagainsttheseagentsbutviaacytochromeP450-dependentmicrosomalethanolappearstoaidinrecovery.Theactionofcarbontetra-oxidizingsystem(MEOS)involvingNADPHandO2.chlorideprobablyinvolvesformationoffreeradicalsThissystemincreasesinactivityinchronicalcoholism
211LIPIDTRANSPORT&STORAGE/213VLDLApoCHDLNascentApoEBLOODVLDLNascentLIVERHEPATOCYTEVLDL–GlycosylGolgicomplexresiduesSmoothOroticacidCarbontetrachlorideendoplasmicDestructionPuromycinofsurplusapoB-100AminoreticulumEthionineCarbonacidstetrachlorideCholesterolApoB-100ProteinCholesterylApoCsynthesisesterApoEM–PolyribosomesMRough–MembraneendoplasmicsynthesisreticulumNascentpolypeptidechainsofTriacylglycerol*PhospholipidapoB-100CholesterolfeedingEFAdeficiency–LipidEFACholineTRIACYLGLYCEROLdeficiency1,2-DiacylglycerolCDP-cholinePhosphocholineCholineGlucagon+Insulin–InsulinEthanol+Acyl-CoAOxidation–Insulin+FFALipogenesisfromcarbohydrateFigure25–6.Thesynthesisofverylowdensitylipoprotein(VLDL)intheliverandthepossiblelociofactionoffactorscausingaccumulationoftriacylglycerolandafattyliver.(EFA,essentialfattyacids;FFA,freefattyacids;HDL,high-densitylipoproteins;Apo,apolipoprotein;M,microsomaltriacylglyceroltransferprotein.)ThepathwaysindicatedformabasisforeventsdepictedinFigure25–2.ThemaintriacylglycerolpoolinliverisnotonthedirectpathwayofVLDLsynthesisfromacyl-CoA.Thus,FFA,insulin,andglucagonhaveimmediateeffectsonVLDLsecre-tionastheireffectsimpingedirectlyonthesmalltriacylglycerol*precursorpool.Inthefullyfedstate,apoB-100issynthesizedinexcessofrequirementsforVLDLsecretionandthesurplusisdestroyedintheliver.Duringtransla-tionofapoB-100,microsomaltransferprotein-mediatedlipidtransportenableslipidtobecomeassociatedwiththenascentpolypeptidechain.Afterreleasefromtheribosomes,theseparticlesfusewithmorelipidsfromthesmoothendoplasmicreticulum,producingnascentVLDL.
212214/CHAPTER25andmayaccountfortheincreasedmetabolicclearanceGlucoseinthiscondition.Ethanolwillalsoinhibitthemetabo-lismofsomedrugs,eg,barbiturates,bycompetingforInsulin+BLOODcytochromeP450-dependentenzymes.ADIPOSETISSUEGlucose6-phosphateMEOSCHCHOH+NADPH+H++O322+NADP++2HOEthanolCH3CHO2GlycolysisAcetaldehydeCO2PPPAcetyl-CoAInsomeAsianpopulationsandNativeAmericans,NADPH+H+alcoholconsumptionresultsinincreasedadversereac-CO2tionstoacetaldehydeowingtoageneticdefectofmito-chondrialaldehydedehydrogenase.GlycerolAcyl-CoA3-phosphateEsterificationADIPOSETISSUEISTHEMAINSTOREOFTRIACYLGLYCEROLINTHEBODYATPCoAThetriacylglycerolstoresinadiposetissuearecontinu-TGallyundergoinglipolysis(hydrolysis)andreesterifica-ACYL-CoASYNTHETASEHORMONE-tion(Figure25–7).Thesetwoprocessesareentirelydif-SENSITIVEferentpathwaysinvolvingdifferentreactantsandLIPASEenzymes.Thisallowstheprocessesofesterificationorlipolysistoberegulatedseparatelybymanynutritional,Lipolysismetabolic,andhormonalfactors.TheresultantofthesetwoprocessesdeterminesthemagnitudeofthefreeFFAFFAGlycerolfattyacidpoolinadiposetissue,whichinturndeter-(pool2)(pool1)minestheleveloffreefattyacidscirculatingintheplasma.SincethelatterhasmostprofoundeffectsuponLIPOPROTEINthemetabolismofothertissues,particularlyliverandLIPASEmuscle,thefactorsoperatinginadiposetissuethatreg-ulatetheoutflowoffreefattyacidsexertaninfluenceFFAGlycerolfarbeyondthetissueitself.TGBLOOD(chylomicrons,VLDL)TheProvisionofGlycerol3-PhosphateRegulatesEsterification:LipolysisIsControlledbyHormone-SensitiveLipaseFFAGlycerol(Figure25–7)Figure25–7.Metabolismofadiposetissue.Hor-Triacylglycerolissynthesizedfromacyl-CoAandglyc-mone-sensitivelipaseisactivatedbyACTH,TSH,erol3-phosphate(Figure24–2).Becausetheenzymeglucagon,epinephrine,norepinephrine,andvaso-glycerolkinaseisnotexpressedinadiposetissue,glyc-pressinandinhibitedbyinsulin,prostaglandinE1,anderolcannotbeutilizedfortheprovisionofglycerolnicotinicacid.Detailsoftheformationofglycerol3-phosphate,whichmustbesuppliedbyglucoseviaglycolysis.3-phosphatefromintermediatesofglycolysisareTriacylglycerolundergoeshydrolysisbyahormone-showninFigure24–2.(PPP,pentosephosphatepath-sensitivelipasetoformfreefattyacidsandglycerol.way;TG,triacylglycerol;FFA,freefattyacids;VLDL,veryThislipaseisdistinctfromlipoproteinlipasethatcat-lowdensitylipoprotein.)alyzeslipoproteintriacylglycerolhydrolysisbeforeitsuptakeintoextrahepatictissues(seeabove).Sinceglyc-erolcannotbeutilized,itdiffusesintotheblood,whenceitisutilizedbytissuessuchasthoseoftheliverandkidney,whichpossessanactiveglycerolkinase.
213LIPIDTRANSPORT&STORAGE/215Thefreefattyacidsformedbylipolysiscanberecon-regulatedinacoordinatemannerbyphosphorylation-vertedinthetissuetoacyl-CoAbyacyl-CoAsyn-dephosphorylationmechanisms.thetaseandreesterifiedwithglycerol3-phosphatetoAprincipalactionofinsulininadiposetissueistoformtriacylglycerol.Thus,thereisacontinuouscycleinhibittheactivityofhormone-sensitivelipase,reduc-oflipolysisandreesterificationwithinthetissue.ingthereleasenotonlyoffreefattyacidsbutofglycerolHowever,whentherateofreesterificationisnotsuffi-aswell.Adiposetissueismuchmoresensitivetoinsulincienttomatchtherateoflipolysis,freefattyacidsaccu-thanaremanyothertissues,whichpointstoadiposemulateanddiffuseintotheplasma,wheretheybindtotissueasamajorsiteofinsulinactioninvivo.albuminandraisetheconcentrationofplasmafreefattyacids.SeveralHormonesPromoteLipolysisIncreasedGlucoseMetabolismReducesOtherhormonesacceleratethereleaseoffreefattyacidstheOutputofFreeFattyAcidsfromadiposetissueandraisetheplasmafreefattyacidconcentrationbyincreasingtherateoflipolysisoftheWhentheutilizationofglucosebyadiposetissueisin-triacylglycerolstores(Figure25–8).Theseincludeepi-creased,thefreefattyacidoutflowdecreases.However,nephrine,norepinephrine,glucagon,adrenocorticotro-thereleaseofglycerolcontinues,demonstratingthatthepichormone(ACTH),α-andβ-melanocyte-stimulat-effectofglucoseisnotmediatedbyreducingtherateofinghormones(MSH),thyroid-stimulatinghormonelipolysis.Theeffectisduetotheprovisionofglycerol(TSH),growthhormone(GH),andvasopressin.Many3-phosphate,whichenhancesesterificationoffreefattyoftheseactivatethehormone-sensitivelipase.Foranacids.Glucosecantakeseveralpathwaysinadiposetis-optimaleffect,mostoftheselipolyticprocessesrequiresue,includingoxidationtoCO2viathecitricacidthepresenceofglucocorticoidsandthyroidhor-cycle,oxidationinthepentosephosphatepathway,mones.Thesehormonesactinafacilitatoryorper-conversiontolong-chainfattyacids,andformationofmissivecapacitywithrespecttootherlipolyticen-acylglycerolviaglycerol3-phosphate(Figure25–7).docrinefactors.Whenglucoseutilizationishigh,alargerproportionofThehormonesthatactrapidlyinpromotinglipoly-theuptakeisoxidizedtoCO2andconvertedtofattysis,ie,catecholamines,dosobystimulatingtheactivityacids.However,astotalglucoseutilizationdecreases,ofadenylylcyclase,theenzymethatconvertsATPtothegreaterproportionoftheglucoseisdirectedtothecAMP.Themechanismisanalogoustothatresponsibleformationofglycerol3-phosphatefortheesterificationforhormonalstimulationofglycogenolysis(Chap-ofacyl-CoA,whichhelpstominimizetheeffluxoffreeter18).cAMP,bystimulatingcAMP-dependentpro-fattyacids.teinkinase,activateshormone-sensitivelipase.Thus,processeswhichdestroyorpreservecAMPinfluenceHORMONESREGULATElipolysis.cAMPisdegradedto5′-AMPbytheenzymeFATMOBILIZATIONcyclic3,5-nucleotidephosphodiesterase.Thisen-InsulinReducestheOutputzymeisinhibitedbymethylxanthinessuchascaffeineandtheophylline.InsulinantagonizestheeffectoftheofFreeFattyAcidslipolytichormones.Lipolysisappearstobemoresensi-Therateofreleaseoffreefattyacidsfromadiposetissuetivetochangesinconcentrationofinsulinthanareglu-isaffectedbymanyhormonesthatinfluenceeitherthecoseutilizationandesterification.Theantilipolyticef-rateofesterificationortherateoflipolysis.Insulinin-fectsofinsulin,nicotinicacid,andprostaglandinE1arehibitsthereleaseoffreefattyacidsfromadiposetissue,accountedforbyinhibitionofthesynthesisofcAMPatwhichisfollowedbyafallincirculatingplasmafreetheadenylylcyclasesite,actingthroughaGiprotein.fattyacids.ItenhanceslipogenesisandthesynthesisofInsulinalsostimulatesphosphodiesteraseandthelipaseacylglycerolandincreasestheoxidationofglucosetophosphatasethatinactivateshormone-sensitivelipase.CO2viathepentosephosphatepathway.Alloftheseef-Theeffectofgrowthhormoneinpromotinglipolysisisfectsaredependentonthepresenceofglucoseandcandependentonsynthesisofproteinsinvolvedinthefor-beexplained,toalargeextent,onthebasisoftheabil-mationofcAMP.Glucocorticoidspromotelipolysisviaityofinsulintoenhancetheuptakeofglucoseintoadi-synthesisofnewlipaseproteinbyacAMP-independentposecellsviatheGLUT4transporter.Insulinalsoin-pathway,whichmaybeinhibitedbyinsulin,andalsocreasestheactivityofpyruvatedehydrogenase,acetyl-bypromotingtranscriptionofgenesinvolvedintheCoAcarboxylase,andglycerolphosphateacyltrans-cAMPsignalcascade.Thesefindingshelptoexplainferase,reinforcingtheeffectsofincreasedglucoseup-theroleofthepituitaryglandandtheadrenalcortexintakeontheenhancementoffattyacidandacylglycerolenhancingfatmobilization.Therecentlydiscoveredsynthesis.Thesethreeenzymesarenowknowntobebodyweightregulatoryhormone,leptin,stimulates
214216/CHAPTER25Epinephrine,ACTH,Insulin,prostaglandinE1,norepinephrine()TSH,nicotinicacidglucagonβ-Adrenergic–blockersATP+++–ThyroidhormoneHormone-sensitiveADENYLYL–lipasebInsulinGTPFFACYCLASE(inactive)ATP+Pi+–Growthhormone–cAMP-–PPi+dependentLipaseInhibitorsofcAMPMg2+proteinphosphataseproteinsynthesisAdenosinekinaseTRIACYL-Methyl-ADPGLYCEROLxanthines–PHOSPHODI-Hormone-sensitive–(eg,caffeine)ESTERASElipasea(active)?FFA+++PDiacylglycerol–ThyroidhormoneHormone-sensitive5′AMP–lipaseInsulinFFA+Insulin2-MonoacylglycerolcAMP-independentpathway–2-MonoacylglycerollipaseInhibitorsofGlucocorticoidsproteinsynthesisFFA+glycerolFigure25–8.Controlofadiposetissuelipolysis.(TSH,thyroid-stimulatinghormone;FFA,freefattyacids.)Notethecascadesequenceofreactionsaffordingamplificationateachstep.Thelipolyticstimulusis“switchedoff”byremovalofthestimulatinghormone;theactionoflipasephosphatase;theinhibitionofthelipaseandadenylylcyclasebyhighconcentrationsofFFA;theinhibitionofadenylylcyclasebyadenosine;andtheremovalofcAMPbytheactionofphosphodiesterase.ACTH,TSH,andglucagonmaynotactivateadenylylcyclaseinvivo,sincetheconcentrationofeachhormonerequiredinvitroismuchhigherthanisfoundinthecirculation.Posi-tive(+)andnegative(−)regulatoryeffectsarerepresentedbybrokenlinesandsubstrateflowbysolidlines.lipolysisandinhibitslipogenesisbyinfluencingtheac-citratelyase,akeyenzymeinlipogenesis,doesnotap-tivityoftheenzymesinthepathwaysforthebreak-peartobepresent,andotherlipogenicenzymes—eg,downandsynthesisoffattyacids.glucose-6-phosphatedehydrogenaseandthemalicen-Thesympatheticnervoussystem,throughliberationzyme—donotundergoadaptivechanges.Indeed,ithasofnorepinephrineinadiposetissue,playsacentralrolebeensuggestedthatinhumansthereisa“carbohydrateinthemobilizationoffreefattyacids.Thus,thein-excesssyndrome”duetoauniquelimitationinabilitycreasedlipolysiscausedbymanyofthefactorsde-todisposeofexcesscarbohydratebylipogenesis.Inscribedabovecanbereducedorabolishedbydenerva-birds,lipogenesisisconfinedtotheliver,whereitistionofadiposetissueorbyganglionicblockade.particularlyimportantinprovidinglipidsforeggfor-mation,stimulatedbyestrogens.HumanadiposetissueAVarietyofMechanismsHaveEvolvedforisunresponsivetomostofthelipolytichormonesapartfromthecatecholamines.FineControlofAdiposeTissueMetabolismOnconsiderationoftheprofoundderangementofHumanadiposetissuemaynotbeanimportantsiteofmetabolismindiabetesmellitus(dueinlargeparttolipogenesis.Thereisnosignificantincorporationofincreasedreleaseoffreefattyacidsfromthedepots)andglucoseorpyruvateintolong-chainfattyacids;ATP-thefactthatinsulintoalargeextentcorrectsthecondi-
215LIPIDTRANSPORT&STORAGE/217INNERtion,itmustbeconcludedthatinsulinplaysapromi-OUTSIDEMITOCHONDRIALINSIDEnentroleintheregulationofadiposetissuemetabolism.MEMBRANEBROWNADIPOSETISSUENorepinephinePROMOTESTHERMOGENESIS+F0F1Brownadiposetissueisinvolvedinmetabolismparticu-ATP+Hlarlyattimeswhenheatgenerationisnecessary.Thus,cAMPsynthasethetissueisextremelyactiveinsomespeciesinarousalF0+fromhibernation,inanimalsexposedtocold(nonshiv-eringthermogenesis),andinheatproductionintheHeatH+newbornanimal.Thoughnotaprominenttissueinhu-Hormone-mans,itispresentinnormalindividuals,whereitcouldsensitivelipaseberesponsiblefor“diet-inducedthermogenesis.”Itisnoteworthythatbrownadiposetissueisreducedorab-+sentinobesepersons.ThetissueischaracterizedbyaRespiratorychainwell-developedbloodsupplyandahighcontentofmi-Triacyl-glyceroltochondriaandcytochromesbutlowactivityofATPsynthase.MetabolicemphasisisplacedonoxidationofH+H+bothglucoseandfattyacids.NorepinephrineliberatedFFAfromsympatheticnerveendingsisimportantinincreas-inglipolysisinthetissueandincreasingsynthesisofThermogeninlipoproteinlipasetoenhanceutilizationoftriacylglyc-+erol-richlipoproteinsfromthecirculation.OxidationAcyl-CoAReducingandphosphorylationarenotcoupledinmitochondria+equivalentsofthistissue,andthephosphorylationthatdoesoccurisatthesubstratelevel,eg,atthesuccinatethiokinase–Heatstepandinglycolysis.Thus,oxidationproducesmuchheat,andlittlefreeenergyistrappedinATP.Ather-Purinemogenicuncouplingprotein,thermogenin,actsasanucleotidesprotonconductancepathwaydissipatingtheelectro-chemicalpotentialacrossthemitochondrialmembrane-Oxidation(Figure25–9).CarnitineβtransporterSUMMARY•Sincenonpolarlipidsareinsolubleinwater,fortransportbetweenthetissuesintheaqueousbloodplasmatheyarecombinedwithamphipathiclipidsFigure25–9.Thermogenesisinbrownadiposetis-andproteinstomakewater-misciblelipoproteins.sue.Activityoftherespiratorychainproducesheatin•Fourmajorgroupsoflipoproteinsarerecognized:additiontotranslocatingprotons(Chapter12).TheseChylomicronstransportlipidsresultingfromdiges-protonsdissipatemoreheatwhenreturnedtothetionandabsorption.Verylowdensitylipoproteinsinnermitochondrialcompartmentviathermogeninin-(VLDL)transporttriacylglycerolfromtheliver.Low-steadofgeneratingATPwhenreturningviatheF1ATP+densitylipoproteins(LDL)delivercholesteroltothesynthase.ThepassageofHviathermogeninisinhib-tissues,andhigh-densitylipoproteins(HDL)removeitedbypurinenucleotideswhenbrownadiposetissuecholesterolfromthetissuesintheprocessknownasisunstimulated.Undertheinfluenceofnorepinephrine,reversecholesteroltransport.theinhibitionisremovedbytheproductionoffree•ChylomicronsandVLDLaremetabolizedbyhydrol-fattyacids(FFA)andacyl-CoA.Notethedualroleofysisoftheirtriacylglycerol,andlipoproteinremnantsacyl-CoAinbothfacilitatingtheactionofthermogeninareleftinthecirculation.Thesearetakenupbyliver,andsupplyingreducingequivalentsfortherespiratorybutsomeoftheremnants(IDL)resultingfromchain.andsignify+−positiveornegativeregulatoryVLDLformLDLwhichistakenupbytheliverandeffects.othertissuesviatheLDLreceptor.
216218/CHAPTER25•ApolipoproteinsconstitutetheproteinmoietyofREFERENCESlipoproteins.Theyactasenzymeactivators(eg,apoC-IIandapoA-I)orasligandsforcellreceptors(eg,ChappellDA,MedhJD:Receptor-mediatedmechanismsoflipoproteinremnantcatabolism.ProgLipidRes1998;37:apoA-I,apoE,andapoB-100).393.•TriacylglycerolisthemainstoragelipidinadiposeEatonSetal:Multiplebiochemicaleffectsinthepathogenesisoftissue.Uponmobilization,freefattyacidsandglyc-fattyliver.EurJClinInvest1997;27:719.erolarereleased.FreefattyacidsareanimportantGoldbergIJ,MerkelM:Lipoproteinlipase:physiology,biochem-fuelsource.istryandmolecularbiology.FrontBiosci2001;6:D388.•Brownadiposetissueisthesiteof“nonshiveringHolmCetal:Molecularmechanismsregulatinghormonesensitivethermogenesis.”Itisfoundinhibernatingandnew-lipaseandlipolysis.AnnuRevNutr2000;20:365.bornanimalsandispresentinsmallquantityinhu-KaikansRM,BassNM,OcknerRK:Functionsoffattyacidbind-mans.Thermogenesisresultsfromthepresenceofaningproteins.Experientia1990;46:617.uncouplingprotein,thermogenin,intheinnermito-LardyH,ShragoE:Biochemicalaspectsofobesity.AnnuRevBiochem1990;59:689.chondrialmembrane.RyeK-Aetal:Overviewofplasmalipidtransport.In:PlasmaLipidsandTheirRoleinDisease.BarterPJ,RyeK-A(editors).HarwoodAcademicPublishers,1999.ShelnessGS,SellersJA:Very-low-densitylipoproteinassemblyandsecretion.CurrOpinLipidol2001;12:151.Variousauthors:BiochemistryofLipids,LipoproteinsandMem-branes.VanceDE,VanceJE(editors).Elsevier,1996.Variousauthors:Brownadiposetissue—roleinnutritionalenerget-ics.(Symposium.)ProcNutrSoc1989;48:165.
217CholesterolSynthesis,Transport,&Excretion26PeterA.Mayes,PhD,DSc,&KathleenM.Botham,PhD,DScBIOMEDICALIMPORTANCEfrommevalonatebylossofCO2(Figure26–2).(3)Sixisoprenoidunitscondensetoformsqualene.(4)Squa-Cholesterolispresentintissuesandinplasmaeitheraslenecyclizestogiverisetotheparentsteroid,lanos-freecholesterolorasastorageform,combinedwithaterol.(5)Cholesterolisformedfromlanosterol(Figurelong-chainfattyacidascholesterylester.Inplasma,26–3).bothformsaretransportedinlipoproteins(Chapter25).CholesterolisanamphipathiclipidandassuchisStep1—BiosynthesisofMevalonate:HMG-CoAanessentialstructuralcomponentofmembranesandof(3-hydroxy-3-methylglutaryl-CoA)isformedbythere-theouterlayerofplasmalipoproteins.Itissynthesizedactionsusedinmitochondriatosynthesizeketonebod-inmanytissuesfromacetyl-CoAandistheprecursorofies(Figure22–7).However,sincecholesterolsynthesisallothersteroidsinthebodysuchascorticosteroids,sexisextramitochondrial,thetwopathwaysaredistinct.hormones,bileacids,andvitaminD.Asatypicalprod-Initially,twomoleculesofacetyl-CoAcondensetouctofanimalmetabolism,cholesteroloccursinfoodsformacetoacetyl-CoAcatalyzedbycytosolicthiolase.ofanimaloriginsuchaseggyolk,meat,liver,andAcetoacetyl-CoAcondenseswithafurthermoleculeofbrain.Plasmalow-densitylipoprotein(LDL)istheve-acetyl-CoAcatalyzedbyHMG-CoAsynthasetoformhicleofuptakeofcholesterolandcholesterylesterintoHMG-CoA,whichisreducedtomevalonatebymanytissues.FreecholesterolisremovedfromtissuesNADPHcatalyzedbyHMG-CoAreductase.Thisisbyplasmahigh-densitylipoprotein(HDL)andtrans-theprincipalregulatorystepinthepathwayofcholes-portedtotheliver,whereitiseliminatedfromthebodyterolsynthesisandisthesiteofactionofthemosteffec-eitherunchangedorafterconversiontobileacidsinthetiveclassofcholesterol-loweringdrugs,theHMG-CoAprocessknownasreversecholesteroltransport.Cho-reductaseinhibitors(statins)(Figure26–1).lesterolisamajorconstituentofgallstones.However,Step2—FormationofIsoprenoidUnits:Meval-itschiefroleinpathologicprocessesisasafactorintheonateisphosphorylatedsequentiallybyATPbythreegenesisofatherosclerosisofvitalarteries,causingcere-kinases,andafterdecarboxylation(Figure26–2)theac-brovascular,coronary,andperipheralvasculardisease.tiveisoprenoidunit,isopentenyldiphosphate,isCHOLESTEROLISDERIVEDformed.Step3—SixIsoprenoidUnitsFormSqualene:ABOUTEQUALLYFROMTHEDIETIsopentenyldiphosphateisisomerizedbyashiftofthe&FROMBIOSYNTHESISdoublebondtoformdimethylallyldiphosphate,thencondensedwithanothermoleculeofisopentenylAlittlemorethanhalfthecholesterolofthebodyarisesdiphosphatetoformtheten-carbonintermediateger-bysynthesis(about700mg/d),andtheremainderisanyldiphosphate(Figure26–2).Afurthercondensa-providedbytheaveragediet.Theliverandintestineac-tionwithisopentenyldiphosphateformsfarnesylcountforapproximately10%eachoftotalsynthesisindiphosphate.Twomoleculesoffarnesyldiphosphatehumans.Virtuallyalltissuescontainingnucleatedcellscondenseatthediphosphateendtoformsqualene.Ini-arecapableofcholesterolsynthesis,whichoccursinthetially,inorganicpyrophosphateiseliminated,formingendoplasmicreticulumandthecytosol.presqualenediphosphate,whichisthenreducedbyAcetyl-CoAIstheSourceofAllCarbonNADPHwitheliminationofafurtherinorganicpy-AtomsinCholesterolrophosphatemolecule.Step4—FormationofLanosterol:SqualenecanThebiosynthesisofcholesterolmaybedividedintofivefoldintoastructurethatcloselyresemblesthesteroidsteps:(1)Synthesisofmevalonateoccursfromacetyl-nucleus(Figure26–3).Beforeringclosureoccurs,squa-CoA(Figure26–1).(2)Isoprenoidunitsareformedleneisconvertedtosqualene2,3-epoxidebyamixed-219
218220/CHAPTER26OFarnesylDiphosphateGivesRiseCH3CSCoAtoDolichol&Ubiquinone2Acetyl-CoAThepolyisoprenoidsdolichol(Figure14–20andChapter47)andubiquinone(Figure12–5)areformedTHIOLASEfromfarnesyldiphosphatebythefurtheradditionofupto16(dolichol)or3–7(ubiquinone)isopentenylCoASHdiphosphateresidues,respectively.SomeGTP-bindingCH3Oproteinsinthecellmembraneareprenylatedwithfar-CCH2CSCoAnesylorgeranylgeranyl(20carbon)residues.ProteinOAcetoacetyl-CoAOprenylationisbelievedtofacilitatetheanchoringofproteinsintolipoidmembranesandmayalsobein-H2OCH3CSCoAvolvedinprotein-proteininteractionsandmembrane-Acetyl-CoAassociatedproteintrafficking.HMG-CoASYNTHASECoASHCHOCHOLESTEROLSYNTHESISIS3–CONTROLLEDBYREGULATIONOOCCH2CCH2CSCoAOFHMG-CoAREDUCTASEOH3-Hydroxy-3-methylglutaryl-CoA(HMG-CoA)RegulationofcholesterolsynthesisisexertedneartheBileacid,cholesterolbeginningofthepathway,attheHMG-CoAreductase2NADPH+2H+step.ThereducedsynthesisofcholesterolinstarvingStatins,eg,animalsisaccompaniedbyadecreaseintheactivityofHMG-CoAREDUCTASEsimvastatintheenzyme.However,itisonlyhepaticsynthesisthatis2NADP++CoASHinhibitedbydietarycholesterol.HMG-CoAreductaseMevalonateCH3inliverisinhibitedbymevalonate,theimmediateprod-–uctofthepathway,andbycholesterol,themainprod-OOCCH2CCH2CH2OHuct.Cholesterol(orametabolite,eg,oxygenatedsterol)OHrepressestranscriptionoftheHMG-CoAreductaseMevalonategeneandisalsobelievedtoinfluencetranslation.Adi-Figure26–1.Biosynthesisofmevalonate.HMG-CoAurnalvariationoccursinbothcholesterolsynthesisandreductaseactivity.Inadditiontothesemechanismsreductaseisinhibitedbyatorvastatin,pravastatin,andregulatingtherateofproteinsynthesis,theenzymeac-simvastatin.Theopenandsolidcirclesindicatethefatetivityisalsomodulatedmorerapidlybyposttransla-ofeachofthecarbonsintheacetylmoietyofacetyl-tionalmodification(Figure26–4).InsulinorthyroidCoA.hormoneincreasesHMG-CoAreductaseactivity,whereasglucagonorglucocorticoidsdecreaseit.Activ-ityisreversiblymodifiedbyphosphorylation-dephos-functionoxidaseintheendoplasmicreticulum,squa-phorylationmechanisms,someofwhichmaybeleneepoxidase.ThemethylgrouponC14istransferredcAMP-dependentandthereforeimmediatelyresponsivetoC13andthatonC8toC14ascyclizationoccurs,cat-toglucagon.Attemptstolowerplasmacholesterolinalyzedbyoxidosqualene:lanosterolcyclase.humansbyreducingtheamountofcholesterolintheStep5—FormationofCholesterol:Theforma-dietproducevariableresults.Generally,adecreaseoftionofcholesterolfromlanosteroltakesplaceinthe100mgindietarycholesterolcausesadecreaseofap-membranesoftheendoplasmicreticulumandinvolvesproximately0.13mmol/Lofserum.changesinthesteroidnucleusandsidechain(Figure26–3).ThemethylgroupsonC14andC4areremovedMANYFACTORSINFLUENCETHEtoform14-desmethyllanosterolandthenzymosterol.CHOLESTEROLBALANCEINTISSUESThedoublebondatC8–C9issubsequentlymovedtoC5–C6intwosteps,formingdesmosterol.Finally,theIntissues,cholesterolbalanceisregulatedasfollows(Fig-doublebondofthesidechainisreduced,producingure26–5):Cellcholesterolincreaseisduetouptakeofcholesterol.Theexactorderinwhichthestepsde-cholesterol-containinglipoproteinsbyreceptors,eg,thescribedactuallytakeplaceisnotknownwithcer-LDLreceptororthescavengerreceptor;uptakeoffreetainty.cholesterolfromcholesterol-richlipoproteinstothecell
219ATPADPCH3OHCH3OHMg2+–OOCCCH–OOCCCH22MEVALONATECH2CH2OHKINASECH2CH2OPMevalonateMevalonate5-phosphateATPPHOSPHOMEVALONATEMg2+KINASEADPADPATPCH3OPCH3OHMg2+–OOCCCH–OOCCCH22DIPHOSPHOMEVALONATECH2CH2OPPKINASECH2CH2OPPMevalonate3-phospho-5-diphosphateMevalonate5-diphosphateCO2+PiHMG-CoADIPHOSPHO-MEVALONATEtrans-Methyl-CH3DECARBOXYLASECH3glutaconateshuntCCH2CCH2CH3CHOPPISOPENTENYL-CH2CH2OPPDIPHOSPHATE3,3-DimethylallylISOMERASEIsopentenyldiphosphatediphosphateIsopentenyltRNACIS-PRENYLTRANSFERASEPPiCH3CH3CCH2CCH2PrenylatedproteinsCH3CHCH2CHOPPGeranyldiphosphateCIS-PRENYLTRANSFERASEPPiTRANS-PRENYLCIS-PRENYLTRANSFERASETRANSFERASESidechainofCH*2DolicholubiquinoneOPPFarnesyldiphosphateHemeaNADPH+H+SQUALENESYNTHETASEMg2+,Mn2+2PPNADP+iCH*2*CH2SqualeneFigure26–2.Biosynthesisofsqualene,ubiquinone,dolichol,andotherpolyisoprenederivatives.(HMG,3-hydroxy-3-methylglutaryl;⋅×⋅⋅⋅,cytokinin.)Afarnesylresidueispresentinhemeaofcytochromeoxidase.ThecarbonmarkedwithasteriskbecomesC11orC12insqualene.Squalenesynthetaseisamicrosomalen-zyme;allotherenzymesindicatedaresolublecytosolicproteins,andsomearefoundinperoxisomes.221
220222/CHAPTER26OCH3CH3CHCSCoA–OOCCHCCHCHOHCHCCHCH322232–OHCO2H2OAcetyl-CoAMevalonateIsoprenoidunitCH3CH2CH3CH2CCH2CCH2121224CH324CH3CH213CHHCC*CH213CHHCCSqualene11CH311*CH3epoxideCH2CHCH2CH2CHCH21CH31CH3CH2CH14CCH2CH2CH14CCH28SQUALENE8H2CCCCH3EPOXIDASEH2CCCCH3CH3CH3HC3CHCHNADPH1HC3CHCHX62/2O22FADCCH2CCH2SqualeneOOXIDOSQUALENE:CH3LANOSTEROLCH3CH3CH3CYCLASEHCOOH2CO214148NADPHO2,NADPH8ONAD+24HOHOHOLanosterol14-DesmethylZymosterollanosterolISOMERASE212218202326252424241217NADPHNADPH1911131627CD141519Δ24-REDUCTASEO22108AB35737465HOHOHO–7,24CholesterolDesmosterolΔ-Cholestadienol(24-dehydrocholesterol)TriparanolFigure26–3.Biosynthesisofcholesterol.Thenumberedpositionsarethoseofthesteroidnucleusandtheopenandsolidcirclesindicatethefateofeachofthecarbonsintheacetylmoietyofacetyl-CoA.Asterisks:RefertolabelingofsqualeneinFigure26–2.
221CHOLESTEROLSYNTHESIS,TRANSPORT,&EXCRETION/223REDUCTASEATPKINASEPi(inactive)+REDUCTASEInsulinKINASEPROTEIN?PHOSPHATASESKINASEP–REDUCTASEGlucagonADPKINASEH2O(active)+ATPADPInhibitor-1-cAMPphosphate*+HMG-CoAHMG-CoAHMG-CoALDL-cholesterolREDUCTASEPREDUCTASE(active)(inactive)CholesterolH2O?InsulinPi+–PROTEINOxysterolsPHOSPHATASES–EnzymesynthesisFigure26–4.PossiblemechanismsintheregulationofcholesterolsynthesisbyHMG-CoAreductase.Insulinhasadominantrolecomparedwithglucagon.Asterisk:SeeFigure18–6.membrane;cholesterolsynthesis;andhydrolysisofcho-ity;anddown-regulatessynthesisoftheLDLreceptor.lesterylestersbytheenzymecholesterylesterhydrolase.Thus,thenumberofLDLreceptorsonthecellsurfaceDecreaseisduetoeffluxofcholesterolfromthemem-isregulatedbythecholesterolrequirementformem-branetoHDL,promotedbyLCAT(lecithin:cholesterolbranes,steroidhormones,orbileacidsynthesis(Figureacyltransferase)(Chapter25);esterificationofcholesterol26–5).TheapoB-100,Ereceptorisa“high-affinity”byACAT(acyl-CoA:cholesterolacyltransferase);anduti-LDLreceptor,whichmaybesaturatedundermostcir-lizationofcholesterolforsynthesisofothersteroids,suchcumstances.Other“low-affinity”LDLreceptorsalsoashormones,orbileacidsintheliver.appeartobepresentinadditiontoascavengerpath-way,whichisnotregulated.TheLDLReceptorIsHighlyRegulatedLDL(apoB-100,E)receptorsoccuronthecellsurfaceCHOLESTEROLISTRANSPORTEDinpitsthatarecoatedonthecytosolicsideofthecellBETWEENTISSUESINPLASMAmembranewithaproteincalledclathrin.Theglycopro-LIPOPROTEINSteinreceptorspansthemembrane,theB-100binding(Figure26–6)regionbeingattheexposedaminoterminalend.Afterbinding,LDListakenupintactbyendocytosis.TheInWesterncountries,thetotalplasmacholesterolinapoproteinandcholesterylesterarethenhydrolyzedinhumansisabout5.2mmol/L,risingwithage,thoughthelysosomes,andcholesterolistranslocatedintothetherearewidevariationsbetweenindividuals.Thecell.Thereceptorsarerecycledtothecellsurface.Thisgreaterpartisfoundintheesterifiedform.Itistrans-influxofcholesterolinhibitsinacoordinatedman-portedinlipoproteinsoftheplasma,andthehighestnerHMG-CoAsynthase,HMG-CoAreductase,and,proportionofcholesterolisfoundintheLDL.Dietarytherefore,cholesterolsynthesis;stimulatesACATactiv-cholesterolequilibrateswithplasmacholesterolindays
222224/CHAPTER26CELLMEMBRANERecyclingReceptor–vesicleLDL(apoB-100,E)synthesisreceptorsDown-regulationCholesterol(incoatedpits)LysosomesynthesisCE–EndosomeC+ACATCELDLCEUnesterifiedCEcholesterolCECoatedpoolvesicle(mainlyinmembranes)ScavengerreceptororLDLCECnonregulatedpathwayCELysosomeHYDROLASELDLCSynthesisVLDLofsteroidsABC-1CEA-1A-1LCATPLPLCPreβ-HDLHDL3Figure26–5.Factorsaffectingcholesterolbalanceatthecellularlevel.ReversecholesteroltransportmaybeinitiatedbypreβHDLbindingtotheABC-1transporterproteinviaapoA-I.Cholesterolisthenmovedoutofthecellviathetransporter,lipidatingtheHDL,andthelargerparticlesthendissociatefromtheABC-1mol-ecule.(C,cholesterol;CE,cholesterylester;PL,phospholipid;ACAT,acyl-CoA:cholesterolacyltransferase;LCAT,lecithin:cholesterolacyltransferase;A-I,apolipoproteinA-I;LDL,low-densitylipoprotein;VLDL,verylowden-sitylipoprotein.)LDLandHDLarenotshowntoscale.andwithtissuecholesterolinweeks.Cholesterylesteratesaconcentrationgradientanddrawsincholesterolinthedietishydrolyzedtocholesterol,whichisthenfromtissuesandfromotherlipoproteins(Figures26–5absorbedbytheintestinetogetherwithdietaryunesteri-and26–6),thusenablingHDLtofunctioninreversefiedcholesterolandotherlipids.Withcholesterolsyn-cholesteroltransport(Figure25–5).thesizedintheintestines,itisthenincorporatedintochylomicrons.Ofthecholesterolabsorbed,80–90%isesterifiedwithlong-chainfattyacidsintheintestinalCholesterylEsterTransferProteinmucosa.Ninety-fivepercentofthechylomicroncholes-FacilitatesTransferofCholesterylEsterterolisdeliveredtotheliverinchylomicronremnants,FromHDLtoOtherLipoproteinsandmostofthecholesterolsecretedbytheliverinVLDLisretainedduringtheformationofIDLandul-ThisproteinisfoundinplasmaofhumansandmanytimatelyLDL,whichistakenupbytheLDLreceptorotherspecies,associatedwithHDL.Itfacilitatestransferinliverandextrahepatictissues(Chapter25).ofcholesterylesterfromHDLtoVLDL,IDL,andLDLinexchangefortriacylglycerol,relievingproductinhibi-PlasmaLCATIsResponsibleforVirtuallytionofLCATactivityinHDL.Thus,inhumans,muchofthecholesterylesterformedbyLCATfindsitswaytoAllPlasmaCholesterylEsterinHumanstheliverviaVLDLremnants(IDL)orLDL(FigureLCATactivityisassociatedwithHDLcontainingapo26–6).Thetriacylglycerol-enrichedHDL2deliversitsA-I.AscholesterolinHDLbecomesesterified,itcre-cholesteroltotheliverintheHDLcycle(Figure25–5).
223CHOLESTEROLSYNTHESIS,TRANSPORT,&EXCRETION/225ENTEROHEPATICCIRCULATIONHEPATICPORTALVEINDiet(0.4g/d)CCEGALLBLADDERSynthesis––Bileacids(totalpool,3–5g)BILEDUCTUnesterifiedcholesterolCEpoolCACATCEBileCCacidsHLVLDLCTG,CETGChylomicronCEILEUMCELDLC(apoB-100,E)TGLIVERreceptorCE98–99%LDLTGCCELRPreceptorCECTGCECETGCCETPCBileacidsTGTGCE(0.6g/d)(0.4g/d)CEA-ICELCATCCIDLHDLFeces(VLDLremnant)ChylomicronremnantLPLCLDL(apoB-100,E)CreceptorEXTRAHEPATICCSynthesisTISSUESCEFigure26–6.Transportofcholesterolbetweenthetissuesinhumans.(C,unesterifiedcholesterol;CE,cho-lesterylester;TG,triacylglycerol;VLDL,verylowdensitylipoprotein;IDL,intermediate-densitylipoprotein;LDL,low-densitylipoprotein;HDL,high-densitylipoprotein;ACAT,acyl-CoA:cholesterolacyltransferase;LCAT,lecithin:cholesterolacyltransferase;A-I,apolipoproteinA-I;CETP,cholesterylestertransferprotein;LPL,lipopro-teinlipase;HL,hepaticlipase;LRP,LDLreceptor-relatedprotein.)CHOLESTEROLISEXCRETEDFROMTHEfeces;itisformedfromcholesterolbythebacteriainBODYINTHEBILEASCHOLESTEROLORthelowerintestine.BILEACIDS(SALTS)BileAcidsAreFormedFromCholesterolAbout1gofcholesteroliseliminatedfromthebodyperday.ApproximatelyhalfisexcretedinthefecesafterTheprimarybileacidsaresynthesizedintheliverfromconversiontobileacids.Theremainderisexcretedascholesterol.Thesearecholicacid(foundinthelargestcholesterol.Coprostanolistheprincipalsterolintheamount)andchenodeoxycholicacid(Figure26–7).
224226/CHAPTER26VitaminC1217NADPH+H+NADP+O2377HO7α-HYDROXYLASEHOOHCholesterol7α-Hydroxycholesterol12α-HYDROX-BileYLASEacidsVitaminCOO22deficiency++NADPH+HNADPH+H(Several2CoASHsteps)2CoASHPropionyl-CoAPropionyl-CoACSCoAOHHOCN(CH2)SO3H2OCoASHOHHOOHHHOOHTaurine12CSCoAChenodeoxycholyl-CoAHOTaurocholicacidGlycine(primarybileacid)CoASHHOOHHTauro-andglyco-Cholyl-CoAchenodeoxycholicacid(primarybileacids)OHHCNCH2COOH*Deconjugation+7α-dehydroxylationOOHHOOHHCOOHCOOHGlycocholicacid(primarybileacid)*DeconjugationHOHOHH+7α-dehydroxylationDeoxycholicacidLithocholicacid(secondarybileacid)(secondarybileacid)Figure26–7.Biosynthesisanddegradationofbileacids.Asecondpathwayinmitochondriainvolveshy-droxylationofcholesterolbysterol27-hydroxylase.Asterisk:Catalyzedbymicrobialenzymes.The7α-hydroxylationofcholesterolisthefirstandchenodeoxycholyl-CoA(Figure26–7).Asecondpath-principalregulatorystepinthebiosynthesisofbileacidswayinmitochondriainvolvingthe27-hydroxylationofcatalyzedby7-hydroxylase,amicrosomalenzyme.Acholesterolbysterol27-hydroxylaseasthefirststepistypicalmonooxygenase,itrequiresoxygen,NADPH,responsibleforasignificantproportionoftheprimaryandcytochromeP450.Subsequenthydroxylationstepsbileacidssynthesized.Theprimarybileacids(Figurearealsocatalyzedbymonooxygenases.Thepathwayof26–7)enterthebileasglycineortaurineconjugates.bileacidbiosynthesisdividesearlyintoonesubpathwayConjugationtakesplaceinperoxisomes.Inhumans,theleadingtocholyl-CoA,characterizedbyanextraα-OHratiooftheglycinetothetaurineconjugatesisnormallygrouponposition12,andanotherpathwayleadingto3:1.Inthealkalinebile,thebileacidsandtheirconju-
225CHOLESTEROLSYNTHESIS,TRANSPORT,&EXCRETION/227gatesareassumedtobeinasaltform—hencethetermizedbythedepositionofcholesterolandcholesteryl“bilesalts.”esterfromtheplasmalipoproteinsintothearterywall.AportionoftheprimarybileacidsintheintestineisDiseasesinwhichprolongedelevatedlevelsofVLDL,subjectedtofurtherchangesbytheactivityofthein-IDL,chylomicronremnants,orLDLoccurinthetestinalbacteria.Theseincludedeconjugationand7α-blood(eg,diabetesmellitus,lipidnephrosis,hypothy-dehydroxylation,whichproducethesecondarybileroidism,andotherconditionsofhyperlipidemia)areacids,deoxycholicacidandlithocholicacid.oftenaccompaniedbyprematureormoresevereather-osclerosis.ThereisalsoaninverserelationshipbetweenMostBileAcidsReturntotheLiverHDL(HDL2)concentrationsandcoronaryheartdis-ease,andsomeconsiderthatthemostpredictiverela-intheEnterohepaticCirculationtionshipistheLDL:HDLcholesterolratio.ThisisAlthoughproductsoffatdigestion,includingcholes-consistentwiththefunctionofHDLinreversecholes-terol,areabsorbedinthefirst100cmofsmallintestine,teroltransport.Susceptibilitytoatherosclerosisvariestheprimaryandsecondarybileacidsareabsorbedal-widelyamongspecies,andhumansareoneofthefewmostexclusivelyintheileum,and98–99%arere-inwhichthediseasecanbeinducedbydietshighinturnedtotheliverviatheportalcirculation.Thisischolesterol.knownastheenterohepaticcirculation(Figure26–6).However,lithocholicacid,becauseofitsinsolubility,isDietCanPlayanImportantRoleinnotreabsorbedtoanysignificantextent.OnlyasmallReducingSerumCholesterolfractionofthebilesaltsescapesabsorptionandisthere-foreeliminatedinthefeces.Nonetheless,thisrepresentsHereditaryfactorsplaythegreatestroleindeterminingamajorpathwayfortheeliminationofcholesterol.individualserumcholesterolconcentrations;however,Eachdaythesmallpoolofbileacids(about3–5g)isdietaryandenvironmentalfactorsalsoplayapart,andcycledthroughtheintestinesixtotentimesandanthemostbeneficialoftheseisthesubstitutionintheamountofbileacidequivalenttothatlostinthefecesisdietofpolyunsaturatedandmonounsaturatedfattysynthesizedfromcholesterol,sothatapoolofbileacidsacidsforsaturatedfattyacids.Plantoilssuchascornoilofconstantsizeismaintained.Thisisaccomplishedbyandsunflowerseedoilcontainahighproportionofasystemoffeedbackcontrols.polyunsaturatedfattyacids,whileoliveoilcontainsahighconcentrationofmonounsaturatedfattyacids.Ontheotherhand,butterfat,beeffat,andpalmoilcontainBileAcidSynthesisIsRegulatedahighproportionofsaturatedfattyacids.Sucroseandatthe7-HydroxylaseStepfructosehaveagreatereffectinraisingbloodlipids,par-Theprincipalrate-limitingstepinthebiosynthesisofticularlytriacylglycerols,thandoothercarbohydrates.bileacidsisatthecholesterol7-hydroxylasereac-Thereasonforthecholesterol-loweringeffectoftion(Figure26–7).Theactivityoftheenzymeisfeed-polyunsaturatedfattyacidsisstillnotfullyunderstood.back-regulatedviathenuclearbileacid-bindingrecep-Itisclear,however,thatoneofthemechanismsin-torfarnesoidXreceptor(FXR).Whenthesizeofthevolvedistheup-regulationofLDLreceptorsbypoly-bileacidpoolintheenterohepaticcirculationincreases,andmonounsaturatedascomparedwithsaturatedfattyFXRisactivatedandtranscriptionofthecholesterolacids,causinganincreaseinthecatabolicrateofLDL,7α-hydroxylasegeneissuppressed.Chenodeoxycholicthemainatherogeniclipoprotein.Inaddition,saturatedacidisparticularlyimportantinactivatingFXR.Cho-fattyacidscausetheformationofsmallerVLDLparti-lesterol7α-hydroxylaseactivityisalsoenhancedbyclesthatcontainrelativelymorecholesterol,andtheycholesterolofendogenousanddietaryoriginandregu-areutilizedbyextrahepatictissuesataslowerratethanlatedbyinsulin,glucagon,glucocorticoids,andthyroidarelargerparticles—tendenciesthatmayberegardedashormone.atherogenic.CLINICALASPECTSLifestyleAffectstheSerumCholesterolLevelTheSerumCholesterolIsCorrelatedWiththeIncidenceofAtherosclerosis&Additionalfactorsconsideredtoplayapartincoronaryheartdiseaseincludehighbloodpressure,smoking,CoronaryHeartDiseasemalegender,obesity(particularlyabdominalobesity),Whilecholesterolisbelievedtobechieflyconcernedinlackofexercise,anddrinkingsoftasopposedtohardtherelationship,otherserumlipidssuchastriacylglyc-water.FactorsassociatedwithelevationofplasmaFFAerolsmayalsoplayarole.Atherosclerosisischaracter-followedbyincreasedoutputoftriacylglycerolandcho-
226228/CHAPTER26lesterolintothecirculationinVLDLincludeemotionalofcoronaryheartdisease.Thismaybeduetoelevationstressandcoffeedrinking.Premenopausalwomenap-ofHDLconcentrationsresultingfromincreasedsyn-peartobeprotectedagainstmanyofthesedeleteriousthesisofapoA-Iandchangesinactivityofcholesterylfactors,andthisisthoughttoberelatedtothebenefi-estertransferprotein.Ithasbeenclaimedthatredwinecialeffectsofestrogen.Thereisanassociationbetweenisparticularlybeneficial,perhapsbecauseofitscontentmoderatealcoholconsumptionandalowerincidenceofantioxidants.RegularexerciselowersplasmaLDLTable26–1.Primarydisordersofplasmalipoproteins(dyslipoproteinemias).NameDefectRemarksHypolipoproteinemiasNochylomicrons,VLDL,orLDLareRare;bloodacylglycerolslow;intestineandliverAbetalipoproteinemiaformedbecauseofdefectintheaccumulateacylglycerols.Intestinalmalabsorp-loadingofapoBwithlipid.tion.Earlydeathavoidablebyadministrationoflargedosesoffat-solublevitamins,particularlyvitaminE.Familialalpha-lipoproteindeficiencyAllhavelowornearabsenceofHDL.TendencytowardhypertriacylglycerolemiaasaTangierdiseaseresultofabsenceofapoC-II,causinginactiveFish-eyediseaseLPL.LowLDLlevels.Atherosclerosisintheel-Apo-A-Ideficienciesderly.HyperlipoproteinemiasHypertriacylglycerolemiaduetode-SlowclearanceofchylomicronsandVLDL.LowFamiliallipoproteinlipaseficiencyofLPL,abnormalLPL,orapolevelsofLDLandHDL.Noincreasedriskofcoro-deficiency(typeI)C-IIdeficiencycausinginactiveLPL.narydisease.FamilialhypercholesterolemiaDefectiveLDLreceptorsormutationElevatedLDLlevelsandhypercholesterolemia,(typeIIa)inligandregionofapoB-100.resultinginatherosclerosisandcoronarydisease.FamilialtypeIIIhyperlipoprotein-DeficiencyinremnantclearancebyIncreaseinchylomicronandVLDLremnantsofemia(broadbetadisease,rem-theliverisduetoabnormalityinapodensity<1.019(β-VLDL).Causeshypercholes-nantremovaldisease,familialE.PatientslackisoformsE3andE4terolemia,xanthomas,andatherosclerosis.dysbetalipoproteinemia)andhaveonlyE2,whichdoesnot1reactwiththeEreceptor.FamilialhypertriacylglycerolemiaOverproductionofVLDLoftenCholesterollevelsrisewiththeVLDLconcentra-(typeIV)associatedwithglucoseintolerancetion.LDLandHDLtendtobesubnormal.Thisandhyperinsulinemia.typeofpatterniscommonlyassociatedwithcoronaryheartdisease,typeIIdiabetesmellitus,obesity,alcoholism,andadministrationofprogestationalhormones.FamilialhyperalphalipoproteinemiaIncreasedconcentrationsofHDL.Arareconditionapparentlybeneficialtohealthandlongevity.HepaticlipasedeficiencyDeficiencyoftheenzymeleadstoPatientshavexanthomasandcoronaryheartaccumulationoflargetriacylgly-disease.cerol-richHDLandVLDLremnants.Familiallecithin:cholesterolAbsenceofLCATleadstoblockinPlasmaconcentrationsofcholesterylestersandacyltransferase(LCAT)deficiencyreversecholesteroltransport.HDLlysolecithinarelow.PresentisanabnormalLDLremainsasnascentdisksincapablefraction,lipoproteinX,foundalsoinpatientsoftakingupandesterifyingcholes-withcholestasis.VLDLisabnormal(β-VLDL).terol.Familiallipoprotein(a)excessLp(a)consistsof1molofLDLPrematurecoronaryheartdiseaseduetoathero-attachedto1molofapo(a).Apo(a)sclerosis,plusthrombosisduetoinhibitionofshowsstructuralhomologiestoplas-fibrinolysis.minogen.1ThereisanassociationbetweenpatientspossessingtheapoE4alleleandtheincidenceofAlzheimer’sdisease.Apparently,apoE4bindsmoreavidlytoβ-amyloidfoundinneuriticplaques.
227CHOLESTEROLSYNTHESIS,TRANSPORT,&EXCRETION/229butraisesHDL.Triacylglycerolconcentrationsarealsoacids,andvitaminD.Italsoplaysanimportantreduced,duemostlikelytoincreasedinsulinsensitivity,structuralroleinmembranesandintheouterlayerofwhichenhancesexpressionoflipoproteinlipase.lipoproteins.•CholesterolissynthesizedinthebodyentirelyfromWhenDietChangesFail,Hypolipidemicacetyl-CoA.Threemoleculesofacetyl-CoAformDrugsWillReduceSerumCholesterolmevalonateviatheimportantregulatoryreactionfor&Triacylglycerolthepathway,catalyzedbyHMG-CoAreductase.Next,afive-carbonisoprenoidunitisformed,andSignificantreductionsofplasmacholesterolcanbeef-sixofthesecondensetoformsqualene.Squaleneun-fectedmedicallybytheuseofcholestyramineresinordergoescyclizationtoformtheparentsteroidlanos-surgicallybytheilealexclusionoperations.Bothproce-terol,which,afterthelossofthreemethylgroups,duresblockthereabsorptionofbileacids,causingin-formscholesterol.creasedbileacidsynthesisintheliver.Thisincreases•Cholesterolsynthesisintheliverisregulatedpartlycholesterolexcretionandup-regulatesLDLreceptors,bycholesterolinthediet.Intissues,cholesterolbal-loweringplasmacholesterol.Sitosterolisahypocholes-anceismaintainedbetweenthefactorscausinggainterolemicagentthatactsbyblockingtheabsorptionofofcholesterol(eg,synthesis,uptakeviatheLDLorcholesterolfromthegastrointestinaltract.scavengerreceptors)andthefactorscausinglossofSeveraldrugsareknowntoblocktheformationofcholesterol(eg,steroidsynthesis,cholesterylesterfor-cholesterolatvariousstagesinthebiosyntheticpath-mation,excretion).TheactivityoftheLDLreceptorway.ThestatinsinhibitHMG-CoAreductase,thusismodulatedbycellularcholesterollevelstoachieveup-regulatingLDLreceptors.Statinscurrentlyinusethisbalance.Inreversecholesteroltransport,HDLincludeatorvastatin,simvastatin,andpravastatin.Fi-(preβ-HDL,discoidal,orHDL3)takesupcholesterolbratessuchasclofibrateandgemfibrozilactmainlytofromthetissuesandLCATesterifiesitanddepositslowerplasmatriacylglycerolsbydecreasingthesecretionitinthecoreofHDL,whichisconvertedtoHDL.2oftriacylglycerolandcholesterol-containingVLDLbyThecholesterylesterinHDListakenupbythe2theliver.Inaddition,theystimulatehydrolysisofliver,eitherdirectlyoraftertransfertoVLDL,IDL,VLDLtriacylglycerolsbylipoproteinlipase.ProbucolorLDLviathecholesterylestertransferprotein.appearstoincreaseLDLcatabolismviareceptor-•Excesscholesterolisexcretedfromtheliverintheindependentpathways,butitsantioxidantpropertiesbileascholesterolorbilesalts.AlargeproportionofmaybemoreimportantinpreventingaccumulationofbilesaltsisabsorbedintotheportalcirculationandoxidizedLDL,whichhasenhancedatherogenicproper-returnedtotheliveraspartoftheenterohepaticcir-ties,inarterialwalls.Nicotinicacidreducesthefluxofculation.FFAbyinhibitingadiposetissuelipolysis,therebyin-hibitingVLDLproductionbytheliver.•ElevatedlevelsofcholesterolpresentinVLDL,IDL,orLDLareassociatedwithatherosclerosis,whereashighlevelsofHDLhaveaprotectiveeffect.PrimaryDisordersofthePlasma•InheriteddefectsinlipoproteinmetabolismleadtoaLipoproteins(Dyslipoproteinemias)primaryconditionofhypo-orhyperlipoproteinemia.AreInheritedConditionssuchasdiabetesmellitus,hypothy-Inheriteddefectsinlipoproteinmetabolismleadtotheroidism,kidneydisease,andatherosclerosisexhibitprimaryconditionofeitherhypo-orhyperlipopro-secondaryabnormallipoproteinpatternsthatresem-teinemia(Table26–1).Inaddition,diseasessuchasblecertainprimaryconditions.diabetesmellitus,hypothyroidism,kidneydisease(nephroticsyndrome),andatherosclerosisareassoci-atedwithsecondaryabnormallipoproteinpatternsthatREFERENCESareverysimilartooneoranotheroftheprimaryinher-itedconditions.VirtuallyalloftheprimaryconditionsIllingworthDR:Managementofhypercholesterolemia.MedClinNorthAm2000;84:23.areduetoadefectatastageinlipoproteinformation,transport,ordestruction(seeFigures25–4,26–5,andNessGC,ChambersCM:Feedbackandhormonalregulationofhepatic3-hydroxy-3-methylglutarylcoenzymeAreductase:26–6).Notalloftheabnormalitiesareharmful.theconceptofcholesterolbufferingcapacity.ProcSocExpBiolMed2000;224:8.SUMMARYParksDJetal:Bileacids:naturalligandsforanuclearorphanre-ceptor.Science1999;284:1365.•CholesterolistheprecursorofallothersteroidsinPrincenHMG:Regulationofbileacidsynthesis.CurrPharmDe-thebody,eg,corticosteroids,sexhormones,bilesign1997;3:59.
228230/CHAPTER26RussellDW:Cholesterolbiosynthesisandmetabolism.Cardiovas-Variousauthors:Thecholesterolfacts.AsummaryoftheevidencecularDrugsTherap1992;6:103.relatingdietaryfats,serumcholesterol,andcoronaryheartSpadyDK,WoollettLA,DietschyJM:RegulationofplasmaLDL-disease.Circulation1990;81:1721.cholesterollevelsbydietarycholesterolandfattyacids.AnnuZhangFL,CaseyPJ:Proteinprenylation:MolecularmechanismsRevNutr1993;13:355.andfunctionalconsequences.AnnuRevBiochem1996;TallA:Plasmalipidtransferproteins.AnnuRevBiochem1995;65:241.64:235.Variousauthors:BiochemistryofLipids,LipoproteinsandMem-branes.VanceDE,VanceJE(editors).Elsevier,1996.
229IntegrationofMetabolism—TheProvisionofMetabolicFuels27DavidABender,PhD,&PeterA.Mayes,PhD,DScBIOMEDICALIMPORTANCEMANYMETABOLICFUELSAREINTERCONVERTIBLEAnadulthumanweighing70kgrequiresabout10–12MJ(2400–2900kcal)frommetabolicfuelseachday.CarbohydrateinexcessofimmediaterequirementsasThisrequirementismetfromcarbohydrates(40–60%),fuelorforsynthesisofglycogeninmuscleandlivermaylipids(mainlytriacylglycerol,30–40%),protein(10–beusedforlipogenesis(Chapter21)andhencetriacyl-15%),andalcoholifconsumed.Themixbeingoxi-glycerolsynthesisinbothadiposetissueandliverdizedvariesdependingonwhetherthesubjectisinthe(whenceitisexportedinverylowdensitylipoprotein).fedorstarvingstateandontheintensityofphysicalTheimportanceoflipogenesisinhumanbeingsisun-work.Therequirementformetabolicfuelsisrelativelyclear;inWesterncountries,dietaryfatprovidesconstantthroughouttheday,sinceaveragephysicalac-35–45%ofenergyintake,whileinlessdevelopedcoun-tivityonlyincreasesmetabolicratebyabout40–50%trieswherecarbohydratemayprovide60–75%ofen-overthebasalmetabolicrate.However,mostpeopleergyintakethetotalintakeoffoodmaybesolowthatconsumetheirdailyintakeofmetabolicfuelsintwoorthereislittlesurplusforlipogenesis.Ahighintakeoffatthreemeals,sothereisaneedtoformreservesofcarbo-inhibitslipogenesis.hydrate(glycogeninliverandmuscle)andlipid(tri-Fattyacids(andketonebodiesformedfromthem)acylglycerolinadiposetissue)forusebetweenmeals.cannotbeusedforthesynthesisofglucose.Thereac-Iftheintakeoffuelsisconsistentlygreaterthanen-tionofpyruvatedehydrogenase,formingacetyl-CoA,isergyexpenditure,thesurplusisstored,largelyasfat,irreversible,andforeverytwo-carbonunitfromacetyl-leadingtothedevelopmentofobesityanditsassociatedCoAthatentersthecitricacidcyclethereisalossofhealthhazards.Iftheintakeoffuelsisconsistentlytwocarbonatomsascarbondioxidebeforeonlyonelowerthanenergyexpenditure,therewillbenegligiblemoleculeofoxaloacetateisre-formed—ie,thereisnofatandcarbohydratereserves,andaminoacidsarisingnetincrease.Thismeansthatacetyl-CoA(andthereforefromproteinturnoverwillbeusedforenergyratheranysubstratesthatyieldacetyl-CoA)canneverbeusedthanreplacementproteinsynthesis,leadingtoemacia-forgluconeogenesis(Chapter19).The(relativelyrare)tionandeventuallydeath.fattyacidswithanoddnumberofcarbonatomsyieldAfteranormalmealthereisanamplesupplyofcar-propionyl-CoAastheproductofthefinalcycleofβ-bohydrate,andthefuelformosttissuesisglucose.Inoxidation(Chapter22),andthiscanbeasubstrateforthestarvingstate,glucosemustbesparedforusebythegluconeogenesis,ascantheglycerolreleasedbylipolysiscentralnervoussystem(whichislargelydependentonofadiposetissuetriacylglycerolreserves.Mostoftheglucose)andtheerythrocytes(whicharewhollyreliantaminoacidsinexcessofrequirementsforproteinsyn-onglucose).Othertissuescanutilizealternativefuelsthesis(arisingfromthedietorfromtissueproteinsuchasfattyacidsandketonebodies.Asglycogenre-turnover)yieldpyruvate,orfive-andfour-carbonin-servesbecomedepleted,soaminoacidsarisingfromtermediatesofthecitricacidcycle.Pyruvatecanbeproteinturnoverandglycerolarisingfromlipolysisarecarboxylatedtooxaloacetate,whichistheprimaryusedforgluconeogenesis.Theseeventsarelargelycon-substrateforgluconeogenesis,andthefive-andtrolledbythehormonesinsulinandglucagon.India-four-carbonintermediatesalsoresultinanetincreaseinbetesmellitusthereiseitherimpairedsynthesisandtheformationofoxaloacetate,whichisthenavailablesecretionofinsulin(type1diabetesmellitus)orim-forgluconeogenesis.Theseaminoacidsareclassifiedaspairedsensitivityoftissuestoinsulinaction(type2di-glucogenic.Lysineandleucineyieldonlyacetyl-CoAabetesmellitus),leadingtoseveremetabolicderange-onoxidationandthuscannotbeusedforgluconeogen-ment.Incattlethedemandsofheavylactationcanleadesis,whilephenylalanine,tyrosine,tryptophan,andtoketosis,ascanthedemandsoftwinpregnancyinisoleucinegiverisetobothacetyl-CoAandtointerme-sheep.diatesofthecitricacidcyclethatcanbeusedforgluco-231
230232/CHAPTER27neogenesis.Thoseaminoacidsthatgiverisetoacetyl-rateofsynthesisofglucose6-phosphate.Thisisinex-CoAareclassifiedasketogenicbecauseinthestarvingcessoftheliver’srequirementforenergyandisusedstatemuchoftheacetyl-CoAwillbeusedforsynthesismainlyforsynthesisofglycogen.Inbothliverandofketonebodiesintheliver.skeletalmuscle,insulinactstostimulateglycogensyn-thaseandinhibitglycogenphosphorylase.Someoftheglucoseenteringthelivermayalsobeusedforlipogene-ASUPPLYOFMETABOLICFUELSsisandsynthesisoftriacylglycerol.Inadiposetissue,in-sulinstimulatesglucoseuptake,itsconversiontofattyISPROVIDEDINBOTHTHEFEDacids,andtheiresterification;andinhibitsintracellular&STARVINGSTATESlipolysisandthereleaseoffreefattyacids.(Figure27–1)Theproductsoflipiddigestionenterthecirculationastriacylglycerol-richchylomicrons(Chapter25).InGlucoseIsAlwaysRequiredbytheCentraladiposetissueandskeletalmuscle,lipoproteinlipaseisNervousSystem&Erythrocytesactivatedinresponsetoinsulin;theresultantfreefattyErythrocyteslackmitochondriaandhencearewhollyacidsarelargelytakenuptoformtriacylglycerolre-reliantonglycolysisandthepentosephosphatepath-serves,whiletheglycerolremainsinthebloodstreamway.Thebraincanmetabolizeketonebodiestomeetandistakenupbytheliverandusedforglycogensyn-about20%ofitsenergyrequirements;theremainderthesisorlipogenesis.Freefattyacidsremaininginthemustbesuppliedbyglucose.Themetabolicchangesbloodstreamaretakenupbytheliverandreesterified.thatoccurinstarvationaretheconsequencesoftheThelipid-depletedchylomicronremnantsarealsoneedtopreserveglucoseandthelimitedreservesofclearedbytheliver,andsurpluslivertriacylglycerol—glycogeninliverforusebythebrainanderythrocytesincludingthatfromlipogenesis—isexportedinveryandtoensuretheprovisionofalternativefuelsforotherlowdensitylipoprotein.tissues.ThefetusandsynthesisoflactoseinmilkalsoUndernormalfeedingpatternstherateoftissuerequireasignificantamountofglucose.proteincatabolismismoreorlessconstantthroughouttheday;itisonlyincachexiathatthereisanincreasedrateofproteincatabolism.Thereisnetproteincatabo-IntheFedState,MetabolicFuellisminthepostabsorptivephaseofthefeedingcycleReservesAreLaidDownandnetproteinsynthesisintheabsorptivephase,whentherateofsynthesisincreasesbyabout20–25%.TheForseveralhoursafterameal,whiletheproductsofdi-increasedrateofproteinsynthesisis,again,aresponsegestionarebeingabsorbed,thereisanabundantsupplytoinsulinaction.Proteinsynthesisisanenergy-expen-ofmetabolicfuels.Undertheseconditions,glucoseissiveprocess,accountingforuptoalmost20%ofenergythemajorfuelforoxidationinmosttissues;thisisob-expenditureinthefedstate,whenthereisanampleservedasanincreaseintherespiratoryquotient(thesupplyofaminoacidsfromthediet,butunder9%inratioofcarbondioxideproducedtooxygenconsumed)thestarvedstate.fromabout0.8inthestarvedstatetonear1(Table27–1).MetabolicFuelReservesAreMobilizedGlucoseuptakeintomuscleandadiposetissueisintheStarvingStatecontrolledbyinsulin,whichissecretedbytheBisletcellsofthepancreasinresponsetoanincreasedconcen-Thereisasmallfallinplasmaglucoseuponstarvation,trationofglucoseintheportalblood.Anearlyresponsethenlittlechangeasstarvationprogresses(Table27–2;toinsulininmuscleandadiposetissueisthemigrationFigure27–2).Plasmafreefattyacidsincreasewithofglucosetransportervesiclestothecellsurface,expos-onsetofstarvationbutthenplateau.Thereisaninitialingactiveglucosetransporters(GLUT4).Thesein-delayinketonebodyproduction,butasstarvationpro-sulin-sensitivetissueswillonlytakeupglucosefromthegressestheplasmaconcentrationofketonebodiesin-bloodstreamtoanysignificantextentinthepresenceofcreasesmarkedly.thehormone.AsinsulinsecretionfallsinthestarvedInthepostabsorptivestate,astheconcentrationofstate,sothetransportersareinternalizedagain,reduc-glucoseintheportalbloodfalls,soinsulinsecretionde-ingglucoseuptake.creases,resultinginskeletalmuscleandadiposetissueTheuptakeofglucoseintotheliverisindependenttakinguplessglucose.Theincreaseinsecretionofofinsulin,butliverhasanisoenzymeofhexokinaseglucagonfromtheAcellsofthepancreasinhibits(glucokinase)withahighKm,sothatastheconcentra-glycogensynthaseandactivatesglycogenphosphorylasetionofglucoseenteringtheliverincreases,sodoestheinliver.Theresultingglucose6-phosphateinliveris
231INTEGRATIONOFMETABOLISM—THEPROVISIONOFMETABOLICFUELS/233Glucose6-phosphateAcyl-CoAGlycerol3-phosphateADIPOSETISSUETRIACYLGLYCEROL(TG)cAMPFFAGlycerolLPLEXTRAHEPATICTISSUE(eg,BLOODFFAGlycerolheartmuscle)GlycerolChylomicronsGASTRO-LPLTGINTESTINAL(lipoproteins)TRACTFFAFFAOxidationGlucoseGlucoseExtraglucosedrain(eg,diabetes,pregnancy,VLDLlactation)KetonebodiesFFATGGlucoseLIVERAcyl-CoAGlycerol3-phosphateAcetyl-CoAGlucose6-phosphateCitricGluconeogenesisacid2CO2Aminoacids,GlycogencyclelactateFigure27–1.Metabolicinterrelationshipsbetweenadiposetissue,theliver,andextrahepatictissues.Inextrahepatictissuessuchasheart,metabolicfuelsareoxidizedinthefollowingorderofpreference:(1)ketonebodies,(2)fattyacids,(3)glucose.(LPL,lipoproteinlipase;FFA,freefattyacids;VLDL,verylowdensitylipoproteins.)
232234/CHAPTER27Table27–1.Energyyields,oxygenconsumption,andcarbondioxideproductionintheoxidationofmetabolicfuels.EnergyYieldO2ConsumedCO2ProducedOxygen(kJ/g)(L/g)(L/g)RQ(kJ/L)Carbohydrate160.8290.8291.0020Protein170.9660.7820.8120Fat372.0161.4270.7120hydrolyzedbyglucose-6-phosphatase,andglucoseisre-Althoughmuscletakesupandpreferentiallyoxidizesleasedintothebloodstreamforusebyothertissues,freefattyacidsinthestarvingstate,itcannotmeetallofparticularlythebrainanderythrocytes.itsenergyrequirementsbyβ-oxidation.Bycontrast,theMuscleglycogencannotcontributedirectlytoliverhasagreatercapacityforβ-oxidationthanitre-plasmaglucose,sincemusclelacksglucose-6-phos-quirestomeetitsownenergyneedsandformsmorephatase,andtheprimarypurposeofmuscleglycogenisacetyl-CoAthancanbeoxidized.Thisacetyl-CoAistoprovideasourceofglucose6-phosphateforenergy-usedtosynthesizeketonebodies(Chapter22),whichyieldingmetabolisminthemuscleitself.However,aremajormetabolicfuelsforskeletalandheartmuscleacetyl-CoAformedbyoxidationoffattyacidsinmuscleandcanmeetsomeofthebrain’senergyneeds.Inpro-inhibitspyruvatedehydrogenaseandleadstocitrateac-longedstarvation,glucosemayrepresentlessthan10%cumulation,whichinturninhibitsphosphofructoki-ofwholebodyenergy-yieldingmetabolism.Further-naseandthereforeglycolysis,thussparingglucose.Anymore,asaresultofproteincatabolism,anincreasingaccumulatedpyruvateistransaminatedtoalanineatthenumberofaminoacidsarereleasedandutilizedintheexpenseofaminoacidsarisingfrombreakdownofpro-liverandkidneysforgluconeogenesis.teinreserves.Thealanine—andmuchoftheketoacidsresultingfromthistransamination—areexportedfrommuscleandtakenupbytheliver,wherethealanineisPlasmaglucagonPlasmatransaminatedtoyieldpyruvate.Theresultantaminoinsulinacidsarelargelyexportedbacktomuscletoprovideaminogroupsforformationofmorealanine,whilethepyruvateisamajorsubstrateforgluconeogenesisintheliver.Inadiposetissue,theeffectofthedecreaseininsulinandincreaseinglucagonresultsininhibitionoflipo-genesis,inactivationoflipoproteinlipase,andactiva-tionofhormone-sensitivelipase(Chapter25).Thisleadstoreleaseofincreasedamountsofglycerol(asub-strateforgluconeogenesisintheliver)andfreefattyPlasmafreeacids,whichareusedbyskeletalmuscleandliverasfattyacidstheirpreferredmetabolicfuels,sosparingglucose.RelativechangeBloodglucoseTable27–2.PlasmaconcentrationsofmetabolicLiverglycogenfuels(mmol/L)inthefedandstarvingstates.iesodebBloodketon40Hours7DaysFedStarvationStarvation012–24Glucose5.53.63.5HoursofstarvationFreefattyacids0.301.151.19Figure27–2.Relativechangesinmetabolicparame-KetonebodiesNegligible2.94.5tersduringtheonsetofstarvation.
233,e,eeee-αnsitived.ese,arginas,7e-snolpyruvateelopenolpyruvate)veee-6-phosphatasenasspiratorychainelldandlyaseee.R,hormon.eeecialistEnzym,phosphoe,fructokinased.,phosphoeee,glucoseeehydrogeinlipasinlipasinlipaselopeeevrolkinaseerolkinaselldemoglobin)espiratorychainweeGlyc(Hs,glycerolLipoprotebodiacids,ecarboxykinasetoninsHMG-CoAsynthasee,VLDL(triacylglyc-Glucokinas)(Alcoholdeeeeeetatfattyacids,glyca,uricacid,bilerol),HDL,keeeeLactatLactatLactat,llGlucoseprincipalorgans.ee-eroleLipoprotLipoproteese(weMajorProductstoneSp,k,Glucoseerol,fructosrol,someintriacyl-eFredfattyacidses,triacylglyc,glyceees,VLDLandchylo-ewtabolismofthe,aminoacid,kee,lipoproteebodieefattyacids,glucoss(instarvation)eonatfattyacids,lactatbodirolefattyacidsfattyacids,lactatrolelipascarboxykinaseeeed),lactateeetoneeeeesofm(Ethanol)Polyunsaturatinn(Acmicrontriacylglycglucosglycfre1-Glucossise1aturacidssis;aminohydroxylases-enursisnoeesis;feneeg,Freng,Kfeeeeephos-eGlucosfored,inclu-neFrreeetogsisFrntogeenntosesesis;lipogeeeeinformation;eplasmaproteogpreepathway.Nomito-a,uricacid,andbilerificationoffattyacidsGlucose.e-oxidation;kerolsynthrobicpathways,e-oxidationandcitricacidtonerobicpathways,e-oxidationandcitricinVLDLandchylomicrons,-oxidationorcitricacidRβlipoproturacidformation;cholteGlycolysis,aminoacidmβcyclβacidcyclGlycolysis,pphatchondriaandthβcyclmajoranduniquereMostrentAsdinggluconeinhumans.eak-eEstntmeGlycolysisGlucoseothemtabolismbodie2emesisglyceryactivforthandbrdmoveneeeetionandglu-ogGluconeervicrvoussysteerolSummaryofthorgansandtissunStoragdownoftriacylglyc-eandlipolysis;lipogconsbutnotveesTransportofOcieetissueeyExcrOrganrSeMajorFunctionMajorPathwaysMainSubstratartPumpingofbloodAeeFasttwitchSlowtwitchSustainRapidmovTable27–3.LivBrainCoordinationofthHAdiposMusclKidnErythrocyt1Inmanysp
234236/CHAPTER27CLINICALASPECTSAsummaryofthemajoranduniquemetabolicfea-turesoftheprincipaltissuesispresentedinTable27–3.Inprolongedstarvation,asadiposetissuereservesaredepletedthereisaveryconsiderableincreaseinthenetrateofproteincatabolismtoprovideaminoacidsnotSUMMARYonlyassubstratesforgluconeogenesisbutalsoasthemainmetabolicfuelofthetissues.Deathresultswhen•Thebodycaninterconvertthemajorityoffoodstuffs.essentialtissueproteinsarecatabolizedbeyondtheHowever,thereisnonetconversionofmostfattypointatwhichtheycansustainthismetabolicdrain.Inacids(orotheracetyl-CoA-formingsubstances)topatientswithcachexiaasaresultofreleaseofcytokinesglucose.Mostaminoacids,arisingfromthedietorinresponsetotumorsandanumberofotherpatho-fromtissueprotein,canbeusedforgluconeogenesis,logicconditions,thereisanincreaseintherateoftissueascantheglycerolfromtriacylglycerol.proteincatabolismaswellasaconsiderablyincreased•Instarvation,glucosemustbeprovidedforthebrainmetabolicrate,resultinginastateofadvancedstarva-anderythrocytes;initially,thisissuppliedfromlivertion.Again,deathresultswhenessentialtissueproteinsglycogenreserves.Tospareglucose,muscleandotherhavebeencatabolized.tissuesreduceglucoseuptakeinresponsetoloweredThehighdemandforglucosebythefetusandforinsulinsecretion;theyalsooxidizefattyacidsandke-synthesisoflactoseinlactationcanleadtoketosis.Thistonebodiespreferentiallytoglucose.maybeseenasmildketosiswithhypoglycemiain•Adiposetissuereleasesfreefattyacidsinstarvation,women,butinlactatingcattleandinewescarryingandtheseareusedbymanytissuesasfuel.Further-twinstheremaybeverypronouncedketosisandpro-more,inthelivertheyarethesubstrateforsynthesisfoundhypoglycemia.ofketonebodies.Inpoorlycontrolledtype1diabetesmellitus,pa-•Ketosis,ametabolicadaptationtostarvation,isexac-tientsmaybecomehyperglycemic,partlyasaresultoferbatedinpathologicconditionssuchasdiabeteslackofinsulintostimulateuptakeandutilizationofmellitusandruminantketosis.glucoseandpartlybecauseofincreasedgluconeogenesisfromaminoacidsintheliver.Atthesametime,thelackofinsulinresultsinincreasedlipolysisinadiposeREFERENCEStissue,andtheresultantfreefattyacidsaresubstratesforketogenesisintheliver.Itispossiblethatinveryse-BenderDA:IntroductiontoNutritionandMetabolism,3rdedition.verediabetesutilizationofketonebodiesinmuscleTaylor&Francis,2002.(andothertissues)isimpairedbecauseoflackofox-CaprioSetal:Oxidativefuelmetabolismduringmildhypo-glycemia:criticalroleoffreefattyacids.AmJPhysiolaloacetate(mosttissueshavearequirementforsome1989;256:E413.glucosemetabolismtomaintainanadequateamountofFellD:UnderstandingtheControlofMetabolism.PortlandPress,oxaloacetateforcitricacidcycleactivity).Inuncon-1997.trolleddiabetes,themagnitudeofketosismaybesuchFraynKN:MetabolicRegulation—AHumanPerspective.Portlandastoresultinsevereacidosis(ketoacidosis)sinceace-Press,1996.toaceticacidand3-hydroxybutyricacidarerelativelyMcNamaraJP:Roleandregulationofmetabolisminadiposetissuestrongacids.Comaresultsfromboththeacidosisandduringlactation.JNutrBiochem1995;6:120.theconsiderablyincreasedosmolarityofextracellularRandlePJ:Theglucose-fattyacidcycle—biochemicalaspects.Ath-fluid(mainlyduetothehyperglycemia).erosclerosisRev1991;22:183.
235SECTIONIIIMetabolismofProteins&AminoAcidsBiosynthesisoftheNutritionallyNonessentialAminoAcids28VictorW.Rodwell,PhDBIOMEDICALIMPORTANCEthosethreeenzymesistotransformammoniumionintotheα-aminonitrogenofvariousaminoacids.All20oftheaminoacidspresentinproteinsareessentialGlutamateandGlutamine.Reductiveaminationofforhealth.WhilecomparativelyrareintheWesternα-ketoglutarateiscatalyzedbyglutamatedehydrogenaseworld,aminoaciddeficiencystatesareendemicincer-(Figure28–1).AminationofglutamatetoglutamineistainregionsofWestAfricawherethedietreliesheavilycatalyzedbyglutaminesynthetase(Figure28–2).ongrainsthatarepoorsourcesofaminoacidssuchasAlanine.Transaminationofpyruvateformsalaninetryptophanandlysine.Thesedisordersincludekwash-(Figure28–3).iorkor,whichresultswhenachildisweanedontoaAspartateandAsparagine.Transaminationofstarchydietpoorinprotein;andmarasmus,inwhichoxaloacetateformsaspartate.Theconversionofaspartatebothcaloricintakeandspecificaminoacidsaredeficient.Humanscansynthesize12ofthe20commonaminoacidsfromtheamphibolicintermediatesofglycolysisandofthecitricacidcycle(Table28–1).WhilenutritionallyTable28–1.Aminoacidrequirementsnonessential,these12aminoacidsarenot“nonessential.”ofhumans.All20aminoacidsarebiologicallyessential.Ofthe12nu-tritionallynonessentialaminoacids,nineareformedfromNutritionallyEssentialNutritionallyNonessentialamphibolicintermediatesandthree(cysteine,tyrosine1andhydroxylysine)fromnutritionallyessentialaminoArginineAlanineacids.Identificationofthetwelveaminoacidsthathu-HistidineAsparagineIsoleucineAspartatemanscansynthesizerestedprimarilyondataderivedfromLeucineCysteinefeedingdietsinwhichpurifiedaminoacidsreplacedpro-LysineGlutamatetein.ThischapterconsidersonlythebiosynthesisoftheMethionineGlutaminetwelveaminoacidsthataresynthesizedinhumantissues,PhenylalanineGlycinenottheothereightthataresynthesizedbyplants.ThreonineHydroxyproline22TryptophanHydroxylysineNUTRITIONALLYNONESSENTIALValineProlineAMINOACIDSHAVESHORTSerineTyrosineBIOSYNTHETICPATHWAYS1“Nutritionallysemiessential.”SynthesizedatratesinadequateTheenzymesglutamatedehydrogenase,glutaminesyn-tosupportgrowthofchildren.thetase,andaminotransferasesoccupycentralpositions2Notnecessaryforproteinsynthesisbutformedduringpost-inaminoacidbiosynthesis.Thecombinedeffectoftranslationalprocessingofcollagen.237
236238/CHAPTER28ONH+ONH+33–OO––OO–O–O–PyruvateAlanineOOOOOOα-KetoglutarateL-GlutamateNH+HO42GluorAspα-KetoglutarateoroxaloacetateNAD(P)H+H+NAD(P)+Figure28–3.Formationofalaninebytransamina-tionofpyruvate.TheaminodonormaybeglutamateorFigure28–1.Theglutamatedehydrogenaseaspartate.Theotherproductthusisα-ketoglutarateorreaction.oxaloacetate.toasparagineiscatalyzedbyasparaginesynthetase(Fig-ure28–4),whichresemblesglutaminesynthetase(Fig-ure28–2)exceptthatglutamine,notammoniumion,++providesthenitrogen.BacterialasparaginesynthetasesONH3ONH3––can,however,alsouseammoniumion.Coupledhy-OO–HNdrolysisofPPitoPibypyrophosphataseensuresthatO2thereactionisstronglyfavored.OOSerine.Oxidationoftheα-hydroxylgroupoftheL-AspartateL-Asparagineglycolyticintermediate3-phosphoglycerateconvertsittoanoxoacid,whosesubsequenttransaminationandGlnGludephosphorylationleadstoserine(Figure28–5).Glycine.GlycineaminotransferasescancatalyzethesynthesisofglycinefromglyoxylateandglutamateorMg-ATPMg-AMP+PPialanine.Unlikemostaminotransferasereactions,thesestronglyfavorglycinesynthesis.AdditionalimportantFigure28–4.Theasparaginesynthetasereaction.mammalianroutesforglycineformationarefromNotesimilaritiestoanddifferencesfromtheglutaminecholine(Figure28–6)andfromserine(Figure28–7).synthetasereaction(Figure28–2).Proline.Prolineisformedfromglutamatebyrever-salofthereactionsofprolinecatabolism(Figure28–8).Cysteine.Cysteine,whilenotnutritionallyessen-tial,isformedfrommethionine,whichisnutritionallyessential.Followingconversionofmethioninetoho-OHOO−O−NADHPOOPOOD-3-PhosphoglyceratePhosphohydroxypyruvateNH+NH+α-AA33–OO–HNO–2α-KAOOOONH+NH+L-GlutamateL-Glutamine33O−PiH2OO−NH+4HOOPOOL-SerinePhospho-L-serineMg-ATPMg-ADP+PiFigure28–5.Serinebiosynthesis.(α-AA,α-aminoFigure28–2.Theglutaminesynthetasereaction.acids;α-KA,α-ketoacids.)
237BIOSYNTHESISOFTHENUTRITIONALLYNONESSENTIALAMINOACIDS/239CH32HCH3OOHCN+CHHCN+CH−NADH−3333OO–OONH3+ONH3+CholineBetainealdehydeH2OOHOL-GlutamateL-Glutamate-γ-semialdehydeNAD+H2OH3C+CH3[CH3]CH3N+HH3CNCH3O−OOO−DimethylglycineOBetaineO−NADHO−ONH+NH+2[CH2O]Δ2-Pyrrolidine-L-Proline5-carboxylateH+CH3[CH2O]+Figure28–8.BiosynthesisofprolinefromglutamateNH3byreversalofreactionsofprolinecatabolism.NH−O−OSarcosineGlycineOOFigure28–6.Formationofglycinefromcholine.+NH3−Omocysteine(seeChapter30),homocysteineandserineHOOformcysteineandhomoserine(Figure28–9).+L-SerineTyrosine.PhenylalaninehydroxylaseconvertsH3N+SHphenylalaninetotyrosine(Figure28–10).Provided−OthatthedietcontainsadequatenutritionallyessentialH2ONH+O3phenylalanine,tyrosineisnutritionallynonessential.O−Butsincethereactionisirreversible,dietarytyrosineL-Homocysteine+cannotreplacephenylalanine.Catalysisbythismixed-H3NSO−functionoxygenaseincorporatesoneatomofO2intoOH2Ophenylalanineandreducestheotheratomtowater.Re-Oducingpower,providedastetrahydrobiopterin,derivesultimatelyfromNADPH.Cystathionine+NH3−OMethyleneH4folateH4folate+HSOH3NOH+NH+−OL-CysteineNH33+O––OOHOOOL-HomoserineSerineGlycineFigure28–9.Conversionofhomocysteineandser-Figure28–7.Theserinehydroxymethyltransferaseinetohomoserineandcysteine.Thesulfurofcysteinereaction.Thereactionisfreelyreversible.(H4folate,derivesfrommethionineandthecarbonskeletonfromtetrahydrofolate.)serine.
238240/CHAPTER28NADP+NADPH+H+18O]Succinateα-Ketoglutarate[IIFe2+18O2Tetrahydro-Dihydro-Ascorbate18OHbiopterinbiopterinProProIO2H2OFigure28–11.Theprolylhydroxylasereaction.TheCH2CHCOO–CH2CHCOO−substrateisaproline-richpeptide.Duringthecourseofthereaction,molecularoxygenisincorporatedintoNH+NH+33bothsuccinateandproline.LysylhydroxylasecatalyzesHOananalogousreaction.L-PhenylalanineL-TyrosineHH2NNNaminoacids,tissueaminotransferasesreversiblyinter-HNNCHCHCH3convertallthreeaminoacidsandtheircorrespondingHHOα-ketoacids.Theseα-ketoacidsthuscanreplacetheirOHOHaminoacidsinthediet.TetrahydrobiopterinSelenocysteine.WhilenotnormallyconsideredFigure28–10.Thephenylalaninehydroxylasereac-anaminoacidpresentinproteins,selenocysteineoc-cursattheactivesitesofseveralenzymes.Examplesin-tion.Twodistinctenzymaticactivitiesareinvolved.Ac-cludethehumanenzymesthioredoxinreductase,glu-tivityIIcatalyzesreductionofdihydrobiopterinbytathioneperoxidase,andthedeiodinasethatconvertsNADPH,andactivityIthereductionofO2toH2Oandofthyroxinetotriiodothyronine.Unlikehydroxyprolinephenylalaninetotyrosine.Thisreactionisassociatedorhydroxylysine,selenocysteinearisesco-translation-withseveraldefectsofphenylalaninemetabolismdis-allyduringitsincorporationintopeptides.TheUGAcussedinChapter30.SecanticodonoftheunusualtRNAdesignatedtRNAnormallysignalsSTOP.Theabilityoftheproteinsyn-HydroxyprolineandHydroxylysine.Hydroxy-theticapparatustoidentifyaselenocysteine-specificprolineandhydroxylysinearepresentprincipallyinUGAcodoninvolvestheselenocysteineinsertionele-collagen.SincethereisnotRNAforeitherhydroxy-ment,astem-loopstructureintheuntranslatedregionSeclatedaminoacid,neitherdietaryhydroxyprolinenorofthemRNA.Selenocysteine-tRNAisfirstchargedSerhydroxylysineisincorporatedintoprotein.BotharewithserinebytheligasethatchargestRNA.Subse-completelydegraded(seeChapter30).Hydroxyprolinequentreplacementoftheserineoxygenbyseleniumandhydroxylysinearisefromprolineandlysine,butinvolvesselenophosphateformedbyselenophosphateonlyaftertheseaminoacidshavebeenincorporatedsynthase(Figure28–12).intopeptides.Hydroxylationofpeptide-boundprolylandlysylresiduesiscatalyzedbyprolylhydroxylaseandlysylhydroxylaseoftissues,includingskinandskeletalmuscle,andofgranulatingwounds(Figure28–11).HThehydroxylasesaremixed-functionoxygenasesthat2+HSeCHCCOO–requiresubstrate,molecularO2,ascorbate,Fe,and2α-ketoglutarate.Foreverymoleofprolineorlysinehy-NH+3droxylated,onemoleofα-ketoglutarateisdecarboxy-Olatedtosuccinate.OneatomofO2isincorporatedintoSe+ATPAMP+P+HSePO–prolineorlysine,theotherintosuccinate(Figurei28–11).AdeficiencyofthevitaminCrequiredforO–thesehydroxylasesresultsinscurvy.Valine,Leucine,andIsoleucine.Whileleucine,Figure28–12.Selenocysteine(top)andthereactionvaline,andisoleucineareallnutritionallyessentialcatalyzedbyselenophosphatesynthetase(bottom).
239BIOSYNTHESISOFTHENUTRITIONALLYNONESSENTIALAMINOACIDS/241SUMMARY•Selenocysteine,anessentialactivesiteresidueinsev-eralmammalianenzymes,arisesbyco-translational•AllvertebratescanformcertainaminoacidsfrominsertionofapreviouslymodifiedtRNA.amphibolicintermediatesorfromotherdietaryaminoacids.Theintermediatesandtheaminoacidstowhichtheygiveriseareα-ketoglutarate(Glu,Gln,Pro,Hyp),oxaloacetate(Asp,Asn)and3-phospho-REFERENCESglycerate(Ser,Gly).•Cysteine,tyrosine,andhydroxylysineareformedBrownKM,ArthurJR:Selenium,selenoproteinsandhumanhealth:areview.PublicHealthNutr2001;4:593.fromnutritionallyessentialaminoacids.Serinepro-CombsGF,GrayWP:Chemopreventiveagents—selenium.Phar-videsthecarbonskeletonandhomocysteinethesul-macolTher1998;79:179.furforcysteinebiosynthesis.Phenylalaninehydroxy-MercerLP,DoddsSJ,SmithDI:Dispensable,indispensable,andlaseconvertsphenylalaninetotyrosine.conditionallyindispensableaminoacidratiosinthediet.In:•NeitherdietaryhydroxyprolinenorhydroxylysineisAbsorptionandUtilizationofAminoAcids.FriedmanM(edi-incorporatedintoproteinsbecausenocodonortor).CRCPress,1989.tRNAdictatestheirinsertionintopeptides.NordbergJetal:Mammalianthioredoxinreductaseisirreversiblyinhibitedbydinitrohalobenzenesbyalkylationofboththe•Peptidylhydroxyprolineandhydroxylysineareredoxactiveselenocysteineanditsneighboringcysteineformedbyhydroxylationofpeptidylprolineorlysineresidue.JBiolChem1998;273:10835.inreactionscatalyzedbymixed-functionoxidasesScriverCRetal(editors):TheMetabolicandMolecularBasesofIn-thatrequirevitaminCascofactor.ThenutritionalheritedDisease,8thed.McGraw-Hill,2001.diseasescurvyreflectsimpairedhydroxylationduetoStGermainDL,GaltonVA:Thedeiodinasefamilyofselenopro-adeficiencyofvitaminC.teins.Thyroid1997;7:655.
240CatabolismofProteins&ofAminoAcidNitrogen29VictorW.Rodwell,PhDBIOMEDICALIMPORTANCEzymeshaveat1/2of0.5–2hours.PESTsequences,re-gionsrichinproline(P),glutamate(E),serine(S),andThischapterdescribeshowthenitrogenofaminoacidsisthreonine(T),targetsomeproteinsforrapiddegrada-convertedtoureaandtheraredisordersthataccompanytion.Intracellularproteaseshydrolyzeinternalpeptidedefectsinureabiosynthesis.Innormaladults,nitrogenbonds.Theresultingpeptidesarethendegradedtointakematchesnitrogenexcreted.Positivenitrogenbal-aminoacidsbyendopeptidasesthatcleaveinternalance,anexcessofingestedoverexcretednitrogen,ac-bondsandbyaminopeptidasesandcarboxypeptidasescompaniesgrowthandpregnancy.Negativenitrogenthatremoveaminoacidssequentiallyfromtheaminobalance,whereoutputexceedsintake,mayfollowandcarboxylterminals,respectively.Degradationofsurgery,advancedcancer,andkwashiorkorormarasmus.circulatingpeptidessuchashormonesfollowslossofaWhileammonia,derivedmainlyfromtheα-aminosialicacidmoietyfromthenonreducingendsoftheirnitrogenofaminoacids,ishighlytoxic,tissuesconvertoligosaccharidechains.Asialoglycoproteinsareinternal-ammoniatotheamidenitrogenofnontoxicglutamine.izedbylivercellasialoglycoproteinreceptorsandde-Subsequentdeaminationofglutamineintheliverre-gradedbylysosomalproteasestermedcathepsins.leasesammonia,whichisthenconvertedtonontoxicExtracellular,membrane-associated,andlong-livedurea.Ifliverfunctioniscompromised,asincirrhosisorintracellularproteinsaredegradedinlysosomesbyhepatitis,elevatedbloodammonialevelsgenerateclini-ATP-independentprocesses.Bycontrast,degradationcalsignsandsymptoms.Raremetabolicdisordersin-ofabnormalandothershort-livedproteinsoccursinvolveeachofthefiveureacycleenzymes.thecytosolandrequiresATPandubiquitin.Ubiquitin,sonamedbecauseitispresentinalleukaryoticcells,isaPROTEINTURNOVEROCCURSsmall(8.5kDa)proteinthattargetsmanyintracellularINALLFORMSOFLIFEproteinsfordegradation.Theprimarystructureofubiquitinishighlyconserved.Only3of76residuesThecontinuousdegradationandsynthesisofcellulardifferbetweenyeastandhumanubiquitin.Severalmol-proteinsoccurinallformsoflife.Eachdayhumanseculesofubiquitinareattachedbynon-α-peptideturnover1–2%oftheirtotalbodyprotein,principallybondsformedbetweenthecarboxylterminalofubiqui-muscleprotein.Highratesofproteindegradationoccurtinandtheε-aminogroupsoflysylresiduesinthetar-intissuesundergoingstructuralrearrangement—eg,getprotein(Figure29–1).Theresiduepresentatitsuterinetissueduringpregnancy,tadpoletailtissuedur-aminoterminalaffectswhetheraproteinisubiquiti-ingmetamorphosis,orskeletalmuscleinstarvation.Ofnated.AminoterminalMetorSerretardswhereasAsptheliberatedaminoacids,approximately75%arereuti-orArgacceleratesubiquitination.Degradationoccurslized.Theexcessnitrogenformsurea.Sinceexcessinamulticatalyticcomplexofproteasesknownastheaminoacidsarenotstored,thosenotimmediatelyin-proteasome.corporatedintonewproteinarerapidlydegraded.ANIMALSCONVERT-AMINONITROGENPROTEASES&PEPTIDASESDEGRADETOVARIEDENDPRODUCTSPROTEINSTOAMINOACIDSDifferentanimalsexcreteexcessnitrogenasammonia,Thesusceptibilityofaproteintodegradationisex-uricacid,orurea.Theaqueousenvironmentofpressedasitshalf-life(t1/2),thetimerequiredtolowerteleosteanfish,whichareammonotelic(excreteammo-itsconcentrationtohalftheinitialvalue.Half-livesofnia),compelsthemtoexcretewatercontinuously,facil-liverproteinsrangefromunder30minutestoover150itatingexcretionofhighlytoxicammonia.Birds,whichhours.Typical“housekeeping”enzymeshavet1/2valuesmustconservewaterandmaintainlowweight,areuri-ofover100hours.Bycontrast,manykeyregulatoryen-cotelicandexcreteuricacidassemisolidguano.Many242
241CATABOLISMOFPROTEINS&OFAMINOACIDNITROGEN/243OO1.–+ESH+ATPAMP+PP+UBCSEUBCO1i1OO2.UBCSE1+E2SHE1SH+UBCSE2OOHE33.UBCSE2+H2NεProteinE2SH+UBCNεProteinFigure29–1.Partialreactionsintheattachmentofubiquitin(UB)toproteins.(1)TheterminalCOOHofubiquitinformsathioesterbondwithan-SHofE1inareactiondrivenbyconversionofATPtoAMPandPPi.Sub-sequenthydrolysisofPPibypyrophosphataseensuresthatreaction1willproceedreadily.(2)Athioesterexchangereactiontransfersactivatedubiq-uitintoE2.(3)E3catalyzestransferofubiquitintoε-aminogroupsoflysylresiduesoftargetproteins.landanimals,includinghumans,areureotelicandex-acidsexceptlysine,threonine,proline,andhydroxypro-cretenontoxic,water-solubleurea.Highbloodurealev-lineparticipateintransamination.Transaminationiselsinrenaldiseaseareaconsequence—notacause—ofreadilyreversible,andaminotransferasesalsofunctionimpairedrenalfunction.inaminoacidbiosynthesis.Thecoenzymepyridoxalphosphate(PLP)ispresentatthecatalyticsiteofBIOSYNTHESISOFUREAaminotransferasesandofmanyotherenzymesthatactonaminoacids.PLP,aderivativeofvitaminB6,formsUreabiosynthesisoccursinfourstages:(1)transamina-anenzyme-boundSchiffbaseintermediatethatcanre-tion,(2)oxidativedeaminationofglutamate,(3)am-arrangeinvariousways.Duringtransamination,boundmoniatransport,and(4)reactionsoftheureacyclePLPservesasacarrierofaminogroups.Rearrangement(Figure29–2).formsanα-ketoacidandenzyme-boundpyridoxaminephosphate,whichformsaSchiffbasewithasecondTransaminationTransfersα-Aminoketoacid.Followingremovalofα-aminonitrogenbyNitrogentoα-Ketoglutarate,transamination,theremainingcarbon“skeleton”isde-gradedbypathwaysdiscussedinChapter30.FormingGlutamateAlanine-pyruvateaminotransferase(alanineamino-Transaminationinterconvertspairsofα-aminoacidstransferase)andglutamate-α-ketoglutarateaminotrans-andα-ketoacids(Figure29–3).Alltheproteinaminoferase(glutamateaminotransferase)catalyzethetransferα-Aminoacidα-KetoacidTRANSAMINATIONNH+O3α-KetoglutarateL-GlutamateCHO–CO–RCRC11OXIDATIVEOODEAMINATIONNH3CO2+ONH3CO–CHO–UREACYCLERCRC22UreaOOFigure29–2.OverallflowofnitrogeninaminoacidFigure29–3.Transamination.Thereactionisfreelycatabolism.reversiblewithanequilibriumconstantclosetounity.
242244/CHAPTER29ofaminogroupstopyruvate(formingalanine)ortoα-NAD(P)+NAD(P)H+H+ketoglutarate(formingglutamate)(Figure29–4).EachaminotransferaseisspecificforonepairofsubstratesNH3butnonspecificfortheotherpair.Sincealanineisalsoasubstrateforglutamateaminotransferase,alltheaminoL-Glutamateα-Ketoglutaratenitrogenfromaminoacidsthatundergotransamina-tioncanbeconcentratedinglutamate.Thisisimpor-Figure29–5.TheL-glutamatedehydrogenasereac-tantbecauseL-glutamateistheonlyaminoacidthattion.NAD(P)+meansthateitherNAD+orNADP+canundergoesoxidativedeaminationatanappreciablerateserveasco-substrate.Thereactionisreversiblebutfa-inmammaliantissues.Theformationofammoniavorsglutamateformation.fromα-aminogroupsthusoccursmainlyviatheα-aminonitrogenofL-glutamate.Transaminationisnotrestrictedtoα-aminogroups.drogenperoxide(H2O2),whichthenissplittoO2andTheδ-aminogroupofornithine—butnottheε-aminoH2Obycatalase.groupoflysine—readilyundergoestransamination.SerumlevelsofaminotransferasesareelevatedinsomeAmmoniaIntoxicationIsLife-Threateningdiseasestates(seeFigure7–11).Theammoniaproducedbyentericbacteriaandab-sorbedintoportalvenousbloodandtheammoniapro-L-GLUTAMATEDEHYDROGENASEducedbytissuesarerapidlyremovedfromcirculationOCCUPIESACENTRALPOSITIONbytheliverandconvertedtourea.Onlytraces(10–20INNITROGENMETABOLISMμg/dL)thusnormallyarepresentinperipheralblood.Thisisessential,sinceammoniaistoxictothecentralTransferofaminonitrogentoα-ketoglutarateformsL-nervoussystem.Shouldportalbloodbypasstheliver,glutamate.Releaseofthisnitrogenasammoniaisthensystemicbloodammonialevelsmayrisetotoxiclevels.catalyzedbyhepaticL-glutamatedehydrogenaseThisoccursinseverelyimpairedhepaticfunctionorthe(GDH),whichcanuseeitherNAD+orNADP+(Fig-developmentofcollaterallinksbetweentheportalandure29–5).Conversionofα-aminonitrogentoammo-systemicveinsincirrhosis.Symptomsofammoniain-niabytheconcertedactionofglutamateaminotrans-toxicationincludetremor,slurredspeech,blurredvi-feraseandGDHisoftentermed“transdeamination.”sion,coma,andultimatelydeath.AmmoniamaybeLiverGDHactivityisallostericallyinhibitedbyATP,toxictothebraininpartbecauseitreactswithα-keto-GTP,andNADHandactivatedbyADP.Thereactionglutaratetoformglutamate.Theresultingdepletedlev-catalyzedbyGDHisfreelyreversibleandfunctionsalsoelsofα-ketoglutaratethenimpairfunctionofthetri-inaminoacidbiosynthesis(seeFigure28–1).carboxylicacid(TCA)cycleinneurons.AminoAcidOxidasesAlsoRemoveNH+NH+32NitrogenasAmmoniaAMINOACIDCO–OXIDASECO–Whiletheirphysiologicroleisuncertain,L-aminoacidRHCRCoxidasesofliverandkidneyconvertaminoacidstoanOOα-iminoacidthatdecomposestoanα-ketoacidwithα-AminoacidFlavinFlavin-H2α-Iminoacidreleaseofammoniumion(Figure29–6).ThereducedHO2flavinisreoxidizedbymolecularoxygen,forminghy-NH+4HOOO222Pyruvateα-AminoacidCO–CATALASERCL-Alanineα-Ketoacid1/2O2OH2Oα-Ketoacidα-Ketoglutarateα-AminoacidFigure29–6.OxidativedeaminationcatalyzedbyL-Glutamateα-KetoacidL-aminoacidoxidase(L-α-aminoacid:O2oxidoreduc-Figure29–4.Alanineaminotransferase(top)andtase).Theα-iminoacid,showninbrackets,isnotaglutamateaminotransferase(bottom).stableintermediate.
243CATABOLISMOFPROTEINS&OFAMINOACIDNITROGEN/245GlutamineSynthaseFixesAmmoniaNH+3asGlutamine–HNCHCHO22CHCC2Formationofglutamineiscatalyzedbymitochondrialglutaminesynthase(Figure29–7).SinceamidebondOOsynthesisiscoupledtothehydrolysisofATPtoADPL-GlutamineandPi,thereactionstronglyfavorsglutaminesynthesis.H2OOnefunctionofglutamineistosequesterammoniainanontoxicform.GLUTAMINASENH+Glutaminase&AsparaginaseDeamidate4NH+Glutamine&Asparagine3–OCHCHO–2CHHydrolyticreleaseoftheamidenitrogenofglutamineC2Casammonia,catalyzedbyglutaminase(Figure29–8),OOstronglyfavorsglutamateformation.Theconcertedac-L-Glutamatetionofglutaminesynthaseandglutaminasethuscat-alyzestheinterconversionoffreeammoniumionandFigure29–8.Theglutaminasereactionproceedses-glutamine.AnanalogousreactioniscatalyzedbyL-as-sentiallyirreversiblyinthedirectionofglutamateandparaginase.NH4+formation.Notethattheamidenitrogen,nottheα-aminonitrogen,isremoved.Formation&SecretionofAmmoniaMaintainsAcid-BaseBalanceExcretionintourineofammoniaproducedbyrenaltubu-reactionsofFigure29–9.Ofthesixparticipatinglarcellsfacilitatescationconservationandregulationofaminoacids,N-acetylglutamatefunctionssolelyasanacid-basebalance.Ammoniaproductionfromintracellu-enzymeactivator.Theothersserveascarriersofthelarrenalaminoacids,especiallyglutamine,increasesinatomsthatultimatelybecomeurea.Themajormeta-metabolicacidosisanddecreasesinmetabolicalkalosis.bolicroleofornithine,citrulline,andargininosucci-nateinmammalsisureasynthesis.UreasynthesisisaUREAISTHEMAJORENDPRODUCTOFcyclicprocess.Sincetheornithineconsumedinreac-tion2isregeneratedinreaction5,thereisnonetlossNITROGENCATABOLISMINHUMANSorgainofornithine,citrulline,argininosuccinate,orSynthesisof1molofurearequires3molofATPplusarginine.Ammoniumion,CO2,ATP,andaspartate1moleachofammoniumionandoftheα-aminonitro-are,however,consumed.Somereactionsofureasyn-genofaspartate.Fiveenzymescatalyzethenumberedthesisoccurinthematrixofthemitochondrion,otherreactionsinthecytosol(Figure29–9).NH+3CarbamoylPhosphateSynthaseI–OCHCHO–C2CHCInitiatesUreaBiosynthesis2OOCondensationofCO2,ammonia,andATPtoformL-GlutamatecarbamoylphosphateiscatalyzedbymitochondrialMg-ATPNH+carbamoylphosphatesynthaseI(reaction1,Figure429–9).Acytosolicformofthisenzyme,carbamoylGLUTAMINEphosphatesynthaseII,usesglutamineratherthanam-SYNTHASEmoniaasthenitrogendonorandfunctionsinpyrimi-Mg-ADPH2Odinebiosynthesis(seeChapter34).Carbamoylphos-+PiNH+phatesynthaseI,therate-limitingenzymeoftheurea3–cycle,isactiveonlyinthepresenceofitsallostericacti-HNCHCHO22CHC2CvatorN-acetylglutamate,whichenhancestheaffinityofthesynthaseforATP.Formationofcarbamoylphos-OOphaterequires2molofATP,oneofwhichservesasaL-Glutaminephosphatedonor.ConversionofthesecondATPtoFigure29–7.TheglutaminesynthasereactionAMPandpyrophosphate,coupledtothehydrolysisofstronglyfavorsglutaminesynthesis.pyrophosphatetoorthophosphate,providesthedriving
244246/CHAPTER29CONH+24CO+NH+24NH2UreaCOCARBAMOYLNH2H2O+2Mg-ATPPHOSPHATENH35SYNTHASEICNHN-Acetyl-glutamate1CH2NH3+ARGINASECH2NHCH2CH22Mg-ADP+PiCH2CH2HNCH3+HNCH3+−−COOCOOL-OrnithineL-Arginine−OOHCCOO−HNCOPO−OOCCH2ORNITHINEO−TRANSCARBAMOYLASE4FumarateCarbamoylphosphate2ARGININOSUCCINASEPi−NH2NHCOOCOCNHCHCH2NHCH2NHCH2−CH2CH2COOCH23CH2ArgininosuccinateHNCH3+HNCH3+ARGININOSUCCINICACID−−COOSYNTHASECOOL-CitrullineMg-ATPAMP+Mg-PPi−COOH2NCHCH2−COOL-AspartateFigure29–9.Reactionsandintermediatesofureabiosynthesis.Thenitrogen-containinggroupsthatcontributetotheformationofureaareshaded.Reactions1and2occurinthematrixoflivermitochon-driaandreactions3,4,and5inlivercytosol.CO2(asbicarbonate),ammoniumion,ornithine,andcit-rullineenterthemitochondrialmatrixviaspecificcarriers(seeheavydots)presentintheinnermembraneoflivermitochondria.forceforsynthesisoftheamidebondandthemixedCarbamoylPhosphatePlusOrnithineacidanhydridebondofcarbamoylphosphate.Thecon-FormsCitrullinecertedactionofGDHandcarbamoylphosphatesyn-thaseIthusshuttlesnitrogenintocarbamoylphos-L-Ornithinetranscarbamoylasecatalyzestransferofphate,acompoundwithhighgrouptransferpotential.thecarbamoylgroupofcarbamoylphosphatetoor-Thereactionproceedsstepwise.Reactionofbicarbo-nithine,formingcitrullineandorthophosphate(reac-natewithATPformscarbonylphosphateandADP.tion2,Figure29–9).WhilethereactionoccursintheAmmoniathendisplacesADP,formingcarbamateandmitochondrialmatrix,boththeformationofornithineorthophosphate.PhosphorylationofcarbamatebytheandthesubsequentmetabolismofcitrullinetakeplacesecondATPthenformscarbamoylphosphate.inthecytosol.Entryofornithineintomitochondria
245CATABOLISMOFPROTEINS&OFAMINOACIDNITROGEN/247andexodusofcitrullinefrommitochondriathereforeofammoniathataccompaniesenhancedproteindegra-involvemitochondrialinnermembranetransportsys-dation.tems(Figure29–9).METABOLICDISORDERSARECitrullinePlusAspartateASSOCIATEDWITHEACHREACTIONFormsArgininosuccinateOFTHEUREACYCLEArgininosuccinatesynthaselinksaspartateandcit-rullineviatheaminogroupofaspartate(reaction3,Metabolicdisordersofureabiosynthesis,whileex-Figure29–9)andprovidesthesecondnitrogenofurea.tremelyrare,illustratefourimportantprinciples:(1)ThereactionrequiresATPandinvolvesintermediateDefectsinanyofseveralenzymesofametabolicpath-formationofcitrullyl-AMP.SubsequentdisplacementwayenzymecanresultinsimilarclinicalsignsandofAMPbyaspartatethenformscitrulline.symptoms.(2)Theidentificationofintermediatesandofancillaryproductsthataccumulatepriortoameta-CleavageofArgininosuccinatebolicblockprovidesinsightintothereactionthatisim-FormsArginine&Fumaratepaired.(3)Precisediagnosisrequiresquantitativeassayoftheactivityoftheenzymethoughttobedefective.Cleavageofargininosuccinate,catalyzedbyargini-(4)Rationaltherapymustbebasedonanunderstand-nosuccinase,proceedswithretentionofnitrogeniningoftheunderlyingbiochemicalreactionsinnormalarginineandreleaseoftheaspartateskeletonasfu-andimpairedindividuals.marate(reaction4,Figure29–9).AdditionofwatertoAlldefectsinureasynthesisresultinammoniain-fumarateformsL-malate,andsubsequentNAD+-toxication.Intoxicationismoreseverewhenthemeta-dependentoxidationofmalateformsoxaloacetate.bolicblockoccursatreactions1or2sincesomecova-Thesetworeactionsareanalogoustoreactionsofthelentlinkingofammoniatocarbonhasalreadyoccurredcitricacidcycle(seeFigure16–3)butarecatalyzedbyifcitrullinecanbesynthesized.Clinicalsymptomscytosolicfumaraseandmalatedehydrogenase.Transami-commontoallureacycledisordersincludevomiting,nationofoxaloacetatebyglutamateaminotransferaseavoidanceofhigh-proteinfoods,intermittentataxia,ir-thenre-formsaspartate.Thecarbonskeletonofaspartate-ritability,lethargy,andmentalretardation.Theclinicalfumaratethusactsasacarrierofthenitrogenofgluta-featuresandtreatmentofallfivedisordersdiscussedmateintoaprecursorofurea.belowaresimilar.Significantimprovementandmini-mizationofbraindamageaccompanyalow-proteinCleavageofArginineReleasesUreadietingestedasfrequentsmallmealstoavoidsudden&Re-formsOrnithineincreasesinbloodammonialevels.HyperammonemiaType1.AconsequenceofHydrolyticcleavageoftheguanidinogroupofarginine,carbamoylphosphatesynthaseIdeficiency(reac-catalyzedbyliverarginase,releasesurea(reaction5,tion1,Figure29–9),thisrelativelyinfrequentconditionFigure29–9).Theotherproduct,ornithine,reenters(estimatedfrequency1:62,000)probablyisfamilial.livermitochondriaforadditionalroundsofureasyn-HyperammonemiaType2.Adeficiencyofor-thesis.Ornithineandlysinearepotentinhibitorsofnithinetranscarbamoylase(reaction2,Figure29–9)arginase,competitivewitharginine.ArgininealsoservesproducesthisXchromosome–linkeddeficiency.Theastheprecursorofthepotentmusclerelaxantnitric2+mothersalsoexhibithyperammonemiaandanaversionoxide(NO)inaCa-dependentreactioncatalyzedbytohigh-proteinfoods.LevelsofglutamineareelevatedNOsynthase(seeFigure49–15).inblood,cerebrospinalfluid,andurine,probablydueCarbamoylPhosphateSynthaseIIsthetoenhancedglutaminesynthesisinresponsetoelevatedlevelsoftissueammonia.PacemakerEnzymeoftheUreaCycleCitrullinemia.Inthisraredisorder,plasmaandTheactivityofcarbamoylphosphatesynthaseIisdeter-cerebrospinalfluidcitrullinelevelsareelevatedandminedbyN-acetylglutamate,whosesteady-statelevelis1–2gofcitrullineareexcreteddaily.Onepatientlackeddictatedbyitsrateofsynthesisfromacetyl-CoAanddetectableargininosuccinatesynthaseactivity(reac-glutamateanditsrateofhydrolysistoacetateandglu-tion3,Figure29–9).Inanother,theKmforcitrullinetamate.ThesereactionsarecatalyzedbyN-acetylglu-was25timeshigherthannormal.Citrullineandargini-tamatesynthaseandN-acetylglutamatehydrolase,re-nosuccinate,whichcontainnitrogendestinedforureaspectively.Majorchangesindietcanincreasethesynthesis,serveasalternativecarriersofexcessnitrogen.concentrationsofindividualureacycleenzymes10-foldFeedingarginineenhancedexcretionofcitrullineintheseto20-fold.Starvation,forexample,elevatesenzymelev-patients.Similarly,feedingbenzoatedivertsammoniaels,presumablytocopewiththeincreasedproductionnitrogentohippurateviaglycine(seeFigure31–1).
246248/CHAPTER29Argininosuccinicaciduria.Ararediseasecharac-•Transaminationchannelsα-aminoacidnitrogenintoterizedbyelevatedlevelsofargininosuccinateinblood,glutamate.L-Glutamatedehydrogenase(GDH)oc-cerebrospinalfluid,andurineisassociatedwithfriable,cupiesacentralpositioninnitrogenmetabolism.tuftedhair(trichorrhexisnodosa).Bothearly-onsetand•GlutaminesynthaseconvertsNH3tonontoxicgluta-late-onsettypesareknown.Themetabolicdefectismine.GlutaminasereleasesNH3foruseinureasyn-theabsenceofargininosuccinase(reaction4,Figurethesis.29–9).Diagnosisbymeasurementoferythrocyteargini-•NH3,CO2,andtheamidenitrogenofaspartatepro-nosuccinaseactivitycanbeperformedonumbilicalvidetheatomsofurea.cordbloodoramnioticfluidcells.Asforcitrullinemia,•Hepaticureasynthesistakesplaceinpartinthemi-feedingarginineandbenzoatepromotesnitrogenexcre-tochondrialmatrixandinpartinthecytosol.Inborntion.errorsofmetabolismareassociatedwitheachreac-Hyperargininemia.Thisdefectischaracterizedbytionoftheureacycle.elevatedbloodandcerebrospinalfluidargininelevels,lowerythrocytelevelsofarginase(reaction5,Figure•Changesinenzymelevelsandallostericregulationof29–9),andaurinaryaminoacidpatternresemblingcarbamoylphosphatesynthasebyN-acetylglutamatethatoflysine-cystinuria.Thispatternmayreflectcom-regulateureabiosynthesis.petitionbyargininewithlysineandcystineforreab-sorptionintherenaltubule.Alow-proteindietlowersREFERENCESplasmaammonialevelsandabolisheslysine-cystinuria.BrooksPetal:Subcellularlocalizationofproteasomesandtheirregulatorycomplexesinmammaliancells.BiochemJ2000;GeneTherapyOffersPromisefor346:155.CorrectingDefectsinUreaBiosynthesisCurthoysNP,WatfordM:Regulationofglutaminaseactivityandglutaminemetabolism.AnnuRevNutr1995;15:133.GenetherapyforrectificationofdefectsintheenzymesHershkoA,CiechanoverA:Theubiquitinsystem.AnnuRevoftheureacycleisanareaofactiveinvestigation.En-Biochem1998;67:425.couragingpreliminaryresultshavebeenobtained,forIyerRetal:Thehumanarginasesandarginasedeficiency.JInheritexample,inanimalmodelsusinganadenoviralvectorMetabDis1998;21:86.totreatcitrullinemia.LimCBetal:Reductionintheratesofproteinandaminoacidca-tabolismtoslowdowntheaccumulationofendogenousam-monia:astrategypotentiallyadoptedbymudskippersduringSUMMARYaerialexposureinconstantdarkness.JExpBiol2001;204:1605.•Humansubjectsdegrade1–2%oftheirbodyproteinPatejunasGetal:Evaluationofgenetherapyforcitrullinaemiadailyatratesthatvarywidelybetweenproteinsandusingmurineandbovinemodels.JInheritMetabDiswithphysiologicstate.Keyregulatoryenzymesoften1998;21:138.haveshorthalf-lives.PickartCM.Mechanismsunderlyingubiquitination.AnnuRev•ProteinsaredegradedbybothATP-dependentandBiochem2001;70:503.ATP-independentpathways.UbiquitintargetsmanyScriverCRetal(editors):TheMetabolicandMolecularBasesofIn-intracellularproteinsfordegradation.Livercellsur-heritedDisease,8thed.McGraw-Hill,2001.facereceptorsbindandinternalizecirculatingasialo-TuchmanMetal:Thebiochemicalandmolecularspectrumofor-nithinetranscarbamoylasedeficiency.JInheritMetabDisglycoproteinsdestinedforlysosomaldegradation.1998;21:40.•Ammoniaishighlytoxic.FishexcreteNH3directly;TurnerMAetal:Humanargininosuccinatelyase:astructuralbasisbirdsconvertNH3touricacid.Highervertebratesforintrageniccomplementation.ProcNatlAcadSciUSAconvertNH3tourea.1997;94:9063.
247CatabolismoftheCarbonSkeletonsofAminoAcids30VictorW.Rodwell,PhDBIOMEDICALIMPORTANCETRANSAMINATIONTYPICALLYINITIATESAMINOACIDCATABOLISMThischapterconsidersconversionofthecarbonskele-tonsofthecommonL-aminoacidstoamphibolicinter-Removalofα-aminonitrogenbytransamination(seemediatesandthemetabolicdiseasesor“inbornerrorsofFigure28–3)isthefirstcatabolicreactionofaminometabolism”associatedwiththeseprocesses.Leftun-acidsexceptinthecaseofproline,hydroxyproline,treated,theycanresultinirreversiblebraindamageandthreonine,andlysine.Theresidualhydrocarbonskele-earlymortality.Prenatalorearlypostnataldetectiontonisthendegradedtoamphibolicintermediatesasandtimelyinitiationoftreatmentthusareessential.outlinedinFigure30–1.Manyoftheenzymesconcernedcanbedetectedincul-Asparagine,Aspartate,Glutamine,andGluta-turedamnioticfluidcells,whichfacilitatesearlydiag-mate.Allfourcarbonsofasparagineandaspartatenosisbyamniocentesis.Treatmentconsistsprimarilyofformoxaloacetate(Figure30–2,top).Analogousreac-feedingdietslowintheaminoacidswhosecatabolismtionsconvertglutamineandglutamateto-ketoglu-isimpaired.Whilemanychangesintheprimarystruc-tarate(Figure30–2,bottom).Sincetheenzymesalsotureofenzymeshavenoadverseeffects,othersmodifyfulfillanabolicfunctions,nometabolicdefectsareasso-thethree-dimensionalstructureofcatalyticorregula-ciatedwiththecatabolismofthesefouraminoacids.torysites,lowercatalyticefficiency(lowerVmaxorele-Proline.Prolineformsdehydroproline,glutamate-vateKm),oraltertheaffinityforanallostericregulatorγ-semialdehyde,glutamate,and,ultimately,-ketoglu-ofactivity.Avarietyofmutationsthusmaygiverisetotarate(Figure30–3,top).Themetabolicblockintypethesameclinicalsignsandsymptoms.Ihyperprolinemiaisatprolinedehydrogenase.AlaCysArgGlyHisHypα-KetoglutarateGlutamateGlnSerProThrlleLeulleTrpCitrateSuccinyl-CoAMetValPyruvateCitratecycleAcetyl-CoAAcetoacetyl-CoATyrOxaloacetateFumaratePheLeu,Lys,Phe,Trp,TyrAspartateAsnFigure30–1.Amphibolicintermediatesformedfromthecarbonskeletonsofaminoacids.249
248250/CHAPTER30OHONH+OO24PYRALACCCNHO–O–2CH2CH2CH2HH+CNH2ASPARAGINASECNH3TRANSAMINASECOCOO–COOCOO–L-AsparagineL-AspartateOxaloacetateOOOCHONH+CCNH24–PYRALAO–2OFigure30–2.CatabolismofL-as-CH2CH2CH2paragine(top)andofL-glutamineCH2CH2CH2(bottom)toamphibolicintermedi-GLUTAMINASETRANSAMINASEHCNH+HCNH+COates.(PYR,pyruvate;ALA,L-alanine.)33–––Inthisandsubsequentfigures,colorCOOCOOCOOhighlightsportionsofthemoleculesL-GlutamineL-Glutamateα-Ketoglutarateundergoingchemicalchange.ThereisnoassociatedimpairmentofhydroxyprolineGlycine.Theglycinesynthasecomplexoflivermi-catabolism.ThemetabolicblockintypeIIhyperpro-tochondriasplitsglycinetoCO2andNH4+andforms510linemiaisatglutamate--semialdehydedehydroge-N,N-methylenetetrahydrofolate(Figure30–5).nase,whichalsofunctionsinhydroxyprolinecatabo-Glycinuriaresultsfromadefectinrenaltubularre-lism.Bothprolineandhydroxyprolinecatabolismthusabsorption.Thedefectinprimaryhyperoxaluriaisthe1areaffectedandΔ-pyrroline-3-hydroxy-5-carboxylatefailuretocatabolizeglyoxylateformedbydeamination(seeFigure30–10)isexcreted.ofglycine.Subsequentoxidationofglyoxylatetoox-ArginineandOrnithine.Arginineisconvertedtoalateresultsinurolithiasis,nephrocalcinosis,andearlyornithine,glutamateγ-semialdehyde,andthen-ke-mortalityfromrenalfailureorhypertension.toglutarate(Figure30–3,bottom).Mutationsinor-Serine.Followingconversiontoglycine,catalyzednithine-aminotransferaseelevateplasmaandurinarybyserinehydroxymethyltransferase(Figure30–5),ornithineandcausegyrateatrophyoftheretina.serinecatabolismmergeswiththatofglycine(FigureTreatmentinvolvesrestrictingdietaryarginine.Inhy-30–6).perornithinemia-hyperammonemiasyndrome,ade-Alanine.Transaminationofalanineformspyru-fectivemitochondrialornithine-citrullineantiportervate.Perhapsforthereasonadvancedunderglutamate(seeFigure29–9)impairstransportofornithineintoandaspartatecatabolism,thereisnoknownmetabolicmitochondriaforuseinureasynthesis.defectofalaninecatabolism.Cysteine.CystineisfirstHistidine.Catabolismofhistidineproceedsviareducedtocysteinebycystinereductase(Figureurocanate,4-imidazolone-5-propionate,andN-for-30–7).Twodifferentpathwaysthenconvertcysteinetomiminoglutamate(Figlu).Formiminogrouptransfertopyruvate(Figure30–8).tetrahydrofolateformsglutamate,then-ketoglu-Therearenumerousabnormalitiesofcysteineme-tarate(Figure30–4).Infolicaciddeficiency,grouptabolism.Cystine,lysine,arginine,andornithinearetransferisimpairedandFigluisexcreted.Excretionofexcretedincystine-lysinuria(cystinuria),adefectinFiglufollowingadoseofhistidinethushasbeenusedrenalreabsorption.Apartfromcystinecalculi,cystin-todetectfolicaciddeficiency.Benigndisordersofhisti-uriaisbenign.ThemixeddisulfideofL-cysteineanddinecatabolismincludehistidinemiaandurocanicL-homocysteine(Figure30–9)excretedbycystinuricaciduriaassociatedwithimpairedhistidase.patientsismoresolublethancystineandreducesfor-mationofcystinecalculi.SeveralmetabolicdefectsSIXAMINOACIDSFORMPYRUVATEresultinvitaminB6-responsiveor-unresponsiveho-mocystinurias.Defectivecarrier-mediatedtransportAllofthecarbonsofglycine,serine,alanine,andcys-ofcystineresultsincystinosis(cystinestoragedis-teineandtwocarbonsofthreonineformpyruvateandease)withdepositionofcystinecrystalsintissuessubsequentlyacetyl-CoA.andearlymortalityfromacuterenalfailure.Despite
249HHFigure30–3.Top:Catabolismofproline.NumeralsindicateHNsitesofthemetabolicdefectsin1typeIand2typeIIhyper-+HO−prolinemias.Bottom:Catabolismofarginine.Glutamate-γ-Csemialdehydeformsα-ketoglutarateasshownabove.3,siteofthemetabolicdefectinhyperargininemia.OL-ProlineNAD+PROLINE1++DEHYDROGENASEH3NHNNHNADH+H+–OCHCCNH+HHOO−L-HistidineCH2OOHISTIDASENH+4+NH3+HHNNHCH2CHO−–OCHCCH2CCCHOOOL-Glutamate-γ-semialdehydeUrocanate+H2ONADGLUTAMATESEMIALDEHYDEUROCANASE2DEHYDROGENASENADH+H++HNNHL-Glutamate–OCH2CCH2Oα-KetoglutarateO+4-Imidazolone-5-propionateNH3HHO−2H2NNCH2CHOIMIDAZOLONEPROPIONATECCH2CH2CHYDROLASENHO++L-ArginineHNNH2H2O–OCH2CHO–3ARGINASECCH2CUreaOONH+N-Formiminoglutamate(Figlu)3CH2CHO−H4folateCH2CH2CGLUTAMATEFORMIMINOTRANSFERASE+N5-FormiminoNH3OH4folateL-OrnithineL-Glutamateα-KGα-KetoglutarateGluFigure30–4.CatabolismofL-histidinetoα-ketoglu-tarate.(H4folate,tetrahydrofolate.)HistidaseistheL-Glutamate-γ-semialdehydeprobablesiteofthemetabolicdefectinhistidinemia.251
250252/CHAPTER30MethyleneNH+3H4folateH4folateCHO−NH+NH+33CysteineH2CCCHO–CHO–2HSOH2CCCHOOO[O]L-SerineGlycineCYSTEINEDIOXYGENASEFigure30–5.Interconversionofserineandglycinecatalyzedbyserinehydroxymethyltransferase.(H4fo-NH+3late,tetrahydrofolate.)CHO−CysteinesulfinateH2CC−OSO2NH+3α-KetoacidCHO–+NAD+2TRANSAMINASECα-AminoacidOOGlycineCO−H4folateSulfinylpyruvateH2CCN5,N10-CH-Hfolate–OSO242CO+NH++NADH+H+24DESULFINASESO2−3Figure30–6.Reversiblecleavageofglycinebythemitochondrialglycinesynthasecomplex.(PLP,pyri-Pyruvatedoxalphosphate.)CYSTEINEα-KANH+TRANSAMINASE3−α-AACHOH2CCOSO3-MercaptopyruvateCO−OS(thiolpyruvate)H2CCCCH2HSO−OCHNADH+2HNH3L-Cystine+H++NADH+HH2SNAD+CYSTINEPyruvateHOHREDUCTASECO−NAD+H2CCHSONH+33-MercaptolactateCHO−2CH2CFigure30–8.CatabolismofL-cysteineviathecys-teinesulfinatepathway(top)andbythe3-mercaptopy-SHOL-Cysteineruvatepathway(bottom).Figure30–7.Thecystinereductasereaction.
251CATABOLISMOFTHECARBONSKELETONSOFAMINOACIDS/253HHCH2SSCH2HN++HHCNH3CH2OHO––NH+COOHC3CCOO–O(Cysteine)(Homocysteine)4-Hydroxy-L-prolineFigure30–9.Mixeddisulfideofcysteineandhomo-1HYDROXYPROLINEcysteine.DEHYDROGENASE2HNH+NH+3OHHCCHO−O–3CHCCOHOO1L-Δ-Pyrroline-3-hydroxy-5-carboxylateL-ThreonineH2OTHREONINENONENZYMATICALDOLASEGlycineOHNH+HC33CH–CHCHOHCCH2COAcetaldehydeOO+γ-Hydroxy-L-glutamate-γ-semialdehydeH2ONADNAD+HO2ALDEHYDEDEHYDROGENASENADH+H+2DEHYDROGENASENADH+H+HCO−3NH+COH3–OCHCHO–OCCH2CAcetateOOCoASHMg-ATPErythro-γ-hydroxy-L-glutamateACETATEα-KATHIOKINASETRANSAMINASEH2OMg-ADPα-AAOHO–OCHCO–H3CSCoACCH2CCOOOα-Keto-γ-hydroxyglutarateAcetyl-CoAFigure30–10.ConversionofthreoninetoglycineANALDOLASE(seeFigure30–6)andacetyl-CoA.OO–OCHCO–CH3CCFigure30–11.IntermediatesinL-hydroxyprolinecatabolism.(α-KA,α-ketoacid;α-AA,α-aminoacid.)NumeralsidentifysitesofmetabolicOOdefectsin1hyperhydroxyprolinemiaand2typeIIhyperprolinemia.GlyoxylatePyruvate
252–OCO2;emia;e3CH9CO2toglutaratIItyrosin8CHee-k72Oαtyp1–O6CH-KG,Fumarylacetoacetateα5.(2COHCCectsin2Ofee3C4fateCHO9–OH47OHHomogentisate–tabolicdOe568irultimateCOmtheCAcetatee32ISOMERASEHsofthCO+e1Glutathionesit+mphasizee22CuMALEYLACETOACETATECIS,TRANSAscorbateSCoAdtoHYDROXYLASE–reOCOeprobabl[O]eAcetyl-CoACOC-HYDROXYPHENYLPYRUVATE3p2Hnumbntth–3CHeesOemia,ortyrosinosis.pre92COe1CO2(rewritten)9–OC22O8CHralsrCHOHMaleylacetoacetateeItyrosin347OC4COe7568-KETOTHIOLASEβtyp56dnum-Hydroxyphenylpyruvate–CoASHe4Glup=O7––OOcatabolism.CarbonsarOe.)Circl1COePLP2COCH6TYROSINE22TRANSAMINASE3CHCH5FumarateC+CH-KGα9OCOsintyrosinCC4Acetoacetatee–alkaptonuria;andOO–OCO–3diat31CO4OHeOC7Maleylacetoacetate+3rm29emia;eNHCH2568Int-Tyrosine3CH47OHL;PLP,pyridoxalphosphate5684+32FeOXIDASEHYDROLASEre30–12.onataltyrosineHOMOGENTISATEO2un[O]HFUMARYLACETOACETATEFigGlu,glutamat2254
253CATABOLISMOFTHECARBONSKELETONSOFAMINOACIDS/255epidemiologicdatasuggestingarelationshipbetweenthoseoftyrosine(Figure30–12).Hyperphenylala-plasmahomocysteineandcardiovasculardisease,ninemiasarisefromdefectsinphenylalaninehydroxy-whetherhomocysteinerepresentsacausalcardiovas-laseitself(typeI,classicphenylketonuriaorPKU),incularriskfactorremainscontroversial.dihydrobiopterinreductase(typesIIandIII),orindi-Threonine.Threonineiscleavedtoacetaldehydehydrobiopterinbiosynthesis(typesIVandV)(Figureandglycine.Oxidationofacetaldehydetoacetateisfol-28–10).Alternativecatabolitesareexcreted(Figurelowedbyformationofacetyl-CoA(Figure30–10).Ca-30–13).DNAprobesfacilitateprenataldiagnosisofde-tabolismofglycineisdiscussedabove.fectsinphenylalaninehydroxylaseordihydrobiopterin4-Hydroxyproline.Catabolismof4-hydroxy-L-pro-reductase.Adietlowinphenylalaninecanpreventthe1lineforms,successively,L-Δ-pyrroline-3-hydroxy-5-car-mentalretardationofPKU(frequency1:10,000boxylate,γ-hydroxy-L-glutamate-γ-semialdehyde,erythro-γ-hydroxy-L-glutamate,andα-keto-γ-hydroxyglutarate.Analdol-typecleavagethenformsglyoxylatepluspyru-CHCOO–vate(Figure30–11).Adefectin4-hydroxyprolinede-2CHhydrogenaseresultsinhyperhydroxyprolinemia,NH+whichisbenign.Thereisnoassociatedimpairmentof3prolinecatabolism.L-Phenylalanineα-KetoglutarateTWELVEAMINOACIDSFORMTRANSAMINASEACETYL-CoAL-GlutamateTyrosine.Figure30–12diagramstheconversionofCHCOO–2tyrosinetoamphibolicintermediates.SinceascorbateisCthereductantforconversionofp-hydroxyphenylpyru-Ovatetohomogentisate,scorbuticpatientsexcretein-Phenylpyruvatecompletelyoxidizedproductsoftyrosinecatabolism.Subsequentcatabolismformsmaleylacetoacetate,fu-NAD++marylacetoacetate,fumarate,acetoacetate,andulti-NADH+Hmatelyacetyl-CoA.H2OTheprobablemetabolicdefectintypeItyrosine-NADH+H++NADmia(tyrosinosis)isatfumarylacetoacetatehydrolaseCO2(reaction4,Figure30–12).Therapyemploysadietlowintyrosineandphenylalanine.UntreatedacuteandCHCHCOO–chronictyrosinosisleadstodeathfromliverfailure.Al-2–2COOCHternatemetabolitesoftyrosinearealsoexcretedintypeIItyrosinemia(Richner-Hanhartsyndrome),ade-OHfectintyrosineaminotransferase(reaction1,FigurePhenylacetatePhenyllactate30–12),andinneonataltyrosinemia,duetoloweredL-Glutaminep-hydroxyphenylpyruvatehydroxylaseactivity(reaction2,Figure30–12).Therapyemploysadietlowinprotein.H2OAlkaptonuriawasfirstdescribedinthe16thcen-COO–tury.Characterizedin1859,itprovidedthebasisforHCHNCHGarrod’sclassicideasconcerningheritablemetabolic2Cdisorders.ThedefectislackofhomogentisateoxidaseCH2(reaction3,Figure30–12).Theurinedarkensonexpo-OCH2suretoairduetooxidationofexcretedhomogentisate.PhenylacetylglutamineLateinthedisease,thereisarthritisandconnectivetis-CONH2suepigmentation(ochronosis)duetooxidationofho-mogentisatetobenzoquinoneacetate,whichpolymer-Figure30–13.Alternativepathwaysofphenylala-izesandbindstoconnectivetissue.ninecatabolisminphenylketonuria.ThereactionsalsoPhenylalanine.Phenylalanineisfirstconvertedtooccurinnormallivertissuebutareofminorsignifi-tyrosine(seeFigure28–10).Subsequentreactionsarecance.
254–O–sOer-COe+COsit3+e3NHCHNHCHdhyp2e2CHCH2probabl–2eOCHCHO2th2-AminoadipateeOC2+CH-αL+H2CH2CHNHC2SaccharopineCOHOC2CHO+–2ralsindicatmiawithoutassociateeCHOCNADH+HrlysinOOdnumee–2H++NADNADSCoA.)Circlnthyp–ee–OOC1,2O2COrsiste+OC+CH+3p+HSACCHAROPINE-LYSINE-FORMING32NADHDEHYDROGENASE,LNHCH2CHNHCH2–22Glutaryl-CoAOCHCHCHCO22+-Glutamate3CHLCH-Aminoadipate--semialdehydeOC-αδmia;andNHCH2LOe2OCCH–CHO–O;PLP,pyridoxalphosphat2HC2e–CHCOrammonOe2OC+CH[O]CNHCdhyp2eH2CoASHCHO2;Glu,glutamat2H–eCHOOC–COOOCOOC–+23toglutaratONHCHCHemiawithassociat2-keH22αCHCH-Ketoadipate22αrlysin–-KG,eOCHCHα–2.(OOCOCeCHCO+O–+3CHNH-lysinHCLriodichypNHCH2ep2CHCH212-LysineCHLPLP–CHOCOctsin2-KGGluTRANSAMINASEOαfeOC+CH–Catabolismofe2+CO+HNHC22HNADH+HCH2tabolicd2emia.eCH+re30–14.mSACCHAROPINEeOC-KetoglutarateNADDEHYDROGENASE,uα-GLUTAMATE-FORMINGLO–Figofthammon256
255O2CHC2CO−CHOO−CC-Ketoadipate−2αOHO−O+3NHCHO22CC2CNHONAD(P)HOXIDASE-Kynurenine+L3-HYDROXYANTHRANILATENAD(P)H+HO2O−2OCNHCHCCOHC−OFormateKYNURENINEFORMYLASEOHO−O2HCC3-HydroxyanthranilateHOOxalocrotonate−++O−4OCONADNH+3O+OCH3NHCHO+.)NHCHeCCNH2CCPLPH3HO-Formylkynurenine2KYNURENINASENADH+HLH+-3NHNHCC-muconate−OCOcis,HCOO+3−cis+2NHCHO2CCHsemialdehydeFeCCCNHHO22(inducible)H2-Amino-OTRYPTOPHANOXYGENASE2OH-Hydroxykynurenine-tryptophan.(PLP,pyridoxalphosphatLCOL3-−OO++3O+3−NHCHOCNHCatabolismofCCNH2HNADPH+H-TryptophanL2KYNURENINEHYDROXYLASECOOCHO−re30–15.Ou2-Acroleyl-3-aminofumarateFig257
256258/CHAPTER30OofnewborninfantsiscompulsoryintheUnitedStatesandmanyothercountries.CLysine.Figure30–14summarizesthecatabolismofCH2lysine.LysinefirstformsaSchiffbasewithα-ketoglu-–tarate,whichisreducedtosaccharopine.InoneformCHONNCofperiodichyperlysinemia,elevatedlysinecompeti-HH+23tivelyinhibitsliverarginase(seeFigure29–9),causingHOOhyperammonemia.Restrictingdietarylysinerelievesthe3-Hydroxykynurenineammonemia,whereasingestionofalysineloadprecipi-tatesseverecrisesandcoma.Inadifferentperiodichy-perlysinemia,lysinecatabolitesaccumulate,butevenaNH+lysineloaddoesnottriggerhyperammonemia.Inaddi-4tiontoimpairedsynthesisofsaccharopine,somepa-HOtientscannotcleavesaccharopine.Tryptophan.Tryptophanisdegradedtoamphi-bolicintermediatesviathekynurenine-anthranilate–pathway(Figure30–15).TryptophanoxygenaseOC(tryptophanpyrrolase)openstheindolering,incor-Nporatesmolecularoxygen,andformsN-formylkynure-HOOnine.Anironporphyrinmetalloproteinthatisin-Xanthurenateducibleinliverbyadrenalcorticosteroidsandbytryptophan,tryptophanoxygenaseisfeedback-Figure30–16.Formationofxanthurenateinvitamininhibitedbynicotinicacidderivatives,includingB6deficiency.ConversionofthetryptophanmetaboliteNADPH.Hydrolyticremovaloftheformylgroupof3-hydroxykynurenineto3-hydroxyanthranilateisim-N-formylkynurenine,catalyzedbykynurenineformy-paired(seeFigure30–15).Alargeportionisthereforelase,produceskynurenine.Sincekynureninasere-convertedtoxanthurenate.quirespyridoxalphosphate,excretionofxanthurenate(Figure30–16)inresponsetoatryptophanloadisdi-agnosticofvitaminB6deficiency.Hartnupdiseasere-births).Elevatedbloodphenylalaninemaynotbede-flectsimpairedintestinalandrenaltransportoftrypto-tectableuntil3–4dayspostpartum.False-positivesinphanandotherneutralaminoacids.Indolederivativesprematureinfantsmayreflectdelayedmaturationofen-ofunabsorbedtryptophanformedbyintestinalbacteriazymesofphenylalaninecatabolism.Alessreliableareexcreted.ThedefectlimitstryptophanavailabilityscreeningtestemploysFeCl3todetecturinaryforniacinbiosynthesisandaccountsforthepellagra-phenylpyruvate.FeCl3screeningforPKUoftheurinelikesignsandsymptoms.COOC–COOC–+H3NCH+H3NCHCH2CH2CHPPPH2OPi+PPiCH22S+CHAdenine+SCHAdenine22OOCH3L-METHIONINECH3ADENOSYLTRANSFERASERiboseRiboseHOOHHOOHL-MethionineATPS-Adenosyl-L-methionine(“activemethionine”)Figure30–17.FormationofS-adenosylmethionine.~CH3representsthehighgrouptransferpotentialof“activemethionine.”
257CATABOLISMOFTHECARBONSKELETONSOFAMINOACIDS/259NH+Methionine.MethioninereactswithATPforming3–S-adenosylmethionine,“activemethionine”(FigureH3CCH2CHOSCH2C30–17).Subsequentreactionsformpropionyl-CoA(Figure30–18)andultimatelysuccinyl-CoA(seeFig-Oure19–2).L-MethionineATPTHEINITIALREACTIONSARECOMMONPi+PPiTOALLTHREEBRANCHED-CHAINS-Adenosyl-L-methionineAMINOACIDSAcceptorReactions1–3ofFigure30–19areanalogoustothoseoffattyacidcatabolism.Followingtransamination,allCH3-Acceptorthreeα-ketoacidsundergooxidativedecarboxylationcatalyzedbymitochondrialbranched-chain-ketoS-Adenosyl-L-homocysteineaciddehydrogenase.ThismultimericenzymecomplexH2Oofadecarboxylase,atransacylase,andadihydrolipoyldehydrogenasecloselyresemblespyruvatedehydroge-Adenosinenase(seeFigure17–5).Itsregulationalsoparallelsthat+ofpyruvatedehydrogenase,beinginactivatedbyphos-NH3phorylationandreactivatedbydephosphorylation(seeCHCHO–2Figure17–6).HSCH2CReaction3isanalogoustothedehydrogenationof+Ofattyacyl-CoAthioesters(seeFigure22–3).Inisova-OOHL-Homocysteinelericacidemia,ingestionofprotein-richfoodsele-CCH2CYSTATHIONINEvatesisovalerate,thedeacylationproductofisovaleryl-–OCHβ-SYNTHASECoA.Figures30–20,30–21,and30–22illustratetheNH+subsequentreactionsuniquetoeachaminoacidskele-3HO2ton.L-SerineNH+3CHCHO–METABOLICDISORDERSOFBRANCHED-2OSCH2CCHAINAMINOACIDCATABOLISMCCH2O–OCHCystathionineAsthenameimplies,theodorofurineinmaplesyrupurinedisease(branched-chainketonuria)suggestsNH+3maplesyruporburntsugar.Thebiochemicaldefectin-HOvolvesthe-ketoaciddecarboxylasecomplex(reac-2OSHOtion2,Figure30–19).Plasmaandurinarylevelsofleucine,isoleucine,valine,α-ketoacids,andα-hydroxyCCHHCCO––23acids(reducedα-ketoacids)areelevated.Themecha-OCHCH2C+nismoftoxicityisunknown.Earlydiagnosis,especiallyNH+NH4O3priorto1weekofage,employsenzymaticanalysis.L-Cysteineα-KetobutyratePromptreplacementofdietaryproteinbyanamino+acidmixturethatlacksleucine,isoleucine,andvalineCoASHNADavertsbraindamageandearlymortality.Mutationofthedihydrolipoatereductasecompo-CONADH+H+2nentimpairsdecarboxylationofbranched-chainα-Oketoacids,ofpyruvate,andofα-ketoglutarate.Inin-termittentbranched-chainketonuria,theα-ketoH3CCaciddecarboxylaseretainssomeactivity,andsymp-CH2SCoAPropionyl-CoAtomsoccurlaterinlife.Theimpairedenzymeiniso-valericacidemiaisisovaleryl-CoAdehydrogenaseFigure30–18.Conversionofmethioninetopropi-(reaction3,Figure30–19).Vomiting,acidosis,andonyl-CoA.comafollowingestionofexcessprotein.Accumulated
258260/CHAPTER30CHNH+NH+NH+3333CHCHOHCCHOCHCHO–32H3CCH2CCHCH3CCHCOCH3OCH3OL-LeucineL-ValineL-Isoleucineα-Ketoacidα-Ketoacidα-Ketoacid111α-Aminoacidα-Aminoacidα-AminoacidCH3OOOCHCO–HCCO–CHCO–32H3CCH2CCHCH3CCHCOCH3OCH3Oα-Ketoisocaproateα-Ketoisovalerateα-Keto-β-methylvalerateCoASHCoASHCoASH222CO2CO2CO2CH3OOOCHCH3CCCHCH3CCH2SCoACHSCoAH3CCHSCoAIsovaleryl-CoACH3CH3Isobutyryl-CoAα-Methylbutyryl-CoA333[2H][2H][2H]CH3OOOHCCH2CCCCH3CCHSCoACSCoAH3CCSCoAβ-Methylcrotonyl-CoACH3CH3Methacrylyl-CoATiglyl-CoAFigure30–19.Theanalogousfirstthreereactionsinthecatabolismofleucine,valine,andisoleucine.Notealsotheanalogyofreactions2and3toreactionsofthecatabolismoffattyacids(seeFigure22–3).Theanalogytofattyacidcatabolismcontinues,asshowninsubsequentfigures.
259CATABOLISMOFTHECARBONSKELETONSOFAMINOACIDS/261CH3OOCCCHCH3CCHSCoAH3CCSCoAβ-Methylcrotonyl-CoACH3Biotinyl-*CO2Tiglyl-CoAH2O4L4IBiotinOHOOCH3OHCCC*CCH3CCHSCoA−OCCHHSCoA2β-Methylglutaconyl-CoACH3α-Methyl-β-hydroxybutyryl-CoAH2O5L5I[2H]OOC*H3COHCOO−OCCHHSCoA22CCβ-Hydroxy-β-methylglutaryl-CoAH3CCHSCoAOO6LOCH3C*CCα-Methylacetoacetyl-CoA−OHCHCHCSCoA233AcetoacetateAcetyl-CoACoASH6IFigure30–20.Catabolismoftheβ-methylcrotonyl-OOCoAformedfromL-leucine.AsterisksindicatecarbonatomsderivedfromCO2.CCH3CSCoA+CH2SCoACH3Acetyl-CoAPropionyl-CoAFigure30–21.Subsequentcatabolismofthetiglyl-CoAformedfromL-isoleucine.
260262/CHAPTER30Oisovaleryl-CoAishydrolyzedtoisovalerateandex-creted.HCC2CSCoACH3SUMMARYMethacrylyl-CoA•Excessaminoacidsarecatabolizedtoamphibolicin-4VH2Otermediatesusedassourcesofenergyorforcarbohy-drateandlipidbiosynthesis.HOO•TransaminationisthemostcommoninitialreactionHCCofaminoacidcatabolism.Subsequentreactionsre-2CHSCoAmoveanyadditionalnitrogenandrestructurethehy-CH3drocarbonskeletonforconversiontooxaloacetate,β-Hydroxyisobutyryl-CoAα-ketoglutarate,pyruvate,andacetyl-CoA.H2O•Metabolicdiseasesassociatedwithglycinecatabolism5Vincludeglycinuriaandprimaryhyperoxaluria.CoASH•Twodistinctpathwaysconvertcysteinetopyruvate.HOOMetabolicdisordersofcysteinecatabolismincludeH2CCcystine-lysinuria,cystinestoragedisease,andtheho-CHO–mocystinurias.CH3•Threoninecatabolismmergeswiththatofglycineβ-HydroxyisobutyrateafterthreoninealdolasecleavesthreoninetoglycineNAD+andacetaldehyde.6VNADH+H+•Followingtransamination,thecarbonskeletonofty-rosineisdegradedtofumarateandacetoacetate.OOMetabolicdiseasesoftyrosinecatabolismincludety-HCCrosinosis,Richner-Hanhartsyndrome,neonatalty-CHO–rosinemia,andalkaptonuria.CH3•Metabolicdisordersofphenylalaninecatabolismin-Methylmalonatesemialdehydecludephenylketonuria(PKU)andseveralhyper-α-AAphenylalaninemias.CoASHNAD+7V•Neithernitrogenoflysineundergoestransamination.8Vα-KAMetabolicdiseasesoflysinecatabolismincludeperi-+NADH+Hodicandpersistentformsofhyperlysinemia-OONH+O3ammonemia.–CCH2CC–•Thecatabolismofleucine,valine,andisoleucinepre-OCHSCoACHOsentsmanyanalogiestofattyacidcatabolism.Meta-CH3CH3bolicdisordersofbranched-chainaminoacidcatabo-Methylmalonyl-CoAβ-Aminoisobutyratelismincludehypervalinemia,maplesyrupurinedisease,intermittentbranched-chainketonuria,iso-9VBCOENZYME12valericacidemia,andmethylmalonicaciduria.OCREFERENCES–OCH2BlacherJ,SafarME:Homocysteine,folicacid,Bvitaminsandcar-HCSCoA2diovascularrisk.JNutrHealthAging2001;5:196.CCooperAJL:Biochemistryofthesulfur-containingaminoacids.OAnnuRevBiochem1983;52:187.Succinyl-CoAGjettingTetal:Aphenylalaninehydroxylaseaminoacidpolymor-phismwithimplicationsformoleculardiagnostics.MolFigure30–22.SubsequentcatabolismoftheGenetMetab2001;73:280.methacrylyl-CoAformedfromL-valine(seeFigureHarrisRAetal:Molecularcloningofthebranched-chainα-ke-30–19).(α-KA,α-ketoacid;α-AA,α-aminoacid.)toaciddehydrogenasekinaseandtheCoA-dependentmethyl-
261CATABOLISMOFTHECARBONSKELETONSOFAMINOACIDS/263malonatesemialdehydedehydrogenase.AdvEnzymeRegulWatersPJ,ScriverCR,ParniakMA:Homomericandheteromeric1993;33:255.interactionsbetweenwild-typeandmutantphenylalaninehy-ScriverCR:Garrod’sforesight;ourhindsight.JInheritMetabDisdroxylasesubunits:evaluationoftwo-hybridapproachesfor2001;24:93.functionalanalysisofmutationscausinghyperphenylalanine-mia.MolGenetMetab2001;73:230.ScriverCRetal(editors):TheMetabolicandMolecularBasesofIn-heritedDisease,8thed.McGraw-Hill,2001.
262ConversionofAminoAcidstoSpecializedProducts31VictorW.Rodwell,PhDBIOMEDICALIMPORTANCEβ-aminoisobutyrateareelevatedintheraremetabolicdisorderhyperbeta-alaninemia.Importantproductsderivedfromaminoacidsincludeheme,purines,pyrimidines,hormones,neurotransmit-ters,andbiologicallyactivepeptides.Inaddition,many-AlanylDipeptidesproteinscontainaminoacidsthathavebeenmodifiedTheβ-alanyldipeptidescarnosineandanserineforaspecificfunctionsuchasbindingcalciumorasin-(N-methylcarnosine)(Figure31–2)activatemyosintermediatesthatservetostabilizeproteins—generallyATPase,chelatecopper,andenhancecopperuptake.structuralproteins—bysubsequentcovalentcross-link-β-Alanyl-imidazolebuffersthepHofanaerobicallying.Theaminoacidresiduesinthoseproteinsserveascontractingskeletalmuscle.Biosynthesisofcarnosineisprecursorsforthesemodifiedresidues.Smallpeptidescatalyzedbycarnosinesynthetaseinatwo-stagereac-orpeptide-likemoleculesnotsynthesizedonribosomestionthatinvolvesinitialformationofanenzyme-boundfulfillspecificfunctionsincells.Histamineplaysacen-acyl-adenylateofβ-alanineandsubsequenttransferoftralroleinmanyallergicreactions.Neurotransmitterstheβ-alanylmoietytoL-histidine.derivedfromaminoacidsincludeγ-aminobutyrate,5-hydroxytryptamine(serotonin),dopamine,norepi-ATP+→−ββ--AlanineAlanylAMP→+PPinephrine,andepinephrine.Manydrugsusedtotreatneurologicandpsychiatricconditionsaffectthemetab-β-AlanylAMP−+L-Histidine→Carnosine+AMPolismoftheseneurotransmitters.Hydrolysisofcarnosinetoβ-alanineandL-histidineiscatalyzedbycarnosinase.Theheritabledisordercarnosinasedeficiencyischaracterizedbycarnosinuria.GlycineHomocarnosine(Figure31–2),presentinhumanMetabolitesandpharmaceuticalsexcretedaswater-brainathigherlevelsthancarnosine,issynthesizedinsolubleglycineconjugatesincludeglycocholicacidbraintissuebycarnosinesynthetase.Serumcarnosinase(Chapter24)andhippuricacidformedfromthefooddoesnothydrolyzehomocarnosine.Homocarnosinosis,additivebenzoate(Figure31–1).Manydrugs,drugararegeneticdisorder,isassociatedwithprogressivemetabolites,andothercompoundswithcarboxylspasticparaplegiaandmentalretardation.groupsareexcretedintheurineasglycineconjugates.Glycineisincorporatedintocreatine(seeFigure31–6),PhosphorylatedSerine,Threonine,thenitrogenandα-carbonofglycineareincorporated&Tyrosineintothepyrroleringsandthemethylenebridgecarbonsofheme(Chapter32),andtheentireglycinemoleculeThephosphorylationanddephosphorylationofseryl,becomesatoms4,5,and7ofpurines(Figure34–1).threonyl,andtyrosylresiduesregulatetheactivityofcertainenzymesoflipidandcarbohydratemetabolismandthepropertiesofproteinsthatparticipateinsignal-Alaninetransductioncascades.β-Alanine,ametaboliteofcysteine(Figure34–9),isMethioninepresentincoenzymeAandasβ-alanyldipeptides,prin-cipallycarnosine(seebelow).MammaliantissuesformS-Adenosylmethionine,theprincipalsourceofmethylβ-alaninefromcytosine(Figure34–9),carnosine,andgroupsinthebody,alsocontributesitscarbonskeletonanserine(Figure31–2).Mammaliantissuestransami-forthebiosynthesisofthe3-diaminopropaneportionsnateβ-alanine,formingmalonatesemialdehyde.Bodyofthepolyaminesspermineandspermidine(Figurefluidandtissuelevelsofβ-alanine,taurine,and31–4).264
263CONVERSIONOFAMINOACIDSTOSPECIALIZEDPRODUCTS/265OSH+C+N(CH3)3O–NNH2CHO–CH2CBenzoateOATPCoASHErgothioneineOCHNH+AMP+PP23iCCH2O+NHNNH2C–CHOSCoACH2COBenzoyl-CoACarnosineGlycineOCHNH+23CoASHCCH2+CH3ONNNHHCHO–CCHO–2CHCNC2HOOAnserineHippurateFigure31–1.Biosynthesisofhippurate.AnalogousOCH2CH2reactionsoccurwithmanyacidicdrugsandcatabolites.CCHNH+23+NHNNH2CysteineCHO–CH2CL-Cysteineisaprecursorofthethioethanolaminepor-tionofcoenzymeAandofthetaurinethatconjugatesOwithbileacidssuchastaurocholicacid(Chapter26).HomocarnosineHistidineFigure31–2.Compoundsrelatedtohistidine.Theboxessurroundthecomponentsnotderivedfromhisti-Decarboxylationofhistidinetohistamineiscatalyzedbydine.TheSHgroupofergothioneinederivesfromcys-abroad-specificityaromaticL-aminoaciddecarboxylaseteine.thatalsocatalyzesthedecarboxylationofdopa,5-hy-droxytryptophan,phenylalanine,tyrosine,andtrypto-phan.α-Methylaminoacids,whichinhibitdecarboxy-tricoxide(NO)thatservesasaneurotransmitter,laseactivity,findapplicationasantihypertensiveagents.smoothmusclerelaxant,andvasodilator.SynthesisofHistidinecompoundspresentinthehumanbodyin-NO,catalyzedbyNOsynthase,involvestheNADPH-cludeergothioneine,carnosine,anddietaryanserinedependentreactionofL-argininewithO2toyieldL-cit-(Figure31–2).Urinarylevelsof3-methylhistidinearerullineandNO.unusuallylowinpatientswithWilson’sdisease.PolyaminesOrnithine&ArginineThepolyaminesspermidineandspermine(FigureArginineistheformamidinedonorforcreatinesynthe-31–4)functionincellproliferationandgrowth,aresis(Figure31–6)andviaornithinetoputrescine,sper-growthfactorsforculturedmammaliancells,andstabi-mine,andspermidine(Figure31–3)Arginineisalsolizeintactcells,subcellularorganelles,andmembranes.theprecursoroftheintercellularsignalingmoleculeni-Pharmacologicdosesofpolyaminesarehypothermic
264266/CHAPTER31PROTEINSNITRICOXIDEUREAPROTEINSCREATINEARGININEPHOSPHATE,CREATININEPROLINEORNITHINEARGININEPHOSPHATEGlutamate-γ-PUTRESCINE,semialdehydeSPERMIDINE,SPERMINEGLUTAMATEFigure31–3.Arginine,ornithine,andprolinemetabolism.Reactionswithsolidar-rowsalloccurinmammaliantissues.Putrescineandsperminesynthesisoccursinbothmammalsandbacteria.Argininephosphateofinvertebratemusclefunctionsasaphosphagenanalogoustocreatinephosphateofmammalianmuscle(seeFigure31–6).andhypotensive.Sincetheybearmultiplepositivemine),apotentvasoconstrictorandstimulatorofcharges,polyaminesassociatereadilywithDNAandsmoothmusclecontraction.CatabolismofserotoninisRNA.Figure31–4summarizespolyaminebiosynthesis.initiatedbymonoamineoxidase-catalyzedoxidativedeaminationto5-hydroxyindoleacetate.ThepsychicstimulationthatfollowsadministrationofiproniazidTryptophanresultsfromitsabilitytoprolongtheactionofsero-Followinghydroxylationoftryptophanto5-hydroxy-toninbyinhibitingmonoamineoxidase.Incarcinoidtryptophanbylivertyrosinehydroxylase,subsequent(argentaffinoma),tumorcellsoverproduceserotonin.decarboxylationformsserotonin(5-hydroxytrypta-Urinarymetabolitesofserotonininpatientswithcarci-+H2H3N+NNH+3SpermidineDecarboxylatedS-adenosylmethionineSPERMINESYNTHASEMethylthio-adenosineFigure31–4.Conversionofspermidinetospermine.+H2Spermidineformedfromputrescine(decarboxylatedH3N+NNNH+L-ornithine)bytransferofapropylaminemoietyfromH+32decarboxylatedS-adenosylmethionineacceptsaSperminesecondpropylaminemoietytoformspermidine.
265CONVERSIONOFAMINOACIDSTOSPECIALIZEDPRODUCTS/267HONH+noidincludeN-acetylserotoninglucuronideandthe3glycineconjugateof5-hydroxyindoleacetate.SerotoninCHO–and5-methoxytryptaminearemetabolizedtothecorre-CH2Cspondingacidsbymonoamineoxidase.N-AcetylationOofserotonin,followedbyO-methylationinthepinealL-Tyrosinebody,formsmelatonin.CirculatingmelatoninistakenH4•biopterinupbyalltissues,includingbrain,butisrapidlymetabo-TYROSINElizedbyhydroxylationfollowedbyconjugationwithHYDROXYLASEsulfateorwithglucuronicacid.H2•biopterinKidneytissue,livertissue,andfecalbacteriaallcon-OHverttryptophantotryptamine,thentoindole3-acetate.HONH+3TheprincipalnormalurinarycatabolitesoftryptophanCHO–are5-hydroxyindoleacetateandindole3-acetate.CH2COTyrosineDopaNeuralcellsconverttyrosinetoepinephrineandnorepi-PLPnephrine(Figure31–5).Whiledopaisalsoaninterme-DOPADECARBOXYLASEdiateintheformationofmelanin,differentenzymesCOhydroxylatetyrosineinmelanocytes.Dopadecarboxy-2OHlase,apyridoxalphosphate-dependentenzyme,formsHOdopamine.Subsequenthydroxylationbydopamineβ-oxidasethenformsnorepinephrine.IntheadrenalCH2+medulla,phenylethanolamine-N-methyltransferaseuti-CH2NH3lizesS-adenosylmethioninetomethylatetheprimaryDopamineamineofnorepinephrine,formingepinephrine(FigureO31–5).Tyrosineisalsoaprecursoroftriiodothyronine2DOPAMINEandthyroxine(Chapter42).Cu2+β-OXIDASEVitaminCCreatinineOHHOCreatinineisformedinmusclefromcreatinephosphatebyirreversible,nonenzymaticdehydrationandlossofCH2+phosphate(Figure31–6).The24-hoururinaryexcre-CHNH3tionofcreatinineisproportionatetomusclemass.OHGlycine,arginine,andmethionineallparticipateincre-Norepinephrineatinebiosynthesis.SynthesisofcreatineiscompletedbymethylationofguanidoacetatebyS-adenosylmethio-PHENYLETHANOL-S-Adenosylmethioninenine(Figure31–6).AMINEN-METHYL-TRANSFERASES-Adenosylhomocysteine-AminobutyrateOHγ-Aminobutyrate(GABA)functionsinbraintissueasHOaninhibitoryneurotransmitterbyalteringtransmem-CHCHbranepotentialdifferences.Itisformedbydecarboxyla-23CHNtionofL-glutamate,areactioncatalyzedbyL-glutamateH+2decarboxylase(Figure31–7).Transaminationofγ-OHaminobutyrateformssuccinatesemialdehyde(FigureEpinephrine31–7),whichmaythenundergoreductiontoγ-hydroxy-Figure31–5.Conversionoftyrosinetoepinephrinebutyrate,areactioncatalyzedbyL-lactatedehydro-andnorepinephrineinneuronalandadrenalcells.(PLP,genase,oroxidationtosuccinateandthenceviathecit-pyridoxalphosphate.)ricacidcycletoCO2andH2O.AraregeneticdisorderofGABAmetabolisminvolvesadefectiveGABAamino-transferase,anenzymethatparticipatesinthecatabo-lismofGABAsubsequenttoitspostsynapticreleaseinbraintissue.
266NH2+H2NC(Kidney)NHARGININE-GLYCINETRANSAMIDINASENH2CH2+H2NCCH2HNCHCOO–2CH2+–H3NCH2COOOrnithineGlycocyamineHCNH+(guanidoacetate)3Glycine(Liver)COO–S-Adenosyl-ATPL-ArgininemethionineGUANIDOACETATEMETHYLTRANSFERASES-Adenosyl-ADPhomocysteineOHNCNONENZYMATICINMUSCLENHPHNCHNCNCHCOO–NCH22CH3Pi+H2OCH3CreatinineCreatinephosphateFigure31–6.Biosynthesisandmetabolismofcreatineandcreatinine.COO–HCNH+3α-KACH2L-GLUTAMATEDECARBOXYLASETRANSAMINASECH2–α-AACOOPLPL-GlutamateCO–2CH2OHCOOCH2CO+HNCHCHCHCOO–CH2CH32222γ-AminobutyrateCOO–CH2[O]γ-HydroxybutyrateCOO–NAD+α-KetoglutaratePLPLACTATEDEHYDROGENASE[NH+]+4NADH+HCO2OSUCCINICSEMIALDEHYDECOO–CHDEHYDROGENASECH2CH2CH2CH2HONAD+NADH+H+–2–COOCOOSuccinatesemialdehydeSuccinateFigure31–7.Metabolismofγ-aminobutyrate.(α-KA,α-ketoacids;α-AA,α-aminoacids;PLP,pyri-doxalphosphate.)268
267CONVERSIONOFAMINOACIDSTOSPECIALIZEDPRODUCTS/269SUMMARY•Decarboxylationofhistidineformshistamine,andseveraldipeptidesarederivedfromhistidineand•Inadditiontotheirrolesinproteinsandpolypep-β-alanine.tides,aminoacidsparticipateinawidevarietyofad-•Arginineservesastheformamidinedonorforcrea-ditionalbiosyntheticprocesses.tinebiosynthesis,participatesinpolyaminebiosyn-•Glycineparticipatesinthebiosynthesisofheme,thesis,andprovidesthenitrogenofnitricoxidepurines,andcreatineandisconjugatedtobileacids(NO).andtotheurinarymetabolitesofmanydrugs.•Importanttryptophanmetabolitesincludeserotonin,•Inadditiontoitsrolesinphospholipidandsphingo-melanin,andmelatonin.sinebiosynthesis,serineprovidescarbons2and8of•Tyrosineformsbothepinephrineandnorepineph-purinesandthemethylgroupofthymine.rine,anditsiodinationformsthyroidhormone.•S-Adenosylmethionine,themethylgroupdonorformanybiosyntheticprocesses,alsoparticipatesdirectlyinspermineandspermidinebiosynthesis.•Glutamateandornithineformtheneurotransmitterγ-aminobutyrate(GABA).REFERENCE•ThethioethanolamineofcoenzymeAandthetau-ScriverCRetal(editors):TheMetabolicandMolecularBasesofrineoftaurocholicacidarisefromcysteine.InheritedDisease,8thed.McGraw-Hill,2001.
268Porphyrins&BilePigments32RobertK.Murray,MD,PhDBIOMEDICALIMPORTANCEsubstituentpositionsnumberedasshowninFigure32–2.VariousporphyrinsarerepresentedinFiguresThebiochemistryoftheporphyrinsandofthebilepig-32–2,32–3,and32–4.mentsispresentedinthischapter.ThesetopicsareThearrangementoftheacetate(A)andpropionatecloselyrelated,becausehemeissynthesizedfrompor-(P)substituentsintheuroporphyrinshowninFigurephyrinsandiron,andtheproductsofdegradationof32–2isasymmetric(inringIV,theexpectedorderofhemearethebilepigmentsandiron.theAandPsubstituentsisreversed).AporphyrinwithKnowledgeofthebiochemistryoftheporphyrinsthistypeofasymmetricsubstitutionisclassifiedasaandofhemeisbasictounderstandingthevariedfunc-typeIIIporphyrin.Aporphyrinwithacompletelysym-tionsofhemoproteins(seebelow)inthebody.Themetricarrangementofthesubstituentsisclassifiedasaporphyriasareagroupofdiseasescausedbyabnormal-typeIporphyrin.OnlytypesIandIIIarefoundinna-itiesinthepathwayofbiosynthesisofthevariouspor-ture,andthetypeIIIseriesisfarmoreabundant(Figurephyrins.Althoughporphyriasarenotveryprevalent,32–3)—andmoreimportantbecauseitincludesheme.physiciansmustbeawareofthem.Amuchmorepreva-Hemeanditsimmediateprecursor,protoporphyrinlentclinicalconditionisjaundice,duetoelevationofIX(Figure32–4),arebothtypeIIIporphyrins(ie,thebilirubinintheplasma.Thiselevationisduetoover-methylgroupsareasymmetricallydistributed,asintypeproductionofbilirubinortofailureofitsexcretionandIIIcoproporphyrin).However,theyaresometimesisseeninnumerousdiseasesrangingfromhemolyticidentifiedasbelongingtoseriesIX,becausetheywereanemiastoviralhepatitisandtocancerofthepancreas.designatedninthinaseriesofisomerspostulatedbyHansFischer,thepioneerworkerinthefieldofpor-METALLOPORPHYRINSphyrinchemistry.&HEMOPROTEINSAREIMPORTANTINNATUREHEMEISSYNTHESIZEDFROMPorphyrinsarecycliccompoundsformedbythelinkageSUCCINYL-COA&GLYCINEoffourpyrroleringsthrough⎯HC⎯⎯methenylHemeissynthesizedinlivingcellsbyapathwaythathasbridges(Figure32–1).Acharacteristicpropertyofthebeenmuchstudied.Thetwostartingmaterialsaresuc-porphyrinsistheformationofcomplexeswithmetalcinyl-CoA,derivedfromthecitricacidcycleinmito-ionsboundtothenitrogenatomofthepyrrolerings.chondria,andtheaminoacidglycine.Pyridoxalphos-Examplesaretheironporphyrinssuchashemeofhe-phateisalsonecessaryinthisreactionto“activate”moglobinandthemagnesium-containingporphyringlycine.Theproductofthecondensationreactionbe-chlorophyll,thephotosyntheticpigmentofplants.tweensuccinyl-CoAandglycineisα-amino-β-ketoadipicProteinsthatcontainheme(hemoproteins)areacid,whichisrapidlydecarboxylatedtoformα-amino-widelydistributedinnature.Examplesoftheirimpor-levulinate(ALA)(Figure32–5).ThisreactionsequencetanceinhumansandanimalsarelistedinTable32–1.iscatalyzedbyALAsynthase,therate-controllingen-zymeinporphyrinbiosynthesisinmammalianliver.NaturalPorphyrinsHaveSubstituentSideSynthesisofALAoccursinmitochondria.Inthecy-ChainsonthePorphinNucleustosol,twomoleculesofALAarecondensedbytheen-zymeALAdehydratasetoformtwomoleculesofwaterTheporphyrinsfoundinnaturearecompoundsinandoneofporphobilinogen(PBG)(Figure32–5).ALAwhichvarioussidechainsaresubstitutedfortheeightdehydrataseisazinc-containingenzymeandissensitivehydrogenatomsnumberedintheporphinnucleustoinhibitionbylead,ascanoccurinleadpoisoning.showninFigure32–1.AsasimplemeansofshowingTheformationofacyclictetrapyrrole—ie,apor-thesesubstitutions,Fischerproposedashorthandfor-phyrin—occursbycondensationoffourmoleculesofmulainwhichthemethenylbridgesareomittedandPBG(Figure32–6).Thesefourmoleculescondenseinaeachpyrroleringisshownasindicatedwiththeeighthead-to-tailmannertoformalineartetrapyrrole,hy-270
269PORPHYRINS&BILEPIGMENTS/271HCCH12APHCCH83IAAINNHIVIIIVIIPyrrole7III4PIIIP1265PAHHCCFigure32–2.UroporphyrinIII.A(acetate)=δIαHCCCCH⎯CH2COOH;P(propionate)=⎯CH2CH2COOH.N38HCCCCHIVNHHNII(⎯CH⎯),whichdonotformaconjugatedringsys-27HCCNCCHtem.Thus,thesecompoundsarecolorless(asareall4HCCCCHporphyrinogens).However,theporphyrinogensareγIIIβreadilyauto-oxidizedtotheirrespectivecoloredpor-CCphyrins.TheseoxidationsarecatalyzedbylightandbyHH65theporphyrinsthatareformed.UroporphyrinogenIIIisconvertedtocopropor-PorphinphyrinogenIIIbydecarboxylationofalloftheacetate(C20H14N4)(A)groups,whichchangesthemtomethyl(M)sub-Figure32–1.Theporphinmolecule.Ringsarela-stituents.Thereactioniscatalyzedbyuroporphyrino-beledI,II,III,andIV.Substituentpositionsontheringsgendecarboxylase,whichisalsocapableofconvertingarelabeled1,2,3,4,5,6,7,and8.ThemethenyluroporphyrinogenItocoproporphyrinogenI(Figurebridges(⎯HC⎯⎯)arelabeledα,β,γ,andδ.32–7).CoproporphyrinogenIIIthenentersthemito-chondria,whereitisconvertedtoprotoporphyrinogenIIIandthentoprotoporphyrinIII.Severalstepsaredroxymethylbilane(HMB).Thereactioniscatalyzedbyinvolvedinthisconversion.ThemitochondrialenzymeuroporphyrinogenIsynthase,alsonamedPBGdeami-coproporphyrinogenoxidasecatalyzesthedecarboxy-naseorHMBsynthase.HMBcyclizesspontaneouslytolationandoxidationoftwopropionicsidechainstoformuroporphyrinogenI(left-handsideofFigureformprotoporphyrinogen.Thisenzymeisabletoact32–6)orisconvertedtouroporphyrinogenIIIbytheonlyontypeIIIcoproporphyrinogen,whichwouldex-actionofuroporphyrinogenIIIsynthase(right-handsideplainwhytypeIprotoporphyrinsdonotgenerallyoccurofFigure32–6).Undernormalconditions,theuropor-innature.Theoxidationofprotoporphyrinogentopro-phyrinogenformedisalmostexclusivelytheIIIisomer,toporphyriniscatalyzedbyanothermitochondrialen-butincertainoftheporphyrias(discussedbelow),thezyme,protoporphyrinogenoxidase.InmammaliantypeIisomersofporphyrinogensareformedinexcess.liver,theconversionofcoproporphyrinogentoproto-Notethatbothoftheseuroporphyrinogenshaveporphyrinrequiresmolecularoxygen.thepyrroleringsconnectedbymethylenebridgesFormationofHemeInvolvesIncorporationofIronIntoProtoporphyrinTable32–1.Examplesofsomeimportanthumanandanimalhemoproteins.1Thefinalstepinhemesynthesisinvolvestheincorpora-tionofferrousironintoprotoporphyrininareactioncatalyzedbyferrochelatase(hemesynthase),anotherProteinFunctionmitochondrialenzyme(Figure32–4).HemoglobinTransportofoxygeninbloodAsummaryofthestepsinthebiosynthesisoftheMyoglobinStorageofoxygeninmuscleporphyrinderivativesfromPBGisgiveninFigureCytochromecInvolvementinelectrontransportchain32–8.ThelastthreeenzymesinthepathwayandALACytochromeP450Hydroxylationofxenobioticssynthasearelocatedinthemitochondrion,whereastheCatalaseDegradationofhydrogenperoxideotherenzymesarecytosolic.Botherythroidandnon-TryptophanOxidationoftrypotophanerythroid(“housekeeping”)formsofthefirstfouren-pyrrolasezymesarefound.Hemebiosynthesisoccursinmost1Thefunctionsoftheaboveproteinsaredescribedinvariousmammaliancellswiththeexceptionofmatureerythro-chaptersofthistext.cytes,whichdonotcontainmitochondria.However,
270272/CHAPTER32APAPPAAAUroporphyrinswerefirstfoundintheurine,buttheyarenotrestrictedtourine.APPPPAPAUroporphyrinIUroporphyrinIIIMPMPPMMMCoproporphyrinswerefirstisolatedfromfeces,buttheyarealsofoundinurine.MPPPPMPMCoproporphyrinICoproporphyrinIIIFigure32–3.Uroporphyrinsandcoproporphyrins.A(acetate);P(propionate);M(methyl)=⎯CH3;V(vinyl)=⎯CH⎯CH2.approximately85%ofhemesynthesisoccursineryth-ALAS1.Thisrepression-derepressionmechanismisde-roidprecursorcellsinthebonemarrowandthemajor-picteddiagrammaticallyinFigure32–9.Thus,therateityoftheremainderinhepatocytes.ofsynthesisofALAS1increasesgreatlyintheabsenceTheporphyrinogensdescribedabovearecolorless,ofhemeandisdiminishedinitspresence.TheturnovercontainingsixextrahydrogenatomsascomparedwithrateofALAS1inratliverisnormallyrapid(half-lifethecorrespondingcoloredporphyrins.Thesereducedabout1hour),acommonfeatureofanenzymecatalyz-porphyrins(theporphyrinogens)andnotthecorre-ingarate-limitingreaction.Hemealsoaffectstransla-spondingporphyrinsaretheactualintermediatesinthetionoftheenzymeanditstransferfromthecytosoltobiosynthesisofprotoporphyrinandofheme.themitochondrion.Manydrugswhenadministeredtohumanscanre-ALASynthaseIstheKeyRegulatorysultinamarkedincreaseinALAS1.Mostofthesedrugsaremetabolizedbyasystemintheliverthatuti-EnzymeinHepaticBiosynthesisofHemelizesaspecifichemoprotein,cytochromeP450(seeALAsynthaseoccursinbothhepatic(ALAS1)andery-Chapter53).Duringtheirmetabolism,theutilizationthroid(ALAS2)forms.Therate-limitingreactionintheofhemebycytochromeP450isgreatlyincreased,synthesisofhemeinliveristhatcatalyzedbyALAS1whichinturndiminishestheintracellularhemecon-(Figure32–5),aregulatoryenzyme.Itappearsthatcentration.Thislattereventeffectsaderepressionofheme,probablyactingthroughanaporepressormole-ALAS1withacorrespondingincreasedrateofhemecule,actsasanegativeregulatorofthesynthesisofsynthesistomeettheneedsofthecells.MVMVFe2+MMMM2+FeFERROCHELATASEPVPVPMPMProtoporphyrinIII(IX)Heme(parentporphyrinofheme)(prostheticgroupofhemoglobin)Figure32–4.Additionofirontoprotoporphyrintoformheme.
271PORPHYRINS&BILEPIGMENTS/273COOHCOOHCOOHCH2ALACH2ALACH2SYNTHASESYNTHASESuccinyl-CoACH2CH2CH2(“active”CoA•SHCO2succinate)COCOCOSCoAHCNH2HCNH2+PyridoxalHphosphateCOOHHGlycineHCNH2α-Amino-β-ketoadipateδ-Aminolevulinate(ALA)COOHCOOHCOOHCOOHCH2COOHCH22H2OCH2CH2CH2CH2CH2OCCCALACOHCHCCHDEHYDRATASECH2HCH2NNHHNH2NH2TwomoleculesofPorphobilinogenδ-aminolevulinate(firstprecursorpyrrole)Figure32–5.Biosynthesisofporphobilinogen.ALAsynthaseoccursinthemitochon-dria,whereasALAdehydrataseispresentinthecytosol.Severalfactorsaffectdrug-mediatedderepressionofsidechainspresent.ThisbandistermedtheSoretbandALAS1inliver—eg,theadministrationofglucosecanafteritsdiscoverer,theFrenchphysicistCharlesSoret.preventit,ascantheadministrationofhematin(anox-Whenporphyrinsdissolvedinstrongmineralacidsidizedformofheme).orinorganicsolventsareilluminatedbyultravioletTheimportanceofsomeoftheseregulatorymecha-light,theyemitastrongredfluorescence.Thisfluores-nismsisfurtherdiscussedbelowwhentheporphyriascenceissocharacteristicthatitisoftenusedtodetectaredescribed.smallamountsoffreeporphyrins.ThedoublebondsRegulationoftheerythroidformofALAS(ALAS2)joiningthepyrroleringsintheporphyrinsareresponsi-differsfromthatofALAS1.Forinstance,itisnotin-bleforthecharacteristicabsorptionandfluorescenceofducedbythedrugsthataffectALAS1,anditdoesnotthesecompounds;thesedoublebondsareabsentintheundergofeedbackregulationbyheme.porphyrinogens.AninterestingapplicationofthephotodynamicpropertiesofporphyrinsistheirpossibleuseinthePORPHYRINSARECOLOREDtreatmentofcertaintypesofcancer,aprocedurecalled&FLUORESCEcancerphototherapy.Tumorsoftentakeupmorepor-phyrinsthandonormaltissues.Thus,hematopor-Thevariousporphyrinogensarecolorless,whereasthephyrinorotherrelatedcompoundsareadministeredtovariousporphyrinsareallcolored.Inthestudyofpor-apatientwithanappropriatetumor.Thetumoristhenphyrinsorporphyrinderivatives,thecharacteristicab-exposedtoanargonlaser,whichexcitestheporphyrins,sorptionspectrumthateachexhibits—inboththevisibleproducingcytotoxiceffects.andtheultravioletregionsofthespectrum—isofgreatvalue.AnexampleistheabsorptioncurveforasolutionSpectrophotometryIsUsedtoTestofporphyrinin5%hydrochloricacid(Figure32–10).forPorphyrins&TheirPrecursorsNoteparticularlythesharpabsorptionbandnear400nm.ThisisadistinguishingfeatureoftheporphinringCoproporphyrinsanduroporphyrinsareofclinicalin-andischaracteristicofallporphyrinsregardlessoftheterestbecausetheyareexcretedinincreasedamountsin
272274/CHAPTER32HOOCCOOHpainandofavarietyofneuropsychiatricfindings);oth-AH2CCH2Perwise,patientswillbesubjectedtoinappropriatetreat-CHments.IthasbeenspeculatedthatKingGeorgeIIIhad2atypeofporphyria,whichmayaccountforhisperiodicCCconfinementsinWindsorCastleandperhapsforsomeCCHofhisviewsregardingAmericancolonists.Also,theH2CNphotosensitivity(favoringnocturnalactivities)andse-HNHveredisfigurementexhibitedbysomevictimsofcon-2genitalerythropoieticporphyriahaveledtothesugges-Fourmoleculesofporphobilinogentionthattheseindividualsmayhavebeentheprototypesofso-calledwerewolves.Noevidencetosup-UROPORPHYRINOGENI4NH3SYNTHASEportthisnotionhasbeenadduced.Hydroxymethylbilane(lineartetrapyrrole)BiochemistryUnderliestheSPONTANEOUSUROPORPHYRINOGENIIICauses,Diagnoses,&TreatmentsCYCLIZATIONSYNTHASEofthePorphyriasSixmajortypesofporphyriahavebeendescribed,re-APAPAPAPsultingfromdepressionsintheactivitiesofenzymes3CCH2CCCCH2CCthrough8showninFigure32–9(seealsoTable32–2).IICIIICIAssayoftheactivityofoneormoreoftheseenzymesCCCCCCCCusinganappropriatesource(eg,redbloodcells)isthusNNNNimportantinmakingadefinitivediagnosisinasus-HHHHCH2CH2CH2CH2pectedcaseofporphyria.IndividualswithlowactivitiesHHHHofenzyme1(ALAS2)developanemia,notporphyriaNNNN(seeTable32–2).PatientswithlowactivitiesofenzymeCCCCCCCCIVIIIIVIII2(ALAdehydratase)havebeenreported,butveryCCCCH2CCCCH2CCrarely;theresultingconditioniscalledALAdehy-PAPAAPPAdratase-deficientporphyria.TypeITypeIIIIngeneral,theporphyriasdescribedareinheritedinuroporphyrinogenuroporphyrinogenanautosomaldominantmanner,withtheexceptionofFigure32–6.Conversionofporphobilinogentouro-congenitalerythropoieticporphyria,whichisinheritedporphyrinogens.UroporphyrinogensynthaseIisalsoinarecessivemode.Thepreciseabnormalitiesinthegenesdirectingsynthesisoftheenzymesinvolvedincalledporphobilinogen(PBG)deaminaseorhydroxy-hemebiosynthesishavebeendeterminedinsomein-methylbilane(HMB)synthase.stances.Thus,theuseofappropriategeneprobeshasmadepossibletheprenataldiagnosisofsomeoftheporphyrias.theporphyrias.Thesecompounds,whenpresentinAsistrueofmostinbornerrors,thesignsandsymp-urineorfeces,canbeseparatedfromeachotherbyex-tomsofporphyriaresultfromeitheradeficiencyoftractionwithappropriatesolventmixtures.Theycanmetabolicproductsbeyondtheenzymaticblockorthenbeidentifiedandquantifiedusingspectrophoto-fromanaccumulationofmetabolitesbehindtheblock.metricmethods.IftheenzymelesionoccursearlyinthepathwayALAandPBGcanalsobemeasuredinurinebyap-priortotheformationofporphyrinogens(eg,enzyme3propriatecolorimetrictests.ofFigure32–9,whichisaffectedinacuteintermittentporphyria),ALAandPBGwillaccumulateinbodytis-THEPORPHYRIASAREGENETICsuesandfluids(Figure32–11).Clinically,patientscomplainofabdominalpainandneuropsychiatricDISORDERSOFHEMEMETABOLISMsymptoms.TheprecisebiochemicalcauseoftheseTheporphyriasareagroupofdisordersduetoabnor-symptomshasnotbeendeterminedbutmayrelatetomalitiesinthepathwayofbiosynthesisofheme;theyelevatedlevelsofALAorPBGortoadeficiencyofcanbegeneticoracquired.Theyarenotprevalent,butheme.itisimportanttoconsiderthemincertaincircum-Ontheotherhand,enzymeblockslaterinthepath-stances(eg,inthedifferentialdiagnosisofabdominalwayresultintheaccumulationoftheporphyrinogens
273PORPHYRINS&BILEPIGMENTS/275APMP4CO2IIPAPMIVIIIVIIAIIIPMIIIPPAPMUroporphyrinogenICoproporphyrinogenIUROPORPHYRINOGENDECARBOXYLASEAPMPIIAAMMIVIIIVIIPIIIPPIIIPFigure32–7.Decarboxylationofuropor-4CO2phyrinogenstocoproporphyrinogensincy-PAPMtosol.(A,acetyl;M,methyl;P,propionyl.)UroporphyrinogenIIICoproporphyrinogenIIIPorphobilinogenUROPORPHYRINOGENISYNTHASEHydroxymethylbilaneUROPORPHYRINOGENIIISYNTHASESPONTANEOUS6H6HUroporphyrinUroporphyrinogenUroporphyrinogenUroporphyrinIIILightIIIILightIUROPORPHYRINOGEN6HDECARBOXYLASE6H4CO24CO2CoproporphyrinCoproporphyrinogenCoproporphyrinogenCoproporphyrinIIILightIIIILightICOPROPORPHYRINOGENOXIDASEProtoporphyrinogenIIIPROTOPORPHYRINOGENOrlightinvitroOXIDASE6HProtoporphyrinIIIMITOCHONDRIACYTOSOLFe2+FERROCHELATASEHemeFigure32–8.Stepsinthebiosynthesisoftheporphyrinderivativesfromporphobilinogen.Uropor-phyrinogenIsynthaseisalsocalledporphobilinogendeaminaseorhydroxymethylbilanesynthase.
274276/CHAPTER32HemoproteinsProteinsHemeAporepressor8.FERROCHELATASEFe2+ProtoporphyrinIII7.PROTOPORPHYRINOGENOXIDASEProtoporphyrinogenIII6.COPROPORPHYRINOGENOXIDASECoproporphyrinogenIII5.UROPORPHYRINOGENDECARBOXYLASEUroporphyrinogenIII4.UROPORPHYRINOGENIIISYNTHASEHydroxymethylbilane3.UROPORPHYRINOGENISYNTHASEPorphobilinogen2.ALADEHYDRATASEALA1.ALASYNTHASESuccinyl-CoA+GlycineFigure32–9.Intermediates,enzymes,andregulationofhemesyn-thesis.Theenzymenumbersarethosereferredtoincolumn1ofTable32–2.Enzymes1,6,7,and8arelocatedinmitochondria,theothersinthecytosol.Mutationsinthegeneencodingenzyme1causesX-linkedsideroblasticanemia.Mutationsinthegenesencodingenzymes2–8causetheporphyrias,thoughonlyafewcasesduetodeficiencyofen-zyme2havebeenreported.Regulationofhepatichemesynthesisoc-cursatALAsynthase(ALAS1)byarepression-derepressionmecha-nismmediatedbyhemeanditshypotheticalaporepressor.Thedottedlinesindicatethenegative(−)regulationbyrepression.En-zyme3isalsocalledporphobilinogendeaminaseorhydroxymethyl-bilanesynthase.
275PORPHYRINS&BILEPIGMENTS/277MutationsinDNA54Abnormalitiesoftheenzymesofhemesynthesis3Logabsorbency2AccumulationofAccumulationofALAandPBGand/orporphyrinogensinskindecreaseinhemein1andtissuescellsandbodyfluids300400500600700SpontaneousoxidationWavelength(nm)NeuropsychiatricsignsofporphyrinogenstoandsymptomsporphyrinsFigure32–10.Absorptionspectrumofhematopor-phyrin(0.01%solutionin5%HCl).PhotosensitivityFigure32–11.Biochemicalcausesofthemajorsignsandsymptomsoftheporphyrias.1Table32–2.Summaryofmajorfindingsintheporphyrias.2EnzymeInvolvedType,Class,andMIMNumberMajorSignsandSymptomsResultsofLaboratoryTests31.ALAsynthaseX-linkedsideroblasticanemiaAnemiaRedcellcountsandhemoglobin(erythroidform)(erythropoietic)(MIMdecreased201300)2.ALAdehydrataseALAdehydratasedeficiencyAbdominalpain,neuropsychiatricUrinaryδ-aminolevulinicacid(hepatic)(MIM125270)symptoms3.UroporphyrinogenIAcuteintermittentporphyriaAbdominalpain,neuropsychiatricUrinaryporphobilinogenpositive,4synthase(hepatic)(MIM176000)symptomsuroporphyrinpositive4.UroporphyrinogenIIICongenitalerythropoieticNophotosensitivityUroporphyrinpositive,porpho-synthase(erythropoietic)(MIMbilinogennegative263700)5.UroporphyrinogenPorphyriacutaneatarda(he-PhotosensitivityUroporphyrinpositive,porpho-decarboxylasepatic)(MIM176100)bilinogennegative6.CoproporphyrinogenHereditarycoproporphyriaPhotosensitivity,abdominalpain,Urinaryporphobilinogenposi-oxidase(hepatic)(MIM121300)neuropsychiatricsymptomstive,urinaryuroporphyrinpositive,fecalprotopor-phyrinpositive7.ProtoporphyrinogenVariegateporphyria(hepatic)Photosensitivity,abdominalpain,Urinaryporphobilinogenposi-oxidase(MIM176200)neuropsychiatricsymptomstive,fecalprotoporphyrinpositive8.FerrochelataseProtoporphyria(erythropoietic)PhotosensitivityFecalprotoporphyrinposi-`(MIM177000)tive,redcellprotoporphyrinpositive1Onlythebiochemicalfindingsintheactivestagesofthesediseasesarelisted.Certainbiochemicalabnormalitiesaredetectableinthela-tentstagesofsomeoftheaboveconditions.Conditions3,5,and8aregenerallythemostprevalentporphyrias.2ThenumberingoftheenzymesinthistablecorrespondstothatusedinFigure32-9.3X-linkedsideroblasticanemiaisnotaporphyriabutisincludedherebecauseδ−aminolevulinicacidsynthaseisinvolved.4Thisenzymeisalsocalledporphobilinogendeaminaseorhydroxymethylbilanesynthase.
276278/CHAPTER32indicatedinFigures32–9and32–11.TheiroxidationchromeP450.Ingestionoflargeamountsofcarbohy-products,thecorrespondingporphyrinderivatives,drates(glucoseloading)oradministrationofhematin(acausephotosensitivity,areactiontovisiblelightofhydroxideofheme)mayrepressALAS1,resultingindi-about400nm.Theporphyrins,whenexposedtolightminishedproductionofharmfulhemeprecursors.Pa-ofthiswavelength,arethoughttobecome“excited”tientsexhibitingphotosensitivitymaybenefitfromad-andthenreactwithmolecularoxygentoformoxygenministrationofβ-carotene;thiscompoundappearstoradicals.Theselatterspeciesinjurelysosomesandotherlessenproductionoffreeradicals,thusdiminishingorganelles.Damagedlysosomesreleasetheirdegradativephotosensitivity.Sunscreensthatfilteroutvisiblelightenzymes,causingvariabledegreesofskindamage,in-canalsobehelpfultosuchpatients.cludingscarring.Theporphyriascanbeclassifiedonthebasisoftheorgansorcellsthataremostaffected.Thesearegener-CATABOLISMOFHEMEallyorgansorcellsinwhichsynthesisofhemeispartic-PRODUCESBILIRUBINularlyactive.Thebonemarrowsynthesizesconsiderablehemoglobin,andtheliverisactiveinthesynthesisofUnderphysiologicconditionsinthehumanadult,1–28anotherhemoprotein,cytochromeP450.Thus,one×10erythrocytesaredestroyedperhour.Thus,in1classificationoftheporphyriasistodesignatethemasday,a70-kghumanturnsoverapproximately6gofhe-predominantlyeithererythropoieticorhepatic;themoglobin.Whenhemoglobinisdestroyedinthebody,typesofporphyriasthatfallintothesetwoclassesaresoglobinisdegradedtoitsconstituentaminoacids,characterizedinTable32–2.Porphyriascanalsobewhicharereused,andtheironofhemeenterstheironclassifiedasacuteorcutaneousonthebasisoftheirpool,alsoforreuse.Theiron-freeporphyrinportionofclinicalfeatures.Whydospecifictypesofporphyriaaf-hemeisalsodegraded,mainlyinthereticuloendothelialfectcertainorgansmoremarkedlythanothers?Apar-cellsoftheliver,spleen,andbonemarrow.tialansweristhatthelevelsofmetabolitesthatcauseThecatabolismofhemefromallofthehemepro-damage(eg,ALA,PBG,specificporphyrins,orlackofteinsappearstobecarriedoutinthemicrosomalfrac-heme)canvarymarkedlyindifferentorgansorcellsde-tionsofcellsbyacomplexenzymesystemcalledhemependinguponthedifferingactivitiesoftheirheme-oxygenase.Bythetimethehemederivedfromhemeformingenzymes.proteinsreachestheoxygenasesystem,theironhasusu-Asdescribedabove,ALAS1isthekeyregulatoryen-allybeenoxidizedtotheferricform,constitutingzymeofthehemebiosyntheticpathwayinliver.Alargehemin.Thehemeoxygenasesystemissubstrate-in-numberofdrugs(eg,barbiturates,griseofulvin)induceducible.AsdepictedinFigure32–12,theheminisre-theenzyme.Mostofthesedrugsdosobyinducingcy-ducedtohemewithNADPH,and,withtheaidoftochromeP450(seeChapter53),whichusesuphememoreNADPH,oxygenisaddedtotheα-methenylandthusderepresses(induces)ALAS1.InpatientswithbridgebetweenpyrrolesIandIIoftheporphyrin.Theporphyria,increasedactivitiesofALAS1resultinin-ferrousironisagainoxidizedtotheferricform.Withcreasedlevelsofpotentiallyharmfulhemeprecursorsthefurtheradditionofoxygen,ferricionisreleased,priortothemetabolicblock.Thus,takingdrugsthatcarbonmonoxideisproduced,andanequimolarcauseinductionofcytochromeP450(so-calledmicro-quantityofbiliverdinresultsfromthesplittingofthesomalinducers)canprecipitateattacksofporphyria.tetrapyrrolering.ThediagnosisofaspecifictypeofporphyriacanInbirdsandamphibia,thegreenbiliverdinIXisex-generallybeestablishedbyconsiderationoftheclinicalcreted;inmammals,asolubleenzymecalledbiliverdinandfamilyhistory,thephysicalexamination,andap-reductasereducesthemethenylbridgebetweenpyrrolepropriatelaboratorytests.ThemajorfindingsinthesixIIIandpyrroleIVtoamethylenegrouptoproduceprincipaltypesofporphyriaarelistedinTable32–2.bilirubin,ayellowpigment(Figure32–12).HighlevelsofleadcanaffecthememetabolismbyItisestimatedthat1gofhemoglobinyields35mgcombiningwithSHgroupsinenzymessuchasfer-ofbilirubin.ThedailybilirubinformationinhumanrochelataseandALAdehydratase.Thisaffectspor-adultsisapproximately250–350mg,derivingmainlyphyrinmetabolism.Elevatedlevelsofprotoporphyrinfromhemoglobinbutalsofromineffectiveerythro-arefoundinredbloodcells,andelevatedlevelsofALApoiesisandfromvariousotherhemeproteinssuchasandofcoproporphyrinarefoundinurine.cytochromeP450.ItishopedthattreatmentoftheporphyriasattheThechemicalconversionofhemetobilirubinbygenelevelwillbecomepossible.Inthemeantime,treat-reticuloendothelialcellscanbeobservedinvivoasthementisessentiallysymptomatic.Itisimportantforpa-purplecolorofthehemeinahematomaisslowlycon-tientstoavoiddrugsthatcauseinductionofcyto-vertedtotheyellowpigmentofbilirubin.
277PORPHYRINS&BILEPIGMENTS/279HemeOIαHeminHNNIVNFe3+NIIHNPNHIIIPPHPNADPHHNNADPHNBilirubinIαHemeONNADPIVNFe2+NIIPNNADPHIIIOMicrosomalhemeoxygenasesystemPHNIINADPHO2NADPHNIIIFe3+(reutilized)OHCO(exhaled)PIPNNIVO2IVNFe3+NIIPNIIIHNIBiliverdinPOFigure32–12.Schematicrepresentationofthemicrosomalhemeoxygenasesystem.(ModifiedfromSchmidR,McDonoughAFin:ThePorphyrins.DolphinD[editor].AcademicPress,1978.)
278280/CHAPTER32Bilirubinformedinperipheraltissuesistransportedconvertedtowater-solublederivativesbyconjugationintotheliverbyplasmaalbumin.Thefurthermetabolismpreparationforexcretion(seeChapter53).ofbilirubinoccursprimarilyintheliver.Itcanbedi-Theconjugationofbilirubiniscatalyzedbyaspe-videdintothreeprocesses:(1)uptakeofbilirubinbycificglucuronosyltransferase.Theenzymeismainlyliverparenchymalcells,(2)conjugationofbilirubinlocatedintheendoplasmicreticulum,usesUDP-withglucuronateintheendoplasmicreticulum,and(3)glucuronicacidastheglucuronosyldonor,andisre-secretionofconjugatedbilirubinintothebile.Eachofferredtoasbilirubin-UGT.Bilirubinmonoglucuronidetheseprocesseswillbeconsideredseparately.isanintermediateandissubsequentlyconvertedtothediglucuronide(Figures32–13and32–14).MostofthebilirubinexcretedinthebileofmammalsisintheformTHELIVERTAKESUPBILIRUBINofbilirubindiglucuronide.However,whenbilirubinBilirubinisonlysparinglysolubleinwater,butitssolu-conjugatesexistabnormallyinhumanplasma(eg,inbilityinplasmaisincreasedbynoncovalentbindingtoobstructivejaundice),theyarepredominantlymono-albumin.Eachmoleculeofalbuminappearstohaveglucuronides.Bilirubin-UGTactivitycanbeinducedonehigh-affinitysiteandonelow-affinitysiteforbyanumberofclinicallyusefuldrugs,includingphe-bilirubin.In100mLofplasma,approximately25mgnobarbital.Moreinformationaboutglucuronosylationofbilirubincanbetightlyboundtoalbuminatitshigh-ispresentedbelowinthediscussionofinheriteddisor-affinitysite.Bilirubininexcessofthisquantitycanbedersofbilirubinconjugation.boundonlylooselyandthuscaneasilybedetachedanddiffuseintotissues.AnumberofcompoundssuchasBilirubinIsSecretedIntoBileantibioticsandotherdrugscompetewithbilirubinforSecretionofconjugatedbilirubinintothebileoccursbythehigh-affinitybindingsiteonalbumin.Thus,theseanactivetransportmechanism,whichisprobablyrate-compoundscandisplacebilirubinfromalbuminandlimitingfortheentireprocessofhepaticbilirubinme-havesignificantclinicaleffects.tabolism.TheproteininvolvedisMRP-2(multidrugIntheliver,thebilirubinisremovedfromalbuminresistance-likeprotein2),alsocalledmultispecificor-andtakenupatthesinusoidalsurfaceofthehepato-ganicaniontransporter(MOAT).Itislocatedinthecytesbyacarrier-mediatedsaturablesystem.Thisfacil-plasmamembraneofthebilecanalicularmembraneitatedtransportsystemhasaverylargecapacity,soandhandlesanumberoforganicanions.ItisamemberthatevenunderpathologicconditionsthesystemdoesofthefamilyofATP-bindingcassette(ABC)trans-notappeartoberate-limitinginthemetabolismofporters.Thehepatictransportofconjugatedbilirubinbilirubin.intothebileisinduciblebythosesamedrugsthatareSincethisfacilitatedtransportsystemallowsthecapableofinducingtheconjugationofbilirubin.Thus,equilibriumofbilirubinacrossthesinusoidalmem-theconjugationandexcretionsystemsforbilirubinbe-braneofthehepatocyte,thenetuptakeofbilirubinwillhaveasacoordinatedfunctionalunit.bedependentupontheremovalofbilirubinviasubse-Figure32–15summarizesthethreemajorprocessesquentmetabolicpathways.involvedinthetransferofbilirubinfrombloodtobile.Oncebilirubinentersthehepatocytes,itcanbindtoSitesthatareaffectedinanumberofconditionscaus-certaincytosolicproteins,whichhelptokeepitsolubi-ingjaundice(seebelow)arealsoindicated.lizedpriortoconjugation.Ligandin(afamilyofglu-tathioneS-transferases)andproteinYaretheinvolvedproteins.Theymayalsohelptopreventeffluxofbiliru-binbackintothebloodstream.OO–OOC(CHO)CO)COO–24OCCOC(CH24ConjugationofBilirubinWithGlucuronicH2CCH2AcidOccursintheLiverH2CCH2MVMMMVBilirubinisnonpolarandwouldpersistincells(eg,IIIIIIVIboundtolipids)ifnotrenderedwater-soluble.Hepato-OCCCOcytesconvertbilirubintoapolarform,whichisreadilyexcretedinthebile,byaddingglucuronicacidmole-Figure32–13.Structureofbilirubindiglucuronideculestoit.Thisprocessiscalledconjugationandcan(conjugated,“direct-reacting”bilirubin).Glucuronicemploypolarmoleculesotherthanglucuronicacid(eg,acidisattachedviaesterlinkagetothetwopropionicsulfate).Manysteroidhormonesanddrugsarealsoacidgroupsofbilirubintoformanacylglucuronide.
279PORPHYRINS&BILEPIGMENTS/281UDP-GLUCOSEDEHYDROGENASEUDP-GlucoseUDP-Glucuronicacid2NAD+2NADH+2H+UDP-GLUCURONOSYL-TRANSFERASEUDP-GlucuronicacidBilirubinmonoglucuronide++BilirubinUDPFigure32–14.Conjugationofbilirubinwithglucuronicacid.Theglucuronatedonor,UDP-GLUCURONOSYL-UDP-glucuronicacid,isformedfromUDP-UDP-GlucuronicacidTRANSFERASEBilirubindiglucuronideglucoseasdepicted.TheUDP-glucuronosyl-++transferaseisalsocalledbilirubin-UGT.BilirubinmonoglucuronideUDPConjugatedBilirubinIsReducedtoUrobilinogenbyIntestinalBacteriaAstheconjugatedbilirubinreachestheterminalileumandthelargeintestine,theglucuronidesareremovedbyBLOODspecificbacterialenzymes(-glucuronidases),andtheBilirubin•Albuminpigmentissubsequentlyreducedbythefecalfloratoagroupofcolorlesstetrapyrroliccompoundscalleduro-1.UPTAKEbilinogens(Figure32–16).Intheterminalileumandlargeintestine,asmallfractionoftheurobilinogensisre-absorbedandreexcretedthroughthelivertoconstituteHEPATOCYTEtheenterohepaticurobilinogencycle.UnderabnormalBilirubinconditions,particularlywhenexcessivebilepigmentis2.CONJUGATIONformedorliverdiseaseinterfereswiththisintrahepaticNeonataljaundicecycle,urobilinogenmayalsobeexcretedintheurine.UDP-GlcUA“Toxic”jaundiceNormally,mostofthecolorlessurobilinogensUDP-GlcUACrigler-NajjarsyndromeformedinthecolonbythefecalfloraareoxidizedthereGilbertsyndrometourobilins(coloredcompounds)andareexcretedinthefeces(Figure32–16).DarkeningoffecesuponBilirubindiglucuronidestandinginairisduetotheoxidationofresidualuro-bilinogenstourobilins.3.SECRETIONDubin-JohnsonsyndromeHYPERBILIRUBINEMIACAUSESJAUNDICEBILEDUCTULEWhenbilirubininthebloodexceeds1mg/dL(17.1Bilirubindiglucuronideμmol/L),hyperbilirubinemiaexists.Hyperbilirubine-miamaybeduetotheproductionofmorebilirubinFigure32–15.Diagrammaticrepresentationofthethanthenormallivercanexcrete,oritmayresultfromthreemajorprocesses(uptake,conjugation,andsecre-thefailureofadamagedlivertoexcretebilirubinpro-tion)involvedinthetransferofbilirubinfrombloodtoducedinnormalamounts.Intheabsenceofhepaticbile.Certainproteinsofhepatocytes,suchasligandin(adamage,obstructionoftheexcretoryductsofthefamilyofglutathioneS-transferase)andYprotein,bindliver—bypreventingtheexcretionofbilirubin—willintracellularbilirubinandmaypreventitseffluxintothealsocausehyperbilirubinemia.Inallthesesituations,bloodstream.Theprocessaffectedinanumberofcon-bilirubinaccumulatesintheblood,andwhenitreachesditionscausingjaundiceisalsoshown.acertainconcentration(approximately2–2.5mg/dL),
280282/CHAPTER32MEMEMEHHIIHIHH2COHH2COHHH2COHHMNOHMMNOHMMNOHMHHHHHNHHNNHHNNHNHHHPEPEPENNNH2CCH2H2CCH2HCCH2PMPMPMMesobilirubinogenStercobilinogenStercobilin(C33H44O6N4)(L-Urobilinogen)(L-Urobilin)Figure32–16.Structureofsomebilepigments.itdiffusesintothetissues,whichthenbecomeyellow.soluble,canreactdirectlywiththediazoreagent,soThatconditioniscalledjaundiceoricterus.thatthe“directbilirubin”ofvandenBerghisactuallyaInclinicalstudiesofjaundice,measurementofbilirubinconjugate(bilirubinglucuronide).bilirubinintheserumisofgreatvalue.AmethodforDependingonthetypeofbilirubinpresentinquantitativelyassayingthebilirubincontentoftheplasma—ie,unconjugatedorconjugated—hyperbiliru-serumwasfirstdevisedbyvandenBerghbyapplicationbinemiamaybeclassifiedasretentionhyperbiliru-ofEhrlich’stestforbilirubininurine.TheEhrlichreac-binemia,duetooverproduction,orregurgitationhy-tionisbasedonthecouplingofdiazotizedsulfanilicperbilirubinemia,duetorefluxintothebloodstreamacid(Ehrlich’sdiazoreagent)andbilirubintoproducebecauseofbiliaryobstruction.areddish-purpleazocompound.Intheoriginalproce-Becauseofitshydrophobicity,onlyunconjugateddureasdescribedbyEhrlich,methanolwasusedtobilirubincancrosstheblood-brainbarrierintothecen-provideasolutioninwhichbothbilirubinandthetralnervoussystem;thus,encephalopathyduetohyper-diazoregentweresoluble.VandenBerghinadvertentlybilirubinemia(kernicterus)canoccuronlyinconnec-omittedthemethanolonanoccasionwhenassayofbiletionwithunconjugatedbilirubin,asfoundinretentionpigmentinhumanbilewasbeingattempted.Tohishyperbilirubinemia.Ontheotherhand,becauseofitssurprise,normaldevelopmentofthecoloroccurred“di-water-solubility,onlyconjugatedbilirubincanappearrectly.”Thisformofbilirubinthatwouldreactwithoutinurine.Accordingly,choluricjaundice(choluriaistheadditionofmethanolwasthustermed“direct-thepresenceofbilepigmentsintheurine)occursonlyreacting.”Itwasthenfoundthatthissamedirectreac-inregurgitationhyperbilirubinemia,andacholurictionwouldalsooccurinserumfromcasesofjaundicejaundiceoccursonlyinthepresenceofanexcessofun-duetobiliaryobstruction.However,itwasstillneces-conjugatedbilirubin.sarytoaddmethanoltodetectbilirubininnormalserumorthatwhichwaspresentinexcessinserumElevatedAmountsofUnconjugatedfromcasesofhemolyticjaundicewherenoevidenceofBilirubininBloodOccurinaNumberobstructionwastobefound.TothatformofbilirubinofConditionswhichcouldbemeasuredonlyaftertheadditionofmethanol,theterm“indirect-reacting”wasapplied.A.HEMOLYTICANEMIASItwassubsequentlydiscoveredthattheindirectHemolyticanemiasareimportantcausesofunconju-bilirubinis“free”(unconjugated)bilirubinenroutetogatedhyperbilirubinemia,thoughunconjugatedhyper-theliverfromthereticuloendothelialtissues,wherethebilirubinemiaisusuallyonlyslight(<4mg/dL;<68.4bilirubinwasoriginallyproducedbythebreakdownofμmol/L)evenintheeventofextensivehemolysisbe-hemeporphyrins.Sincethisbilirubinisnotwater-solu-causeofthehealthyliver’slargecapacityforhandlingble,itrequiresmethanoltoinitiatecouplingwiththebilirubin.diazoreagent.Intheliver,thefreebilirubinbecomesconjugatedwithglucuronicacid,andtheconjugate,B.NEONATAL“PHYSIOLOGICJAUNDICE”bilirubinglucuronide,canthenbeexcretedintotheThistransientconditionisthemostcommoncauseofbile.Furthermore,conjugatedbilirubin,beingwater-unconjugatedhyperbilirubinemia.Itresultsfromanac-
281PORPHYRINS&BILEPIGMENTS/283celeratedhemolysisaroundthetimeofbirthandanim-mushroompoisoning.Theseacquireddisordersareduematurehepaticsystemfortheuptake,conjugation,andtohepaticparenchymalcelldamage,whichimpairssecretionofbilirubin.Notonlyisthebilirubin-UGTconjugation.activityreduced,butthereprobablyisreducedsynthesisofthesubstrateforthatenzyme,UDP-glucuronicacid.ObstructionintheBiliaryTreeIstheSincetheincreasedamountofbilirubinisunconju-gated,itiscapableofpenetratingtheblood-brainbar-CommonestCauseofConjugatedrierwhenitsconcentrationinplasmaexceedsthatHyperbilirubinemiawhichcanbetightlyboundbyalbumin(20–25A.OBSTRUCTIONOFTHEBILIARYTREEmg/dL).Thiscanresultinahyperbilirubinemictoxicencephalopathy,orkernicterus,whichcancausemen-Conjugatedhyperbilirubinemiacommonlyresultsfromtalretardation.Becauseoftherecognizedinducibilityblockageofthehepaticorcommonbileducts,mostofthisbilirubin-metabolizingsystem,phenobarbitaloftenduetoagallstoneortocanceroftheheadofthehasbeenadministeredtojaundicedneonatesandisef-pancreas.Becauseoftheobstruction,bilirubindiglu-fectiveinthisdisorder.Inaddition,exposuretobluecuronidecannotbeexcreted.Itthusregurgitatesintolight(phototherapy)promotesthehepaticexcretionofthehepaticveinsandlymphatics,andconjugatedunconjugatedbilirubinbyconvertingsomeofthebilirubinappearsinthebloodandurine(choluricjaun-bilirubintootherderivativessuchasmaleimidefrag-dice).mentsandgeometricisomersthatareexcretedintheThetermcholestaticjaundiceisusedtoincludeallbile.casesofextrahepaticobstructivejaundice.Italsocoversthosecasesofjaundicethatexhibitconjugatedhyper-C.CRIGLER-NAJJARSYNDROME,TYPEI;bilirubinemiaduetomicro-obstructionofintrahepaticCONGENITALNONHEMOLYTICJAUNDICEbiliaryductulesbyswollen,damagedhepatocytes(eg,asTypeICrigler-Najjarsyndromeisarareautosomalre-mayoccurininfectioushepatitis).cessivedisorder.Itischaracterizedbyseverecongenitaljaundice(serumbilirubinusuallyexceeds20mg/dL)B.DUBIN-JOHNSONSYNDROMEduetomutationsinthegeneencodingbilirubin-UGTThisbenignautosomalrecessivedisorderconsistsofactivityinhepatictissues.Thediseaseisoftenfatalconjugatedhyperbilirubinemiainchildhoodorduringwithinthefirst15monthsoflife.Childrenwiththisadultlife.Thehyperbilirubinemiaiscausedbymuta-conditionhavebeentreatedwithphototherapy,result-tionsinthegeneencodingMRP-2(seeabove),thepro-inginsomereductioninplasmabilirubinlevels.Phe-teininvolvedinthesecretionofconjugatedbilirubinnobarbitalhasnoeffectontheformationofbilirubinintobile.Thecentrilobularhepatocytescontainanab-glucuronidesinpatientswithtypeICrigler-Najjarsyn-normalblackpigmentthatmaybederivedfromepi-drome.Alivertransplantmaybecurative.nephrine.D.CRIGLER-NAJJARSYNDROME,TYPEIIC.ROTORSYNDROMEThisrareinheriteddisorderalsoresultsfrommutationsThisisararebenignconditioncharacterizedbychronicinthegeneencodingbilirubin-UGT,butsomeactivityconjugatedhyperbilirubinemiaandnormalliverhistol-oftheenzymeisretainedandtheconditionhasamoreogy.Itsprecisecausehasnotbeenidentified,butitisbenigncoursethantypeI.Serumbilirubinconcentra-thoughttobeduetoanabnormalityinhepaticstorage.tionsusuallydonotexceed20mg/dL.Patientswiththisconditioncanrespondtotreatmentwithlargedosesofphenobarbital.SomeConjugatedBilirubinCanBindCovalentlytoAlbuminE.GILBERTSYNDROMEAgain,thisiscausedbymutationsinthegeneencodingWhenlevelsofconjugatedbilirubinremainhighinbilirubin-UGT,butapproximately30%oftheen-plasma,afractioncanbindcovalentlytoalbumin(deltazyme’sactivityispreservedandtheconditionisentirelybilirubin).Becauseitisboundcovalentlytoalbumin,harmless.thisfractionhasalongerhalf-lifeinplasmathandoesconventionalconjugatedbilirubin.Thus,itremainsele-F.TOXICHYPERBILIRUBINEMIAvatedduringtherecoveryphaseofobstructivejaundiceUnconjugatedhyperbilirubinemiacanresultfromaftertheremainderoftheconjugatedbilirubinhasde-toxin-inducedliverdysfunctionsuchasthatcausedbyclinedtonormallevels;thisexplainswhysomepatientschloroform,arsphenamines,carbontetrachloride,ace-continuetoappearjaundicedafterconjugatedbilirubintaminophen,hepatitisvirus,cirrhosis,andAmanitalevelshavereturnedtonormal.
282284/CHAPTER32Table32–3.Laboratoryresultsinnormalpatientsandpatientswiththreedifferentcausesofjaundice.ConditionSerumBilirubinUrineUrobilinogenUrineBilirubinFecalUrobilinogenNormalDirect:0.1–0.4mg/dL0–4mg/24hAbsent40–280mg/24hIndirect:0.2–0.7mg/dLHemolyticanemia↑IndirectIncreasedAbsentIncreasedHepatitis↑DirectandindirectDecreasedifmicro-Presentifmicro-Decreasedobstructionisobstructionoccurspresent1Obstructivejaundice↑DirectAbsentPresentTracetoabsent1Thecommonestcausesofobstructive(posthepatic)jaundicearecanceroftheheadofthepancreasandagallstonelodgedinthecom-monbileduct.Thepresenceofbilirubinintheurineissometimesreferredtoascholuria—therefore,hepatitisandobstructionofthecommonbileductcausecholuricjaundice,whereasthejaundiceofhemolyticanemiaisreferredtoasacholuric.Thelaboratoryresultsinpatientswithhepatitisarevariable,dependingontheextentofdamagetoparenchymalcellsandtheextentofmicro-obstructiontobileductules.SerumlevelsofALTandASTareusuallymarkedlyelevatedinhepatitis,whereasserumlevelsofalkalinephosphataseareele-vatedinobstructiveliverdisease.Urobilinogen&BilirubininUrinewhichfourpyrroleringsarejoinedbymethenylAreClinicalIndicatorsbridges.Theeightsidegroups(methyl,vinyl,andpropionylsubstituents)onthefourpyrroleringsofNormally,therearemeretracesofurobilinogeninthehemearearrangedinaspecificsequence.urine.Incompleteobstructionofthebileduct,no•Biosynthesisofthehemeringoccursinmitochondriaurobilinogenisfoundintheurine,sincebilirubinhasandcytosolviaeightenzymaticsteps.Itcommencesnoaccesstotheintestine,whereitcanbeconvertedtowithformationofδ-aminolevulinate(ALA)fromurobilinogen.Inthiscase,thepresenceofbilirubinsuccinyl-CoAandglycineinareactioncatalyzedby(conjugated)intheurinewithouturobilinogensuggestsALAsynthase,theregulatoryenzymeofthepathway.obstructivejaundice,eitherintrahepaticorposthepatic.Injaundicesecondarytohemolysis,theincreased•Geneticallydeterminedabnormalitiesofsevenoftheproductionofbilirubinleadstoincreasedproductionofeightenzymesinvolvedinhemebiosynthesisresultinurobilinogen,whichappearsintheurineinlargetheinheritedporphyrias.Redbloodcellsandliveramounts.Bilirubinisnotusuallyfoundintheurineinarethemajorsitesofmetabolicexpressionofthepor-hemolyticjaundice(becauseunconjugatedbilirubinphyrias.Photosensitivityandneurologicproblemsdoesnotpassintotheurine),sothatthecombinationarecommoncomplaints.Intakeofcertaincom-ofincreasedurobilinogenandabsenceofbilirubinispounds(suchaslead)cancauseacquiredporphyrias.suggestiveofhemolyticjaundice.Increasedbloodde-Increasedamountsofporphyrinsortheirprecursorsstructionfromanycausebringsaboutanincreaseincanbedetectedinbloodandurine,facilitatingdiag-urineurobilinogen.nosis.Table32–3summarizeslaboratoryresultsobtained•Catabolismofthehemeringisinitiatedbytheen-onpatientswiththreedifferentcausesofjaundice—he-zymehemeoxygenase,producingalineartetrapyr-molyticanemia(aprehepaticcause),hepatitis(ahepaticrole.cause),andobstructionofthecommonbileduct(a•Biliverdinisanearlyproductofcatabolismandonposthepaticcause).Laboratorytestsonblood(evalua-reductionyieldsbilirubin.Thelatteristransportedtionofthepossibilityofahemolyticanemiaandmea-byalbuminfromperipheraltissuestotheliver,wheresurementofprothrombintime)andonserum(eg,elec-itistakenupbyhepatocytes.Theironofhemeandtrophoresisofproteins;activitiesoftheenzymesALT,theaminoacidsofglobinareconservedandreuti-AST,andalkalinephosphatase)arealsoimportantinlized.helpingtodistinguishbetweenprehepatic,hepatic,and•Intheliver,bilirubinismadewater-solublebyconju-posthepaticcausesofjaundice.gationwithtwomoleculesofglucuronicacidandissecretedintothebile.Theactionofbacterialen-SUMMARYzymesinthegutproducesurobilinogenandurobilin,•Hemoproteins,suchashemoglobinandthecy-whichareexcretedinthefecesandurine.tochromes,containheme.Hemeisaniron-por-•Jaundiceisduetoelevationofthelevelofbilirubin2+intheblood.Thecausesofjaundicecanbeclassifiedphyrincompound(Fe-protoporphyrinIX)in
283PORPHYRINS&BILEPIGMENTS/285asprehepatic(eg,hemolyticanemias),hepatic(eg,BerkPD,WolkoffAW:Bilirubinmetabolismandthehyperbiliru-hepatitis),andposthepatic(eg,obstructionofthebinemias.In:Harrison’sPrinciplesofInternalMedicine,15thed.BraunwaldEetal(editors).McGraw-Hill,2001.commonbileduct).Measurementsofplasmatotalandnonconjugatedbilirubin,ofurinaryurobilino-ChowdhuryJRetal:Hereditaryjaundiceanddisordersofbilirubinmetabolism.In:TheMetabolicandMolecularBasesofInher-genandbilirubin,andofcertainserumenzymesasitedDisease,8thed.ScriverCRetal(editors).McGraw-Hill,wellasinspectionofstoolsampleshelpdistinguish2001.betweenthesecauses.DesnickRJ:Theporphyrias.In:Harrison’sPrinciplesofInternalMedicine,15thed.BraunwaldEetal(editors).McGraw-Hill,REFERENCES2001.ElderGH:Haemsynthesisandtheporphyrias.In:ScientificFoun-AndersonKEetal:Disordersofhemebiosynthesis:X-linkedsid-dationsofBiochemistryinClinicalPractice,2nded.Williamseroblasticanemiaandtheporphyrias.In:TheMetabolicandDL,MarksV(editors).Butterworth-Heinemann,1994.MolecularBasesofInheritedDisease,8thed.ScriverCRetal(editors).McGraw-Hill,2001.
284SECTIONIVStructure,Function,&ReplicationofInformationalMacromoleculesNucleotides33VictorW.Rodwell,PhDBIOMEDICALIMPORTANCEHH674C5NC5Nucleotides—themonomerunitsorbuildingblocksof1C3CHN8Nnucleicacids—servemultipleadditionalfunctions.TheyCH2CCHCCHformapartofmanycoenzymesandserveasdonorsofHN4N92N63H1phosphorylgroups(eg,ATPorGTP),ofsugars(eg,UDP-orGDP-sugars),oroflipid(eg,CDP-acylglyc-PurinePyrimidineerol).Regulatorynucleotidesincludethesecondmes-Figure33–1.Purineandpyrimidine.TheatomsaresengerscAMPandcGMP,thecontrolbyADPofox-idativephosphorylation,andallostericregulationofnumberedaccordingtotheinternationalsystem.enzymeactivitybyATP,AMP,andCTP.Syntheticpurineandpyrimidineanalogsthatcontainhalogens,thiols,oradditionalnitrogenareemployedforchemo-Nucleosides&NucleotidestherapyofcancerandAIDSandassuppressorsoftheimmuneresponseduringorgantransplantation.Nucleosidesarederivativesofpurinesandpyrimidinesthathaveasugarlinkedtoaringnitrogen.NumeralsPURINES,PYRIMIDINES,NUCLEOSIDES,withaprime(eg,2′or3′)distinguishatomsofthe&NUCLEOTIDESsugarfromthoseoftheheterocyclicbase.ThesugarinribonucleosidesisD-ribose,andindeoxyribonucleo-Purinesandpyrimidinesarenitrogen-containinghete-sidesitis2-deoxy-D-ribose.Thesugarislinkedtotherocycles,cycliccompoundswhoseringscontainbothheterocyclicbaseviaa-N-glycosidicbond,almostal-carbonandotherelements(heteroatoms).NotethatwaystoN-1ofapyrimidineortoN-9ofapurine(Fig-thesmallerpyrimidinehasthelongernameandtheure33–3).largerpurinetheshorternameandthattheirsix-atomringsarenumberedinoppositedirections(Figure33–1).Theplanarcharacterofpurinesandpyrimidinesfacilitatestheircloseassociation,or“stacking,”whichNH2NHOOHstabilizesdouble-strandedDNA(Chapter36).Theoxoandaminogroupsofpurinesandpyrimidinesexhibitketo-enolandamine-iminetautomerism(Figure33–2),butphysiologicconditionsstronglyfavortheaminoFigure33–2.Tautomerismoftheoxoandaminoandoxoforms.functionalgroupsofpurinesandpyrimidines.286
285NUCLEOTIDES/287NHNH2OO2NNNHNNHN9191NONH2NNONNNHOHOHOHOOOOOOHOHOHOHOHOHOHOHAdenosineCytidineGuanosineUridineFigure33–3.Ribonucleosides,drawnasthesynconformers.Mononucleotidesarenucleosideswithaphosphorylcleotides.Boththereforeexistassynoranticonformersgroupesterifiedtoahydroxylgroupofthesugar.The(Figure33–5).Whilebothconformersoccurinnature,3′-and5′-nucleotidesarenucleosideswithaphospho-anticonformerspredominate.Table33–1liststherylgrouponthe3′-or5′-hydroxylgroupofthesugar,majorpurinesandpyrimidinesandtheirnucleosiderespectively.Sincemostnucleotidesare5′-,theprefixandnucleotidederivatives.Single-letterabbreviations“5′-”isusuallyomittedwhennamingthem.UMPandareusedtoidentifyadenine(A),guanine(G),cytosinedAMPthusrepresentnucleotideswithaphosphoryl(C),thymine(T),anduracil(U),whetherfreeorpre-grouponC-5ofthepentose.Additionalphosphorylsentinnucleosidesornucleotides.Theprefix“d”groupslinkedbyacidanhydridebondstothephos-(deoxy)indicatesthatthesugaris2′-deoxy-D-ribosephorylgroupofamononucleotideformnucleoside(eg,dGTP)(Figure33–6).diphosphatesandtriphosphates(Figure33–4).SterichindrancebythebaserestrictsrotationaboutNucleicAcidsAlsoContaintheβ-N-glycosidicbondofnucleosidesandnu-AdditionalBasesSmallquantitiesofadditionalpurinesandpyrimidinesNHoccurinDNAandRNAs.Examplesinclude5-methyl-2cytosineofbacterialandhumanDNA,5-hydroxy-NNAdeninemethylcytosineofbacterialandviralnucleicacids,andmono-anddi-N-methylatedadenineandguanineofNNCH2ONHNH22––ORiboseNNOOONNHOPOPOPHOOHOO–ONNNNHOHOAdenosine5′-monophosphate(AMP)OOAdenosine5′-diphosphate(ADP)SynAntiOHOHOHOHAdenosine5′-triphosphate(ATP)Figure33–5.Thesynandanticonformersofadeno-Figure33–4.ATP,itsdiphosphate,anditssinedifferwithrespecttoorientationabouttheN-gly-monophosphate.cosidicbond.
286288/CHAPTER33Table33–1.Bases,nucleosides,andnucleotides.NucleosideBaseBaseX=RiboseorNucleotide,WhereFormulaX=HDeoxyriboseX=RibosePhosphateNH2NNAdenineAdenosineAdenosinemonophosphateAAAMPNNXOHNNGuanineGuanosineGuanosinemonophosphateGGGMPH2NNNXNH2NCytosineCytidineCytidinemonophosphateCCCMPONXOHNUracilUridineUridinemonophosphateUUUMPONXOHCH3NThymineThymidineThymidinemonophosphateTTTMPONdXNH2NH2OONNCH3NNHNHNNNNNONONOOOOOOOOOOOOPPPP–OO––OO––OO––OO–OHOHOHHOHOHOHHAMPdAMPUMPTMPFigure33–6.AMP,dAMP,UMP,andTMP.
287NUCLEOTIDES/289NH2NH2NH2OCH3CH2OHNNNNNHNOONNH2NNNNNHHOCH2OCH25-Methylcytosine5-HydroxymethylcytosineOO–OPO–OPOH3CCH3OONOCH3OHOHNNFigure33–9.cAMP,3′,5′-cyclicAMP,andcGMP.NHN7NNH2NNNHNucleotidesServeDiverseDimethylaminoadenine7-MethylguaninePhysiologicFunctionsFigure33–7.FouruncommonnaturallyoccurringNucleotidesparticipateinreactionsthatfulfillphysio-pyrimidinesandpurines.logicfunctionsasdiverseasproteinsynthesis,nucleicacidsynthesis,regulatorycascades,andsignaltransduc-tionpathways.mammalianmessengerRNAs(Figure33–7).Theseatypicalbasesfunctioninoligonucleotiderecognitionandinregulatingthehalf-livesofRNAs.Freenu-NucleosideTriphosphatesHaveHighcleotidesincludehypoxanthine,xanthine,anduricacidGroupTransferPotential(seeFigure34–8),intermediatesinthecatabolismofAcidanhydrides,unlikephosphateesters,havehighadenineandguanine.Methylatedheterocyclicbasesofgrouptransferpotential.Δ0′forthehydrolysisofeachplantsincludethexanthinederivativescaffeineofcof-oftheterminalphosphatesofnucleosidetriphosphatesfee,theophyllineoftea,andtheobromineofcocoa(Fig-isabout−7kcal/mol(−30kJ/mol).Thehighgroupure33–8).transferpotentialofpurineandpyrimidinenucleosidePosttranslationalmodificationofpreformedpolynu-triphosphatespermitsthemtofunctionasgrouptrans-cleotidescangenerateadditionalbasessuchasferreagents.Cleavageofanacidanhydridebondtypi-pseudouridine,inwhichD-riboseislinkedtoC-5ofcallyiscoupledwithahighlyendergonicprocesssuchuracilbyacarbon-to-carbonbondratherthanbyaascovalentbondsynthesis—eg,polymerizationofnu-β-N-glycosidicbond.Thenucleotidepseudouridyliccleosidetriphosphatestoformanucleicacid.acidΨarisesbyrearrangementofUMPofapreformedInadditiontotheirrolesasprecursorsofnucleictRNA.Similarly,methylationbyS-adenosylmethionineacids,ATP,GTP,UTP,CTP,andtheirderivativesofaUMPofpreformedtRNAformsTMP(thymidineeachserveuniquephysiologicfunctionsdiscussedinmonophosphate),whichcontainsriboseratherthande-otherchapters.Selectedexamplesincludetheroleofoxyribose.ATPastheprincipalbiologictransduceroffreeenergy;thesecondmessengercAMP(Figure33–9);adenosineCH3′-phosphate-5′-phosphosulfate(Figure33–10),theO3sulfatedonorforsulfatedproteoglycans(Chapter48)H3CNandforsulfateconjugatesofdrugs;andthemethylNgroupdonorS-adenosylmethionine(Figure33–11).ONNCH3PFigure33–8.Caffeine,atrimethylxanthine.Thedi-2–AdenineRibosePOSO3methylxanthinestheobromineandtheophyllinearesimilarbutlackthemethylgroupatN-1andatN-7,re-Figure33–10.Adenosine3′-phosphate-5′-phos-spectively.phosulfate.
288290/CHAPTER33NH2Table33–2.ManycoenzymesandrelatedNcompoundsarederivativesofadenosineNmonophosphate.NNNH2COO–CHCH2NNAdenine3OCHCH2CH2SNN++ONH3ROPOCH2HOOH–OnOMethionineAdenosineR''OOR'Figure33–11.S-Adenosylmethionine.D-RiboseCoenzymeRRRnGTPservesasanallostericregulatorandasanenergyActivemethionineMethionine*HH0sourceforproteinsynthesis,andcGMP(Figure33–9)AminoacidadenylatesAminoacidHH1servesasasecondmessengerinresponsetonitricoxide2−2−ActivesulfateSO3HPO312−(NO)duringrelaxationofsmoothmuscle(Chapter3′,5′-CyclicAMPHPO31†48).UDP-sugarderivativesparticipateinsugarepimer-NAD*HH2†2−izationsandinbiosynthesisofglycogen,glucosyldisac-NADP*PO3H2†charides,andtheoligosaccharidesofglycoproteinsandFADHH2†2−proteoglycans(Chapters47and48).UDP-glucuronicCoASHHPO32acidformstheurinaryglucuronideconjugatesofbiliru-*Replacesphosphorylgroup.bin(Chapter32)andofdrugssuchasaspirin.CTP†RisaBvitaminderivative.participatesinbiosynthesisofphosphoglycerides,sphingomyelin,andothersubstitutedsphingosines(Chapter24).Finally,manycoenzymesincorporatenu-cleotidesaswellasstructuressimilartopurineandnucleicacidsthusoftenisexpressedintermsof“ab-pyrimidinenucleotides(seeTable33–2).sorbanceat260nm.”NucleotidesArePolyfunctionalAcidsSYNTHETICNUCLEOTIDEANALOGSAREUSEDINCHEMOTHERAPYNucleosidesorfreepurineorpyrimidinebasesareun-chargedatphysiologicpH.Bycontrast,theprimarySyntheticanalogsofpurines,pyrimidines,nucleosides,phosphorylgroups(pKabout1.0)andsecondaryphos-andnucleotidesalteredineithertheheterocyclicringorphorylgroups(pKabout6.2)ofnucleotidesensurethatthesugarmoietyhavenumerousapplicationsinclinicaltheybearanegativechargeatphysiologicpH.Nu-medicine.Theirtoxiceffectsreflecteitherinhibitionofcleotidescan,however,actasprotondonorsoraccep-enzymesessentialfornucleicacidsynthesisortheirin-torsatpHvaluestwoormoreunitsaboveorbelowcorporationintonucleicacidswithresultingdisruptionneutrality.ofbase-pairing.Oncologistsemploy5-fluoro-or5-iodouracil,3-deoxyuridine,6-thioguanineand6-mer-captopurine,5-or6-azauridine,5-or6-azacytidine,NucleotidesAbsorbUltravioletLightand8-azaguanine(Figure33–12),whichareincorpo-TheconjugateddoublebondsofpurineandpyrimidineratedintoDNApriortocelldivision.Thepurineana-derivativesabsorbultravioletlight.Themutagenicef-logallopurinol,usedintreatmentofhyperuricemiaandfectofultravioletlightresultsfromitsabsorptionbygout,inhibitspurinebiosynthesisandxanthineoxidasenucleotidesinDNAwithaccompanyingchemicalactivity.Cytarabineisusedinchemotherapyofcancer.changes.WhilespectraarepH-dependent,atpH7.0allFinally,azathioprine,whichiscatabolizedto6-mercap-thecommonnucleotidesabsorblightatawavelengthtopurine,isemployedduringorgantransplantationtocloseto260nm.Theconcentrationofnucleotidesandsuppressimmunologicrejection.
289NUCLEOTIDES/291OOIHN5HN6NOONNHOHOOOFONHNOHN58NH2NN2′ONHNHOHHHOOH5-Iodo-2′-deoxyuridine5-Fluorouracil6-Azauridine8-AzaguanineSHSHOH6N6N6NNN15N243NNH2NNNNNHHH6-Mercaptopurine6-ThioguanineAlloburinolFigure33–12.Selectedsyntheticpyrimidineandpurineanalogs.NonhydrolyzableNucleoside(absentfromDNA)functionsasanucleophileduringTriphosphateAnalogsServeashydrolysisofthe3′,5′-phosphodiesterbond.ResearchToolsSyntheticnonhydrolyzableanalogsofnucleosidePolynucleotidesAreDirectionaltriphosphates(Figure33–13)allowinvestigatorstodis-MacromoleculestinguishtheeffectsofnucleotidesduetophosphorylPhosphodiesterbondslinkthe3′-and5′-carbonsofad-transferfromeffectsmediatedbyoccupancyofal-jacentmonomers.Eachendofanucleotidepolymerlostericnucleotide-bindingsitesonregulatedenzymes.thusisdistinct.Wethereforerefertothe“5′-end”orthe“3′-end”ofpolynucleotides,the5′-endbeingthePOLYNUCLEOTIDESonewithafreeorphosphorylated5′-hydroxyl.The5′-phosphorylgroupofamononucleotidecanes-terifyasecond⎯OHgroup,formingaphosphodi-PolynucleotidesHavePrimaryStructureester.Mostcommonly,thissecond⎯OHgroupisthe3′-OHofthepentoseofasecondnucleotide.ThisThebasesequenceorprimarystructureofapolynu-formsadinucleotideinwhichthepentosemoietiesarecleotidecanberepresentedasshownbelow.Thephos-linkedbya3′→5′phosphodiesterbondtoformthephodiesterbondisrepresentedbyPorp,basesbyasin-“backbone”ofRNAandDNA.gleletter,andpentosesbyaverticalline.Whileformationofadinucleotidemayberepre-ATCAsentedastheeliminationofwaterbetweentwomonomers,thereactioninfactstronglyfavorsphos-phodiesterhydrolysis.Phosphodiesterasesrapidlycat-alyzethehydrolysisofphosphodiesterbondswhosespontaneoushydrolysisisanextremelyslowprocess.Consequently,DNApersistsforconsiderableperiodsPPPPOHandhasbeendetectedeveninfossils.RNAsarefarlessstablethanDNAsincethe2′-hydroxylgroupofRNA
290292/CHAPTER33OOOSUMMARYPu/PyROPOPOPO–•Underphysiologicconditions,theaminoandoxoO–O–O–tautomersofpurines,pyrimidines,andtheirderiva-tivespredominate.Parentnucleosidetriphosphate•Nucleicacidscontain,inadditiontoA,G,C,T,andOOOU,tracesof5-methylcytosine,5-hydroxymethylcyto-–sine,pseudouridine(Ψ),orN-methylatedbases.Pu/PyROPOPCH2PO•MostnucleosidescontainD-riboseor2-deoxy-D-O–O–O–riboselinkedtoN-1ofapyrimidineortoN-9ofaβ,γ-Methylenederivativepurinebyaβ-glycosidicbondwhosesynconformerspredominate.OOOH•Aprimednumerallocatesthepositionofthephos-Pu/PyROPOPNPO–phateonthesugarsofmononucleotides(eg,3′-O–O–O–GMP,5′-dCMP).Additionalphosphorylgroupslinkedtothefirstbyacidanhydridebondsformnu-β,γ-Iminoderivativecleosidediphosphatesandtriphosphates.Figure33–13.Syntheticderivativesofnucleoside•Nucleosidetriphosphateshavehighgrouptransfertriphosphatesincapableofundergoinghydrolyticre-potentialandparticipateincovalentbondsyntheses.leaseoftheterminalphosphorylgroup.(Pu/Py,aThecyclicphosphodiesterscAMPandcGMPfunc-purineorpyrimidinebase;R,riboseordeoxyribose.)tionasintracellularsecondmessengers.Shownaretheparent(hydrolyzable)nucleoside•Mononucleotideslinkedby3′→5′-phosphodiestertriphosphate(top)andtheunhydrolyzableβ-methyl-bondsformpolynucleotides,directionalmacromole-ene(center)andγ-iminoderivatives(bottom).culeswithdistinct3′-and5′-ends.ForpTpGpTporTGCATCA,the5′-endisattheleft,andallphos-phodiesterbondsare3′→5′.Whereallthephosphodiesterbondsare5′→3′,a•Syntheticanalogsofpurineandpyrimidinebasesandmorecompactnotationispossible:theirderivativesserveasanticancerdrugseitherbyinhibitinganenzymeofnucleotidebiosynthesisorpGpGpApTpCpAbybeingincorporatedintoDNAorRNA.Thisrepresentationindicatesthatthe5′-hydroxyl—butnotthe3′-hydroxyl—isphosphorylated.REFERENCESThemostcompactrepresentationshowsonlytheAdamsRLP,KnowlerJT,LeaderDP:TheBiochemistryoftheNu-basesequencewiththe5′-endontheleftandthe3′-cleicAcids,11thed.Chapman&Hall,1992.endontheright.ThephosphorylgroupsareassumedBlackburnGM,GaitMJ:NucleicAcidsinChemistry&Biology.IRLbutnotshown:Press,1990.BuggCE,CarsonWM,MontgomeryJA:Drugsbydesign.SciAmGGATCA1992;269(6):92.
291MetabolismofPurine&PyrimidineNucleotides34VictorW.Rodwell,PhDBIOMEDICALIMPORTANCE(synthesisdenovo),(2)phosphoribosylationofpurines,and(3)phosphorylationofpurinenucleosides.Thebiosynthesisofpurinesandpyrimidinesisstrin-gentlyregulatedandcoordinatedbyfeedbackmecha-nismsthatensuretheirproductioninquantitiesandatINOSINEMONOPHOSPHATE(IMP)timesappropriatetovaryingphysiologicdemand.Ge-neticdiseasesofpurinemetabolismincludegout,ISSYNTHESIZEDFROMAMPHIBOLICLesch-Nyhansyndrome,adenosinedeaminasedefi-INTERMEDIATESciency,andpurinenucleosidephosphorylasedeficiency.Figure34–2illustratestheintermediatesandreactionsBycontrast,apartfromtheoroticacidurias,thereareforconversionofα-D-ribose5-phosphatetoinosinefewclinicallysignificantdisordersofpyrimidinecatab-monophosphate(IMP).Separatebranchesthenleadtoolism.AMPandGMP(Figure34–3).SubsequentphosphoryltransferfromATPconvertsAMPandGMPtoADPPURINES&PYRIMIDINESAREandGDP.ConversionofGDPtoGTPinvolvesasec-ondphosphoryltransferfromATP,whereasconversionDIETARILYNONESSENTIALofADPtoATPisachievedprimarilybyoxidativeHumantissuescansynthesizepurinesandpyrimidinesphosphorylation(seeChapter12).fromamphibolicintermediates.Ingestednucleicacidsandnucleotides,whichthereforearedietarilynonessen-tial,aredegradedintheintestinaltracttomononu-MultifunctionalCatalystsParticipateincleotides,whichmaybeabsorbedorconvertedtoPurineNucleotideBiosynthesispurineandpyrimidinebases.ThepurinebasesarethenInprokaryotes,eachreactionofFigure34–2iscat-oxidizedtouricacid,whichmaybeabsorbedandex-alyzedbyadifferentpolypeptide.Bycontrast,ineu-cretedintheurine.Whilelittleornodietarypurineorkaryotes,theenzymesarepolypeptideswithmultiplepyrimidineisincorporatedintotissuenucleicacids,in-catalyticactivitieswhoseadjacentcatalyticsitesfacili-jectedcompoundsareincorporated.Theincorporation3tatechannelingofintermediatesbetweensites.Threeofinjected[H]thymidineintonewlysynthesizedDNAdistinctmultifunctionalenzymescatalyzereactions3,thusisusedtomeasuretherateofDNAsynthesis.4,and6,reactions7and8,andreactions10and11ofFigure34–2.BIOSYNTHESISOFPURINENUCLEOTIDESPurineandpyrimidinenucleotidesaresynthesizedinAntifolateDrugsorGlutamineAnalogsvivoatratesconsistentwithphysiologicneed.Intracel-BlockPurineNucleotideBiosynthesislularmechanismssenseandregulatethepoolsizesofnucleotidetriphosphates(NTPs),whichriseduringThecarbonsaddedinreactions4and5ofFigure34–2growthortissueregenerationwhencellsarerapidlydi-arecontributedbyderivativesoftetrahydrofolate.viding.EarlyinvestigationsofnucleotidebiosynthesisPurinedeficiencystates,whicharerareinhumans,gen-employedbirds,andlateronesusedEscherichiacoli.erallyreflectadeficiencyoffolicacid.CompoundsthatIsotopicprecursorsfedtopigeonsestablishedthesourceinhibitformationoftetrahydrofolatesandthereforeofeachatomofapurinebase(Figure34–1)andiniti-blockpurinesynthesishavebeenusedincanceratedstudyoftheintermediatesofpurinebiosynthesis.chemotherapy.InhibitorycompoundsandthereactionsThreeprocessescontributetopurinenucleotidetheyinhibitincludeazaserine(reaction5,Figure34–2),biosynthesis.Theseare,inorderofdecreasingimpor-diazanorleucine(reaction2),6-mercaptopurine(reac-tance:(1)synthesisfromamphibolicintermediatestions13and14),andmycophenolicacid(reaction14).293
292294/CHAPTER34RespiratoryCOandthereforeutilizeexogenouspurinestoformnu-2Glycinecleotides.AspartateCAMP&GMPFeedback-RegulatePRPP6NNC715GlutamylAmidotransferase8C24N5,N10-Methenyl-SincebiosynthesisofIMPconsumesglycine,gluta-CC93N10-Formyl-NNtetrahydrofolatemine,tetrahydrofolatederivatives,aspartate,andATP,Htetrahydro-itisadvantageoustoregulatepurinebiosynthesis.Thefolatemajordeterminantoftherateofdenovopurinenu-cleotidebiosynthesisistheconcentrationofPRPP,whosepoolsizedependsonitsratesofsynthesis,uti-Amidenitrogenofglutaminelization,anddegradation.TherateofPRPPsynthesisdependsontheavailabilityofribose5-phosphateandFigure34–1.SourcesofthenitrogenandcarbonontheactivityofPRPPsynthase,anenzymesensitiveatomsofthepurinering.Atoms4,5,and7(shaded)de-tofeedbackinhibitionbyAMP,ADP,GMP,andrivefromglycine.GDP.AMP&GMPFeedback-Regulate“SALVAGEREACTIONS”CONVERTTheirFormationFromIMPPURINES&THEIRNUCLEOSIDESTOTwomechanismsregulateconversionofIMPtoGMPMONONUCLEOTIDESandAMP.AMPandGMPfeedback-inhibitadenylo-Conversionofpurines,theirribonucleosides,andtheirsuccinatesynthaseandIMPdehydrogenase(reactionsdeoxyribonucleosidestomononucleotidesinvolvesso-12and14,Figure34–3),respectively.Furthermore,called“salvagereactions”thatrequirefarlessenergyconversionofIMPtoadenylosuccinateenroutetothandenovosynthesis.Themoreimportantmecha-AMPrequiresGTP,andconversionofxanthinylatenisminvolvesphosphoribosylationbyPRPP(structure(XMP)toGMPrequiresATP.Thiscross-regulationII,Figure34–2)ofafreepurine(Pu)toformapurinebetweenthepathwaysofIMPmetabolismthusserves5′-mononucleotide(Pu-RP).todecreasesynthesisofonepurinenucleotidewhenthereisadeficiencyoftheothernucleotide.AMPandGMPalsoinhibithypoxanthine-guaninephosphoribo-PuPRPP+−→+PRPPPisyltransferase,whichconvertshypoxanthineandgua-ninetoIMPandGMP(Figure34–4),andGMPfeed-back-inhibitsPRPPglutamylamidotransferase(reactionTwophosphoribosyltransferasesthenconvertadenine2,Figure34–2).toAMPandhypoxanthineandguaninetoIMPorGMP(Figure34–4).Asecondsalvagemechanismin-volvesphosphoryltransferfromATPtoapurineri-REDUCTIONOFRIBONUCLEOSIDEbonucleoside(PuR):DIPHOSPHATESFORMSDEOXYRIBONUCLEOSIDEPuRATP+→−PuRPADP+DIPHOSPHATESReductionofthe2′-hydroxylofpurineandpyrimidineAdenosinekinasecatalyzesphosphorylationofadeno-ribonucleotides,catalyzedbytheribonucleotidere-sineanddeoxyadenosinetoAMPanddAMP,andde-ductasecomplex(Figure34–5),formsdeoxyribonu-oxycytidinekinasephosphorylatesdeoxycytidineandcleosidediphosphates(dNDPs).Theenzymecomplex2′-deoxyguanosinetodCMPanddGMP.isactiveonlywhencellsareactivelysynthesizingDNA.Liver,themajorsiteofpurinenucleotidebiosynthe-Reductionrequiresthioredoxin,thioredoxinreductase,sis,providespurinesandpurinenucleosidesforsalvageandNADPH.Theimmediatereductant,reducedandutilizationbytissuesincapableoftheirbiosynthe-thioredoxin,isproducedbyNADPH:thioredoxinre-sis.Forexample,humanbrainhasalowlevelofPRPPductase(Figure34–5).Reductionofribonucleosideamidotransferase(reaction2,Figure34–2)andhencediphosphates(NDPs)todeoxyribonucleosidediphos-dependsinpartonexogenouspurines.Erythrocytesphates(dNDPs)issubjecttocomplexregulatorycon-andpolymorphonuclearleukocytescannotsynthesizetrolsthatachievebalancedproductionofdeoxyribonu-5-phosphoribosylamine(structureIII,Figure34–2)cleotidesforsynthesisofDNA(Figure34–6).
293ePGln+2GluPCHOCHO8ATPMg8HN79NHR-5-(V)NH79NHR-5-(VI)54554CCCC22HHO3Formylglycinamideribosyl-5-phosphateHNFormylglycinamidinribosyl-5-phosphate+2folate4HVISYNTHETASE4ATP,Mg6-Ringclosure10N,folateO254NMethenyl-HHVIISYNTHETASEFORMYLTRANSFERASE+3P7NHNHHCH54HOHNNR-5-PC2CO(IV)(VII)CH.)HHOHHCCNNR-5-–OCHGlycinamide22NH2AminoimidazoleCCiribosyl-5-phosphateH(XII)ribosyl-5-phosphateOCNorPOO22–3P+2COHNHC+33,PO–Mg7P7NHOInosinemonophosphate(IMP)54ATPADP+PGlycineCC2H+3VIICARBOXYLASEOO9NHH2HOHPH-ribosylamine2OD-(III)CHHOHβNNR-5-11xplanations.(CHHe54(VIII)RingclosureOOC63N2IMPCYCLOHYDROLASExtforiHeP5-Phospho-–OAminoimidazoletPP3Pee+2CH2carboxylateribosyl-5-phosphateNNR-5-CHHCNHOOCOOC8CC(XI)OPRPPGLUTAMYL––2HAMIDOTRANSFERASEOCNHandATP.SGlutamineGlutamateO2eHIXSYNTHETASENCH2PAspartateHribosyl-5-phosphateOPCHFormimidoimidazolecarboxamidePONNR-5-5-phosphatfolateeHOCC41OH54HHOC63N2O32(II)12(IX)104HOHPRPPNHH5CHH-Formyl-folateC104OHC2HOOCHOOCAminoimidazolesuccinylNFORMYLTRANSFERASEsisfromribose––PP+carboxamideribosyl-5-phosphate21CHMgPRPPNNR-5-SYNTHASEbiosynthATPAMPCC(X)e–OC29HNPurinHOHNHOHCOOCH2ribosyl-5-phosphateHCH2O(I)OOCHOH–Aminoimidazolecarboxamide5CHHFumarateO-Ribose5-phosphatere34–2.-DuαPADENYLOSUCCINASEFig
294296/CHAPTER34HH––––OOCCCCOOOOCCCCOOHH2O2––HHNH+H2ONHOOCCCCOONH23NNNHN12N13N2+GTP,MgNNNNADENYLOSUCCINASENNADENYLOSUCCINATER-5-PSYNTHASER-5-PR-5-PInosinemonophosphateAdenylosuccinateAdenosinemonophosphate(IMP)(AMPS)(AMP)+NADH2O14IMPDEHYDROGENASENADH+H+OOGlutamineGlutamateNNHN15HNATPONNH2NNNHTRANSAMIDINASER-5-PPR-5-XanthosinemonophosphateGuanosinemonophosphate(XMP)(GMP)Figure34–3.ConversionofIMPtoAMPandGMP.BIOSYNTHESISOFPYRIMIDINETHEDEOXYRIBONUCLEOSIDESOFNUCLEOTIDESURACIL&CYTOSINEARESALVAGEDFigure34–7summarizestherolesoftheintermediatesWhilemammaliancellsreutilizefewfreepyrimidines,andenzymesofpyrimidinenucleotidebiosynthesis.“salvagereactions”converttheribonucleosidesuridineThecatalystfortheinitialreactioniscytosoliccarbamoylandcytidineandthedeoxyribonucleosidesthymidinephosphatesynthaseII,adifferentenzymefromthemi-anddeoxycytidinetotheirrespectivenucleotides.ATP-tochondrialcarbamoylphosphatesynthaseIofureasyn-dependentphosphoryltransferases(kinases)catalyzethethesis(Figure29–9).Compartmentationthusprovidesphosphorylationofthenucleosidediphosphates2′-de-twoindependentpoolsofcarbamoylphosphate.PRPP,oxycytidine,2′-deoxyguanosine,and2′-deoxyadenosineanearlyparticipantinpurinenucleotidesynthesis(Fig-totheircorrespondingnucleosidetriphosphates.Inad-ure34–2),isamuchlaterparticipantinpyrimidinedition,orotatephosphoribosyltransferase(reaction5,biosynthesis.Figure34–7),anenzymeofpyrimidinenucleotidesyn-thesis,salvagesoroticacidbyconvertingittoorotidineMultifunctionalProteinsmonophosphate(OMP).CatalyzetheEarlyReactionsofPyrimidineBiosynthesisMethotrexateBlocksReductionofDihydrofolateFiveofthefirstsixenzymeactivitiesofpyrimidinebiosynthesisresideonmultifunctionalpolypeptides.Reaction12ofFigure34–7istheonlyreactionofpyrimi-Onesuchpolypeptidecatalyzesthefirstthreereactionsdinenucleotidebiosynthesisthatrequiresatetrahydrofo-510ofFigure34–2andensuresefficientchannelingofcar-latederivative.ThemethylenegroupofN,N-methyl-bamoylphosphatetopyrimidinebiosynthesis.Asecondene-tetrahydrofolateisreducedtothemethylgroupthatbifunctionalenzymecatalyzesreactions5and6.istransferred,andtetrahydrofolateisoxidizedtodihydro-
295METABOLISMOFPURINE&PYRIMIDINENUCLEOTIDES/297NH2NH2RIBONUCLEOTIDEPRPPPPiREDUCTASENNNNRibonucleoside2′-DeoxyribonucleosidediphosphatediphosphateNNNNHPOH2CAdenineOReducedOxidizedADENINEPHOSPHORIBOSYLthioredoxinthioredoxinTRANSFERASEHHHHTHIOREDOXINREDUCTASEOHOHNADP+NADPH+H+AMPFigure34–5.Reductionofribonucleosidediphos-OOPRPPPPiphatesto2′-deoxyribonucleosidediphosphates.NNHNHNNNNNanucleotideinwhichtheribosylphosphateisattachedHHypoxanthinePOH2CtoN-1ofthepyrimidinering.TheanticancerdrugO5-fluorouracil(Figure33–12)isalsophosphoribosy-latedbyorotatephosphoribosyltransferase.HHHHHYPOXANTHINE-GUANINEPHOSPHORIBOSYLTRANSFERASEOHOHREGULATIONOFPYRIMIDINEIMPNUCLEOTIDEBIOSYNTHESISOOGeneExpression&EnzymeActivityNNBothAreRegulatedHNHNTheactivitiesofthefirstandsecondenzymesofpyrim-H2NNNH2NNNHidinenucleotidebiosynthesisarecontrolledbyallostericGuaninePRPPPPiPOH2CCDP2′dCDP2′dCTPO–––+HHHHATPOHOHGMP+Figure34–4.Phosphoribosylationofadenine,hy-UDP2′dUDP2′dTTPpoxanthine,andguaninetoformAMP,IMP,andGMP,–––respectively.+folate.Forfurtherpyrimidinesynthesistooccur,dihydro-folatemustbereducedbacktotetrahydrofolate,areac-GDP2′dGDP2′dGTPtioncatalyzedbydihydrofolatereductase.Dividingcells,–whichmustgenerateTMPanddihydrofolate,thusarees-peciallysensitivetoinhibitorsofdihydrofolatereductase+suchastheanticancerdrugmethotrexate.ADP2′dADP2′dATPCertainPyrimidineAnalogsAreFigure34–6.RegulationofthereductionofpurineSubstratesforEnzymesofPyrimidineandpyrimidineribonucleotidestotheirrespectiveNucleotideBiosynthesis2′-deoxyribonucleotides.SolidlinesrepresentchemicalOrotatephosphoribosyltransferase(reaction5,Figureflow.Brokenlinesshownegative(–)orpositive(+)34–7)convertsthedrugallopurinol(Figure33–12)tofeedbackregulation.
296298/CHAPTER34CO+Glutamine+ATP2CARBAMOYLPHOSPHATE1SYNTHASEIIOASPARTATEOO–4TRANSCAR-–DIHYDRO-+HN3OCBAMOYLASEOCOROTASEC3+5CH24CHHNCH2H2N3522C6HO1C2C26CH3CCH+HN–1ON–OP3COOONCOO–COOHPiHH2OCarbamoylAsparticCarbamoylDihydrooroticphosphateacidasparticacid+acid(DHOA)NAD(CAP)(CAA)DIHYDROOROTATEDEHYDROGENASENADH+H+4OOOCO2PPiPRPPHN46HN5HN35261––ONOROTIDYLICACIDONCOOOROTATEONCOODECARBOXYLASEPHOSPHORIBOSYL-HR-5-PR-5-PTRANSFERASEOroticacidUMPOMP(OA)ATP7NADPH+H+NADP+ADP10UDPdUDP(deoxyuridinediphosphate)H2OATPRIBONUCLEOTIDEREDUCTASE811ADPPiUTPdUMP510N,N-MethyleneHfolateATP4GlutamineTHYMIDYLATE12CTPSYNTHASESYNTHASE9Hfolate2NH2OCH3NHNONONR-5-PPP--dR-5-PCTPTMPFigure34–7.Thebiosyntheticpathwayforpyrimidinenucleotides.
297METABOLISMOFPURINE&PYRIMIDINENUCLEOTIDES/299regulation.CarbamoylphosphatesynthaseII(reactionNH21,Figure34–7)isinhibitedbyUTPandpurinenu-NcleotidesbutactivatedbyPRPP.Aspartatetranscar-Nbamoylase(reaction2,Figure34–7)isinhibitedbyCTPbutactivatedbyATP.Inaddition,thefirstthreeNNandthelasttwoenzymesofthepathwayareregulatedHOH2Cbycoordinaterepressionandderepression.OHHPurine&PyrimidineNucleotideHHBiosynthesisAreCoordinatelyRegulatedOHOHAdenosinePurineandpyrimidinebiosynthesisparallelonean-H2Oothermoleformole,suggestingcoordinatedcontroloftheirbiosynthesis.Severalsitesofcross-regulationchar-acterizepurineandpyrimidinenucleotidebiosynthesis.NH4+ThePRPPsynthasereaction(reaction1,Figure34–2),Owhichformsaprecursoressentialforbothprocesses,isONfeedback-inhibitedbybothpurineandpyrimidinenu-HNNHNcleotides.NH2NNNNHOH2CHOH2CHUMANSCATABOLIZEPURINESOOTOURICACIDHHHHHHHHHumansconvertadenosineandguanosinetouricacid(Figure34–8).AdenosineisfirstconvertedtoinosineOHOHOHOHbyadenosinedeaminase.InmammalsotherthanInosineGuanosinehigherprimates,uricaseconvertsuricacidtothewater-PiPisolubleproductallantoin.However,sincehumanslackuricase,theendproductofpurinecatabolisminhu-Ribose1-phosphatemansisuricacid.OONNGOUTISAMETABOLICDISORDERHNHNOFPURINECATABOLISMNNHH2NNNHVariousgeneticdefectsinPRPPsynthetase(reaction1,HypoxanthineGuanineFigure34–2)presentclinicallyasgout.Eachdefect—eg,anelevatedVmax,increasedaffinityforribose5-H2O+O2phosphate,orresistancetofeedbackinhibition—resultsHOOHN3inoverproductionandoverexcretionofpurinecatabo-22lites.WhenserumuratelevelsexceedthesolubilityHNNlimit,sodiumuratecrystalizesinsofttissuesandjointsandcausesaninflammatoryreaction,goutyarthritis.ONHNHHowever,mostcasesofgoutreflectabnormalitiesinXanthinerenalhandlingofuricacid.HO+O22H2O2OFigure34–8.FormationofuricacidfrompurinenucleosidesHNbywayofthepurinebaseshypoxanthine,xanthine,andgua-HN71Onine.Purinedeoxyribonucleosidesaredegradedbythesame9O3NHcatabolicpathwayandenzymes,allofwhichexistinthemucosaNHofthemammaliangastrointestinaltract.Uricacid
298300/CHAPTER34OTHERDISORDERSOFCATABOLISMOFPYRIMIDINESPURINECATABOLISMPRODUCESWATER-SOLUBLEWhilepurinedeficiencystatesarerareinhumansub-METABOLITESjects,therearenumerousgeneticdisordersofpurineca-Unliketheendproductsofpurinecatabolism,thosetabolism.Hyperuricemiasmaybedifferentiatedbasedofpyrimidinecatabolismarehighlywater-soluble:onwhetherpatientsexcretenormalorexcessivequanti-CO2,NH3,β-alanine,andβ-aminoisobutyrate(Figuretiesoftotalurates.Somehyperuricemiasreflectspecific34–9).Excretionofβ-aminoisobutyrateincreasesinenzymedefects.Othersaresecondarytodiseasessuchleukemiaandseverex-rayradiationexposureduetoin-ascancerorpsoriasisthatenhancetissueturnover.creaseddestructionofDNA.However,manypersonsofChineseorJapaneseancestryroutinelyexcreteβ-aminoisobutyrate.HumansprobablytransaminateLesch-NyhanSyndromeβ-aminoisobutyratetomethylmalonatesemialdehyde,Lesch-Nyhansyndrome,anoverproductionhyper-whichthenformssuccinyl-CoA(Figure19–2).uricemiacharacterizedbyfrequentepisodesofuricacidlithiasisandabizarresyndromeofself-mutilation,re-PseudouridineIsExcretedUnchangedflectsadefectinhypoxanthine-guaninephosphoribo-syltransferase,anenzymeofpurinesalvage(FigureSincenohumanenzymecatalyzeshydrolysisorphos-34–4).TheaccompanyingriseinintracellularPRPPre-phorolysisofpseudouridine,thisunusualnucleosideissultsinpurineoverproduction.Mutationsthatdecreaseexcretedunchangedintheurineofnormalsubjects.orabolishhypoxanthine-guaninephosphoribosyltrans-feraseactivityincludedeletions,frameshiftmutations,OVERPRODUCTIONOFPYRIMIDINEbasesubstitutions,andaberrantmRNAsplicing.CATABOLITESISONLYRARELYASSOCIATEDWITHCLINICALLYVonGierke’sDiseaseSIGNIFICANTABNORMALITIESPurineoverproductionandhyperuricemiainvonSincetheendproductsofpyrimidinecatabolismareGierke’sdisease(glucose-6-phosphatasedeficiency)highlywater-soluble,pyrimidineoverproductionresultsoccurssecondarytoenhancedgenerationofthePRPPinfewclinicalsignsorsymptoms.Inhyperuricemiaas-precursorribose5-phosphate.Anassociatedlacticaci-sociatedwithsevereoverproductionofPRPP,thereisdosiselevatestherenalthresholdforurate,elevatingoverproductionofpyrimidinenucleotidesandin-510totalbodyurates.creasedexcretionofβ-alanine.SinceN,N-methyl-ene-tetrahydrofolateisrequiredforthymidylatesynthe-sis,disordersoffolateandvitaminB12metabolismHypouricemiaresultindeficienciesofTMP.Hypouricemiaandincreasedexcretionofhypoxanthineandxanthineareassociatedwithxanthineoxidasede-OroticAciduriasficiencyduetoageneticdefectortosevereliverdam-TheoroticaciduriathataccompaniesReye’ssyndromeage.Patientswithasevereenzymedeficiencymayex-probablyisaconsequenceoftheinabilityofseverelyhibitxanthinuriaandxanthinelithiasis.damagedmitochondriatoutilizecarbamoylphosphate,whichthenbecomesavailableforcytosolicoverproduc-AdenosineDeaminase&Purinetionoforoticacid.TypeIoroticaciduriareflectsade-NucleosidePhosphorylaseDeficiencyficiencyofbothorotatephosphoribosyltransferaseandorotidylatedecarboxylase(reactions5and6,FigureAdenosinedeaminasedeficiencyisassociatedwithan34–7);therarertypeIIoroticaciduriaisduetoadefi-immunodeficiencydiseaseinwhichboththymus-ciencyonlyoforotidylatedecarboxylase(reaction6,derivedlymphocytes(Tcells)andbonemarrow-de-Figure34–7).rivedlymphocytes(Bcells)aresparseanddysfunc-tional.PurinenucleosidephosphorylasedeficiencyisDeficiencyofaUreaCycleEnzymeResultsassociatedwithaseveredeficiencyofTcellsbutappar-inExcretionofPyrimidinePrecursorsentlynormalBcellfunction.Immunedysfunctionsap-peartoresultfromaccumulationofdGTPanddATP,Increasedexcretionoforoticacid,uracil,anduridinewhichinhibitribonucleotidereductaseandtherebyde-accompaniesadeficiencyinlivermitochondrialor-pletecellsofDNAprecursors.nithinetranscarbamoylase(reaction2,Figure29–9).
299METABOLISMOFPURINE&PYRIMIDINENUCLEOTIDES/301NH2Excesscarbamoylphosphateexitstothecytosol,whereitstimulatespyrimidinenucleotidebiosynthesis.TheNresultingmildoroticaciduriaisincreasedbyhigh-nitrogenfoods.ONHCytosineDrugsMayPrecipitateOroticAciduria1/2OAllopurinol(Figure33–12),analternativesubstratefor2orotatephosphoribosyltransferase(reaction5,Figure34–7),competeswithoroticacid.Theresultingnu-NH3Ocleotideproductalsoinhibitsorotidylatedecarboxylase(reaction6,Figure34–7),resultinginoroticaciduriaOHNCH3andorotidinuria.6-Azauridine,followingconversionHNto6-azauridylate,alsocompetitivelyinhibitsorotidylateONdecarboxylase(reaction6,Figure34–7),enhancingex-ONHcretionoforoticacidandorotidine.HThymineUracilNADPH+H+SUMMARY•IngestednucleicacidsaredegradedtopurinesandNADP+pyrimidines.NewpurinesandpyrimidinesareformedfromamphibolicintermediatesandthusareOdietarilynonessential.OCH•SeveralreactionsofIMPbiosynthesisrequirefolateHNHHN3HHderivativesandglutamine.Consequently,antifolateHHdrugsandglutamineanalogsinhibitpurinebiosyn-ONHONHthesis.HHDihydrouracilDihydrothymine•OxidationandaminationofIMPformsAMPandGMP,andsubsequentphosphoryltransferfromH2OH2OATPformsADPandGDP.FurtherphosphoryltransferfromATPtoGDPformsGTP.ADPiscon-vertedtoATPbyoxidativephosphorylation.Reduc-COO−COO−tionofNDPsformsdNDPs.H2NCH2HNCCH3•Hepaticpurinenucleotidebiosynthesisisstringently2CCHHregulatedbythepoolsizeofPRPPandbyfeedback2CCH2ONONinhibitionofPRPP-glutamylamidotransferasebyHHAMPandGMP.β-Ureidopropionateβ-Ureidoisobutyrate(N-carbamoyl-β-alanine)(N-carbamoyl-β-amino-•Coordinatedregulationofpurineandpyrimidineisobutyrate)nucleotidebiosynthesisensurestheirpresenceinpro-portionsappropriatefornucleicacidbiosynthesisandothermetabolicneeds.CO+NH•Humanscatabolizepurinestouricacid(pKa5.8),23presentastherelativelyinsolubleacidatacidicpHor+−+−asitsmoresolublesodiumuratesaltatapHnearH3NCH2CH2COOH3NCH2CHCOOneutrality.Uratecrystalsarediagnosticofgout.β-AlanineCH3OtherdisordersofpurinecatabolismincludeLesch-β-AminoisobutyrateNyhansyndrome,vonGierke’sdisease,andhypo-uricemias.Figure34–9.Catabolismofpyrimidines.•Sincepyrimidinecatabolitesarewater-soluble,theiroverproductiondoesnotresultinclinicalabnormali-ties.Excretionofpyrimidineprecursorscan,how-ever,resultfromadeficiencyofornithinetranscar-bamoylasebecauseexcesscarbamoylphosphateisavailableforpyrimidinebiosynthesis.
300302/CHAPTER34REFERENCESMartinezJetal:Humangeneticdisorders,aphylogeneticperspec-tive.JMolBiol2001;308:587.BenkovicSJ:ThetransformylaseenzymesindenovopurinePuigJGetal:Gout:newquestionsforanancientdisease.AdvExpbiosynthesis.TrendsBiochemSci1994;9:320.MedBiol1998;431:1.BrooksEMetal:Moleculardescriptionofthreemacro-deletionsScriverCRetal(editors):TheMetabolicandMolecularBasesofIn-andanAlu-Alurecombination-mediatedduplicationintheheritedDisease,8thed.McGraw-Hill,2001.HPRTgeneinfourpatientswithLesch-Nyhandisease.TvrdikTetal:MolecularcharacterizationoftwodeletioneventsMutatRes2001;476:43.involvingAlu-sequences,onenovelbasesubstitutionandtwoCurtoR,VoitEO,CascanteM:Analysisofabnormalitiesinpurinetentativehotspotmutationsinthehypoxanthinephosphori-metabolismleadingtogoutandtoneurologicaldysfunctionsbosyltransferasegeneinfivepatientswithLesch-Nyhan-inman.BiochemJ1998;329:477.syndrome.HumGenet1998;103:311.HarrisMD,SiegelLB,AllowayJA:Goutandhyperuricemia.AmZalkinH,DixonJE:Denovopurinenucleotidesynthesis.ProgFamilyPhysician1999;59:925.NucleicAcidResMolBiol1992;42:259.LipkowitzMSetal:Functionalreconstitution,membranetarget-ing,genomicstructure,andchromosomallocalizationofahumanuratetransporter.JClinInvest2001;107:1103.
301NucleicAcidStructure&Function35DarylK.Granner,MDBIOMEDICALIMPORTANCETheinformationalcontentofDNA(thegeneticcode)residesinthesequenceinwhichthesemonomers—Thediscoverythatgeneticinformationiscodedalongpurineandpyrimidinedeoxyribonucleotides—areor-thelengthofapolymericmoleculecomposedofonlydered.Thepolymerasdepictedpossessesapolarity;fourtypesofmonomericunitswasoneofthemajorsci-oneendhasa5′-hydroxylorphosphateterminalwhileentificachievementsofthetwentiethcentury.Thistheotherhasa3′-phosphateorhydroxylterminal.Thepolymericmolecule,DNA,isthechemicalbasisofimportanceofthispolaritywillbecomeevident.Sinceheredityandisorganizedintogenes,thefundamentalthegeneticinformationresidesintheorderoftheunitsofgeneticinformation.Thebasicinformationmonomericunitswithinthepolymers,theremustexistpathway—ie,DNAdirectsthesynthesisofRNA,amechanismofreproducingorreplicatingthisspecificwhichinturndirectsproteinsynthesis—hasbeeneluci-informationwithahighdegreeoffidelity.Thatre-dated.Genesdonotfunctionautonomously;theirquirement,togetherwithx-raydiffractiondatafromreplicationandfunctionarecontrolledbyvariousgenetheDNAmoleculeandtheobservationofChargaffproducts,oftenincollaborationwithcomponentsofthatinDNAmoleculestheconcentrationofde-varioussignaltransductionpathways.Knowledgeoftheoxyadenosine(A)nucleotidesequalsthatofthymidinestructureandfunctionofnucleicacidsisessentialin(T)nucleotides(A=T),whiletheconcentrationofde-understandinggeneticsandmanyaspectsofpathophys-oxyguanosine(G)nucleotidesequalsthatofdeoxycyti-iologyaswellasthegeneticbasisofdisease.dine(C)nucleotides(G=C),ledWatson,Crick,andWilkinstoproposeintheearly1950samodelofadou-DNACONTAINSTHEble-strandedDNAmolecule.ThemodeltheyproposedGENETICINFORMATIONisdepictedinFigure35–2.Thetwostrandsofthisdouble-strandedhelixareheldinregisterbyhydrogenThedemonstrationthatDNAcontainedthegeneticin-bondsbetweenthepurineandpyrimidinebasesoftheformationwasfirstmadein1944inaseriesofexperi-respectivelinearmolecules.ThepairingsbetweenthementsbyAvery,MacLeod,andMcCarty.Theyshowedpurineandpyrimidinenucleotidesontheoppositethatthegeneticdeterminationofthecharacter(type)ofstrandsareveryspecificandaredependentuponhydro-thecapsuleofaspecificpneumococcuscouldbetrans-genbondingofAwithTandGwithC(Figure35–3).mittedtoanotherofadifferentcapsulartypebyintro-ThiscommonformofDNAissaidtoberight-ducingpurifiedDNAfromtheformercoccusintothehandedbecauseasonelooksdownthedoublehelixthelatter.Theseauthorsreferredtotheagent(latershownbaseresiduesformaspiralinaclockwisedirection.IntobeDNA)accomplishingthechangeas“transformingthedouble-strandedmolecule,restrictionsimposedbyfactor.”Subsequently,thistypeofgeneticmanipulationtherotationaboutthephosphodiesterbond,thefa-hasbecomecommonplace.Similarexperimentshavevoredanticonfigurationoftheglycosidicbond(Figurerecentlybeenperformedutilizingyeast,culturedmam-33–8),andthepredominanttautomers(seeFiguremaliancells,andinsectandmammalianembryosasre-33–3)ofthefourbases(A,G,T,andC)allowAtopaircipientsandclonedDNAasthedonorofgeneticinfor-onlywithTandGonlywithC,asdepictedinFiguremation.35–3.Thisbase-pairingrestrictionexplainstheearlierobservationthatinadouble-strandedDNAmoleculeDNAContainsFourDeoxynucleotidesthecontentofAequalsthatofTandthecontentofGequalsthatofC.Thetwostrandsofthedouble-helicalThechemicalnatureofthemonomericdeoxynucleo-molecule,eachofwhichpossessesapolarity,arean-tideunitsofDNA—deoxyadenylate,deoxyguanylate,tiparallel;ie,onestrandrunsinthe5′to3′directiondeoxycytidylate,andthymidylate—isdescribedinandtheotherinthe3′to5′direction.ThisisanalogousChapter33.ThesemonomericunitsofDNAareheldtotwoparallelstreets,eachrunningonewaybutcarry-inpolymericformby3′,5′-phosphodiesterbridgescon-ingtrafficinoppositedirections.Inthedouble-stitutingasinglestrand,asdepictedinFigure35–1.strandedDNAmolecules,thegeneticinformationre-303
302304/CHAPTER35ONNH5′GCHNNH22NNH2ONOCPOOHHCH2NHHH3CONHHOTPOHHCH2NNH2HHONONOHAPHHCH2NNHHOOOHPHHHHO3′HPOFigure35–1.AsegmentofonestrandofaDNAmoleculeinwhichthepurineandpyrimidinebasesguanine(G),cytosine(C),thymine(T),andadenine(A)areheldtogetherbyaphosphodiesterbackbonebetween2′-de-oxyribosylmoietiesattachedtothenucleobasesbyanN-glycosidicbond.Notethatthebackbonehasapolarity(ie,adirection).Conventiondictatesthatasingle-strandedDNAsequenceiswritteninthe5′to3′direction(ie,pGpCpTpA,whereG,C,T,andArepresentthefourbasesandprepresentstheinterconnectingphosphates).sidesinthesequenceofnucleotidesononestrand,thedinenucleotide,whereastheotherpair,theA–Tpair,istemplatestrand.ThisisthestrandofDNAthatisheldtogetherbytwohydrogenbonds.Thus,theG–Ccopiedduringnucleicacidsynthesis.Itissometimesre-bondsaremuchmoreresistanttodenaturation,orferredtoasthenoncodingstrand.Theoppositestrand“melting,”thanA–T-richregions.isconsideredthecodingstrandbecauseitmatchestheRNAtranscriptthatencodestheprotein.TheDenaturation(Melting)ofDNAThetwostrands,inwhichopposingbasesareheldIsUsedtoAnalyzeItsStructuretogetherbyhydrogenbonds,windaroundacentralaxisintheformofadoublehelix.Double-strandedDNAThedouble-strandedstructureofDNAcanbesepa-existsinatleastsixforms(A–EandZ).TheBformisratedintotwocomponentstrands(melted)insolutionusuallyfoundunderphysiologicconditions(lowsalt,byincreasingthetemperatureordecreasingthesalthighdegreeofhydration).AsingleturnofB-DNAconcentration.Notonlydothetwostacksofbasespullabouttheaxisofthemoleculecontainstenbasepairs.apartbutthebasesthemselvesunstackwhilestillcon-ThedistancespannedbyoneturnofB-DNAis3.4nectedinthepolymerbythephosphodiesterbackbone.nm.Thewidth(helicaldiameter)ofthedoublehelixinConcomitantwiththisdenaturationoftheDNAmole-B-DNAis2nm.culeisanincreaseintheopticalabsorbanceoftheAsdepictedinFigure35–3,threehydrogenbondspurineandpyrimidinebases—aphenomenonreferredholdthedeoxyguanosinenucleotidetothedeoxycyti-toashyperchromicityofdenaturation.Becauseofthe
303NUCLEICACIDSTRUCTURE&FUNCTION/305CH3OHHNNNHNONThymidineMinorgrooveNNSATSoAdenosinePP34ASTASPPSCGSHPPSGCSNMajorgrooveHNNOHNNOHCytosineNNNHGuanosineo20AFigure35–3.BasepairingbetweendeoxyadenosineFigure35–2.AdiagrammaticrepresentationoftheandthymidineinvolvestheformationoftwohydrogenWatsonandCrickmodelofthedouble-helicalstructurebonds.ThreesuchbondsformbetweendeoxycytidineoftheBformofDNA.Thehorizontalarrowindicatesanddeoxyguanosine.Thebrokenlinesrepresenthy-thewidthofthedoublehelix(20Å),andtheverticaldrogenbonds.arrowindicatesthedistancespannedbyonecompleteturnofthedoublehelix(34Å).OneturnofB-DNAin-cludestenbasepairs(bp),sotheriseis3.4Åperbp.orDNA-RNAhybridstobeseparatedatmuchlowerThecentralaxisofthedoublehelixisindicatedbythetemperaturesandminimizesthephosphodiesterbondverticalrod.Theshortarrowsdesignatethepolarityofbreakagethatoccursathightemperatures.theantiparallelstrands.Themajorandminorgroovesaredepicted.(A,adenine;C,cytosine;G,guanine;RenaturationofDNARequiresT,thymine;P,phosphate;S,sugar[deoxyribose].)BasePairMatchingSeparatedstrandsofDNAwillrenatureorreassociatewhenappropriatephysiologictemperatureandsaltcon-stackingofthebasesandthehydrogenbondingbe-ditionsareachieved.Therateofreassociationdependstweenthestacks,thedouble-strandedDNAmoleculeupontheconcentrationofthecomplementarystrands.exhibitspropertiesofarigidrodandinsolutionisavis-ReassociationofthetwocomplementaryDNAstrandscousmaterialthatlosesitsviscosityupondenaturation.ofachromosomeafterDNAreplicationisaphysiologicThestrandsofagivenmoleculeofDNAseparateexampleofrenaturation(seebelow).Atagiventemper-overatemperaturerange.Themidpointiscalledtheatureandsaltconcentration,aparticularnucleicacidmeltingtemperature,orTm.TheTmisinfluencedbystrandwillassociatetightlyonlywithacomplementarythebasecompositionoftheDNAandbythesaltcon-strand.Hybridmoleculeswillalsoformunderappro-centrationofthesolution.DNArichinG–Cpairs,priateconditions.Forexample,DNAwillformahy-whichhavethreehydrogenbonds,meltsatahighertem-bridwithacomplementaryDNA(cDNA)orwithaperaturethanthatrichinA–Tpairs,whichhavetwohy-cognatemessengerRNA(mRNA;seebelow).Whendrogenbonds.Atenfoldincreaseofmonovalentcationcombinedwithgelelectrophoresistechniquesthatsepa-concentrationincreasestheTmby16.6°C.Formamide,ratehybridmoleculesbysizeandradioactivelabelingtowhichiscommonlyusedinrecombinantDNAexperi-provideadetectablesignal,theresultinganalytictech-ments,destabilizeshydrogenbondingbetweenbases,niquesarecalledSouthern(DNA/cDNA)andNorth-therebyloweringtheTm.ThisallowsthestrandsofDNAernblotting(DNA/RNA),respectively.Theseproce-
304306/CHAPTER35duresallowforveryspecificidentificationofhybridsthecellandorganism,anditprovidestheinformationfrommixturesofDNAorRNA(seeChapter40).inheritedbydaughtercellsoroffspring.BothofthesefunctionsrequirethattheDNAmoleculeserveasaThereAreGroovesintheDNAMoleculetemplate—inthefirstcaseforthetranscriptionoftheinformationintoRNAandinthesecondcasefortheCarefulexaminationofthemodeldepictedinFigurereplicationoftheinformationintodaughterDNAmol-35–2revealsamajorgrooveandaminorgroovewind-ecules.ingalongthemoleculeparalleltothephosphodiesterThecomplementarityoftheWatsonandCrickdou-backbones.Inthesegrooves,proteinscaninteractspecif-ble-strandedmodelofDNAstronglysuggeststhaticallywithexposedatomsofthenucleotides(usuallybyreplicationoftheDNAmoleculeoccursinasemicon-Hbonding)andtherebyrecognizeandbindtospecificservativemanner.Thus,wheneachstrandofthedou-nucleotidesequenceswithoutdisruptingthebasepair-ble-strandedparentalDNAmoleculeseparatesfromitsingofthedouble-helicalDNAmolecule.Asdiscussedincomplementduringreplication,eachservesasatem-Chapters37and39,regulatoryproteinscontroltheex-plateonwhichanewcomplementarystrandissynthe-pressionofspecificgenesviasuchinteractions.sized(Figure35–4).Thetwonewlyformeddouble-strandeddaughterDNAmolecules,eachcontainingDNAExistsinRelaxedonestrand(butcomplementaryratherthanidentical)&SupercoiledFormsfromtheparentdouble-strandedDNAmolecule,arethensortedbetweenthetwodaughtercells(FigureInsomeorganismssuchasbacteria,bacteriophages,and35–5).EachdaughtercellcontainsDNAmoleculesmanyDNA-containinganimalviruses,theendsofthewithinformationidenticaltothatwhichtheparentDNAmoleculesarejoinedtocreateaclosedcirclewithpossessed;yetineachdaughtercelltheDNAmoleculenocovalentlyfreeends.Thisofcoursedoesnotdestroyoftheparentcellhasbeenonlysemiconserved.thepolarityofthemolecules,butiteliminatesallfree3′and5′hydroxylandphosphorylgroups.Closedcirclesexistinrelaxedorsupercoiledforms.Supercoilsareintro-THECHEMICALNATUREOFRNADIFFERSducedwhenaclosedcircleistwistedarounditsownaxisFROMTHATOFDNAorwhenalinearpieceofduplexDNA,whoseendsarefixed,istwisted.Thisenergy-requiringprocessputstheRibonucleicacid(RNA)isapolymerofpurineandmoleculeunderstress,andthegreaterthenumberofsu-pyrimidineribonucleotideslinkedtogetherby3′,5′-percoils,thegreaterthestressortorsion(testthisbyphosphodiesterbridgesanalogoustothoseinDNAtwistingarubberband).Negativesupercoilsareformed(Figure35–6).AlthoughsharingmanyfeatureswithwhenthemoleculeistwistedinthedirectionoppositeDNA,RNApossessesseveralspecificdifferences:fromtheclockwiseturnsoftheright-handeddoublehelixfoundinB-DNA.SuchDNAissaidtobeunder-(1)InRNA,thesugarmoietytowhichthephos-wound.Theenergyrequiredtoachievethisstateis,inaphatesandpurineandpyrimidinebasesareattachedissense,storedinthesupercoils.Thetransitiontoanotherriboseratherthanthe2′-deoxyriboseofDNA.formthatrequiresenergyistherebyfacilitatedbytheun-(2)ThepyrimidinecomponentsofRNAdifferfromderwinding.Onesuchtransitionisstrandseparation,thoseofDNA.AlthoughRNAcontainstheribonu-whichisaprerequisiteforDNAreplicationandtran-cleotidesofadenine,guanine,andcytosine,itdoesnotscription.SupercoiledDNAisthereforeapreferredformpossessthymineexceptintherarecasementionedinbiologicsystems.Enzymesthatcatalyzetopologicbelow.Insteadofthymine,RNAcontainstheribonu-changesofDNAarecalledtopoisomerases.Topoisom-cleotideofuracil.erasescanrelaxorinsertsupercoils.Thebest-character-(3)RNAexistsasasinglestrand,whereasDNAex-izedexampleisbacterialgyrase,whichinducesnegativeistsasadouble-strandedhelicalmolecule.However,supercoilinginDNAusingATPasenergysource.Ho-giventhepropercomplementarybasesequencewithmologsofthisenzymeexistinallorganismsandareim-oppositepolarity,thesinglestrandofRNA—asportanttargetsforcancerchemotherapy.demonstratedinFigure35–7—iscapableoffoldingbackonitselflikeahairpinandthusacquiringdouble-DNAPROVIDESATEMPLATEFORstrandedcharacteristics.(4)SincetheRNAmoleculeisasinglestrandcom-REPLICATION&TRANSCRIPTIONplementarytoonlyoneofthetwostrandsofagene,itsThegeneticinformationstoredinthenucleotidese-guaninecontentdoesnotnecessarilyequalitscytosinequenceofDNAservestwopurposes.Itisthesourceofcontent,nordoesitsadeninecontentnecessarilyequalinformationforthesynthesisofallproteinmoleculesofitsuracilcontent.
305NUCLEICACIDSTRUCTURE&FUNCTION/307GCOLDOLD5′GC3′GCOriginalCGparentmoleculeATAATGCGCAFirst-generationATdaughtermoleculesTAGCGC3′5′TTAACGGCCCSecond-generationdaughtermoleculesATATATTATAATGCCGFigure35–5.DNAreplicationissemiconservative.GGDuringaroundofreplication,eachofthetwostrandsATATofDNAisusedasatemplateforsynthesisofanew,complementarystrand.3′TA5′3′TA5′OLDNEWNEWOLDTATAFigure35–4.Thedouble-strandedstructureofDNAandthetemplatefunctionofeacholdstrand(darkcomplementarity,anRNAmoleculecanbindspecifi-shading)onwhichanew(lightshading)complemen-callyviathebase-pairingrulestoitstemplateDNAtarystrandissynthesized.strand;itwillnotbind(“hybridize”)withtheother(coding)strandofitsgene.ThesequenceoftheRNAmolecule(exceptforUreplacingT)isthesameasthatofthecodingstrandofthegene(Figure35–8).(5)RNAcanbehydrolyzedbyalkalito2′,3′cyclicdiestersofthemononucleotides,compoundsthatcan-NearlyAlloftheSeveralSpeciesofRNAnotbeformedfromalkali-treatedDNAbecauseoftheAreInvolvedinSomeAspectofProteinabsenceofa2′-hydroxylgroup.ThealkalilabilityofSynthesisRNAisusefulbothdiagnosticallyandanalytically.ThosecytoplasmicRNAmoleculesthatserveastem-InformationwithinthesinglestrandofRNAiscon-platesforproteinsynthesis(ie,thattransfergeneticin-tainedinitssequence(“primarystructure”)ofpurineformationfromDNAtotheprotein-synthesizingma-andpyrimidinenucleotideswithinthepolymer.Thechinery)aredesignatedmessengerRNAs,ormRNAs.sequenceiscomplementarytothetemplatestrandofManyothercytoplasmicRNAmolecules(ribosomalthegenefromwhichitwastranscribed.BecauseofthisRNAs;rRNAs)havestructuralroleswhereintheycon-
306308/CHAPTER35ONNH5′GCH2NNHNH2N2ONOCPOOHHCH2NHHONHHOOUPOHHCH2NNH2HHONONOHOAPHHCH2NNHHOOOHOPHHHHO3′HOPOFigure35–6.Asegmentofaribonucleicacid(RNA)moleculeinwhichthepurineandpyrimidinebases—guanine(G),cytosine(C),uracil(U),andadenine(A)—areheldtogetherbyphosphodiesterbondsbetweenribo-sylmoietiesattachedtothenucleobasesbyN-glycosidicbonds.Notethatthepolymerhasapolarityasindi-catedbythelabeled3′-and5′-attachedphosphates.tributetotheformationandfunctionofribosomes(theThegeneticmaterialforsomeanimalandplantorganellarmachineryforproteinsynthesis)orserveasvirusesisRNAratherthanDNA.AlthoughsomeRNAadaptermolecules(transferRNAs;tRNAs)forthevirusesneverhavetheirinformationtranscribedintoatranslationofRNAinformationintospecificsequencesDNAmolecule,manyanimalRNAviruses—specifi-ofpolymerizedaminoacids.cally,theretroviruses(theHIVvirus,forexample)—areSomeRNAmoleculeshaveintrinsiccatalyticactiv-transcribedbyanRNA-dependentDNApolymerase,ity.Theactivityoftheseribozymesofteninvolvesthetheso-calledreversetranscriptase,toproduceadou-cleavageofanucleicacid.Anexampleistheroleofble-strandedDNAcopyoftheirRNAgenome.InRNAincatalyzingtheprocessingoftheprimarytran-manycases,theresultingdouble-strandedDNAtran-scriptofageneintomaturemessengerRNA.scriptisintegratedintothehostgenomeandsubse-MuchoftheRNAsynthesizedfromDNAtemplatesquentlyservesasatemplateforgeneexpressionandineukaryoticcells,includingmammaliancells,isde-fromwhichnewviralRNAgenomescanbetran-gradedwithinthenucleus,anditneverservesaseitherascribed.structuraloraninformationalentitywithinthecellularcytoplasm.RNAIsOrganizedinSeveralInalleukaryoticcellstherearesmallnuclearRNAUniqueStructures(snRNA)speciesthatarenotdirectlyinvolvedinpro-teinsynthesisbutplaypivotalrolesinRNAprocessing.Inallprokaryoticandeukaryoticorganisms,threemainTheserelativelysmallmoleculesvaryinsizefrom90toclassesofRNAmoleculesexist:messengerRNAabout300nucleotides(Table35–1).(mRNA),transferRNA(tRNA),andribosomalRNA
307NUCLEICACIDSTRUCTURE&FUNCTION/309Table35–1.SomeofthespeciesofsmallstableRNAsfoundinmammaliancells.LoopLengthMoleculesCGName(nucleotides)perCellLocalizationCGU11651×106Nucleoplasm/hnRNAGCU21885×105NucleoplasmAUU32163×105NucleolusAUU41391×105NucleoplasmAUU51182×105NucleoplasmUGU61063×105PerichromatingranulesUG4.5S91–953x105NucleusandcytoplasmStemCC7S2805×105NucleusandcytoplasmGC57-22901×10NucleusandcytoplasmUA57-33002×10NucleusUAUCUAMessengerRNAs,particularlyineukaryotes,haveCGsomeuniquechemicalcharacteristics.The5′terminalGCofmRNAis“capped”bya7-methylguanosinetriphos-phatethatislinkedtoanadjacent2′-O-methylribonu-cleosideatits5′-hydroxylthroughthethreephosphates5′3′(Figure35–10).ThemRNAmoleculesfrequentlycon-taininternal6-methyladenylatesandother2′-O-riboseFigure35–7.Diagrammaticrepresentationofthemethylatednucleotides.Thecapisinvolvedinthesecondarystructureofasingle-strandedRNAmoleculerecognitionofmRNAbythetranslatingmachinery,inwhichastemloop,or“hairpin,”hasbeenformedandanditprobablyhelpsstabilizethemRNAbypreventingisdependentupontheintramolecularbasepairing.theattackof5′-exonucleases.Theprotein-synthesizingNotethatAformshydrogenbondswithUinRNA.machinerybeginstranslatingthemRNAintoproteinsbeginningdownstreamofthe5′orcappedterminal.TheotherendofmostmRNAmolecules,the3′-hy-(rRNA).Eachdiffersfromtheothersbysize,function,droxylterminal,hasanattachedpolymerofadenylateandgeneralstability.residues20–250nucleotidesinlength.Thespecificfunctionofthepoly(A)“tail”atthe3′-hydroxyltermi-nalofmRNAsisnotfullyunderstood,butitseemsthatA.MESSENGERRNA(MRNA)itmaintainstheintracellularstabilityofthespecificThisclassisthemostheterogeneousinsizeandstabil-mRNAbypreventingtheattackof3′-exonucleases.ity.AllmembersoftheclassfunctionasmessengersSomemRNAs,includingthoseforsomehistones,doconveyingtheinformationinagenetotheprotein-notcontainpoly(A).Thepoly(A)tail,becauseitwillsynthesizingmachinery,whereeachservesasatemplateformabasepairwitholigodeoxythymidinepolymersonwhichaspecificsequenceofaminoacidsispolymer-attachedtoasolidsubstratelikecellulose,canbeusedizedtoformaspecificproteinmolecule,theultimatetoseparatemRNAfromotherspeciesofRNA,includ-geneproduct(Figure35–9).ingmRNAmoleculesthatlackthistail.DNAstrands:Coding5′—TGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATG—3′Template3′—ACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGATACTGGTAC—5′RNA5′pAUUGUGAGCGGAUAACAAUUUCACACAGGAAACAGCUAUGACCAUG3′transcriptFigure35–8.TherelationshipbetweenthesequencesofanRNAtranscriptanditsgene,inwhichthecod-ingandtemplatestrandsareshownwiththeirpolarities.TheRNAtranscriptwitha5′to3′polarityiscomple-mentarytothetemplatestrandwithits3′to5′polarity.NotethatthesequenceintheRNAtranscriptanditspolarityisthesameasthatinthecodingstrand,exceptthattheUofthetranscriptreplacestheTofthegene.
308310/CHAPTER35DNA5′3′3′5′mRNA5′3′ProteinsynthesisonmRNAtemplate3′5′Figure35–9.Theexpressionofgeneticin-CompletedRibosomeproteinformationinDNAintotheformofanmRNAmoleculetranscript.Thisissubsequentlytranslatedbyribosomesintoaspecificproteinmolecule.Inmammaliancells,includingcellsofhumans,theTheD,TC,andextraarmshelpdefineaspecificmRNAmoleculespresentinthecytoplasmarenotthetRNA.RNAproductsimmediatelysynthesizedfromtheDNAAlthoughtRNAsarequitestableinprokaryotes,theytemplatebutmustbeformedbyprocessingfromapre-aresomewhatlessstableineukaryotes.Theoppositeiscursormoleculebeforeenteringthecytoplasm.Thus,trueformRNAs,whicharequiteunstableinprokary-inmammaliannuclei,theimmediateproductsofgeneotesbutgenerallystableineukaryoticorganisms.transcriptionconstituteafourthclassofRNAmole-C.RIBOSOMALRNA(RRNA)cules.ThesenuclearRNAmoleculesareveryheteroge-neousinsizeandarequitelarge.TheheterogeneousAribosomeisacytoplasmicnucleoproteinstructurenuclearRNA(hnRNA)moleculesmayhaveamolecu-thatactsasthemachineryforthesynthesisofproteinslarweightinexcessof107,whereasthemolecularfromthemRNAtemplates.Ontheribosomes,theweightofmRNAmoleculesisgenerallylessthan2×mRNAandtRNAmoleculesinteracttotranslateintoa106.AsdiscussedinChapter37,hnRNAmoleculesarespecificproteinmoleculeinformationtranscribedfromprocessedtogeneratethemRNAmoleculeswhichthenthegene.Inactiveproteinsynthesis,manyribosomesenterthecytoplasmtoserveastemplatesforproteinareassociatedwithanmRNAmoleculeinanassemblysynthesis.calledthepolysome.Thecomponentsofthemammalianribosome,B.TRANSFERRNA(TRNA)whichhasamolecularweightofabout4.2×106andatRNAmoleculesvaryinlengthfrom74to95nu-sedimentationvelocityof80S(Svedbergunits),arecleotides.TheyalsoaregeneratedbynuclearprocessingshowninTable35–2.Themammalianribosomecon-ofaprecursormolecule(Chapter37).ThetRNAmole-tainstwomajornucleoproteinsubunits—alargeroneculesserveasadaptersforthetranslationoftheinfor-6withamolecularweightof2.8×10(60S)andamationinthesequenceofnucleotidesofthemRNA6smallersubunitwithamolecularweightof1.4×10intospecificaminoacids.Thereareatleast20species(40S).The60Ssubunitcontainsa5SribosomalRNAoftRNAmoleculesineverycell,atleastone(andoften(rRNA),a5.8SrRNA,anda28SrRNA;therearealsoseveral)correspondingtoeachofthe20aminoacidsre-probablymorethan50specificpolypeptides.The40Squiredforproteinsynthesis.Althougheachspecificsubunitissmallerandcontainsasingle18SrRNAandtRNAdiffersfromtheothersinitssequenceofnu-approximately30distinctpolypeptidechains.Allofthecleotides,thetRNAmoleculesasaclasshavemanyfea-ribosomalRNAmoleculesexceptthe5SrRNAareturesincommon.Theprimarystructure—ie,thenu-processedfromasingle45SprecursorRNAmoleculeincleotidesequence—ofalltRNAmoleculesallowsthenucleolus(Chapter37).5SrRNAisindependentlyextensivefoldingandintrastrandcomplementaritytotranscribed.ThehighlymethylatedribosomalRNAgenerateasecondarystructurethatappearslikeamoleculesarepackagedinthenucleoluswiththespe-cloverleaf(Figure35–11).cificribosomalproteins.Inthecytoplasm,theribo-AlltRNAmoleculescontainfourmainarms.Thesomesremainquitestableandcapableofmanytransla-acceptorarmterminatesinthenucleotidesCpCpAOH.tioncycles.ThefunctionsoftheribosomalRNAThesethreenucleotidesareaddedposttranscription-moleculesintheribosomalparticlearenotfullyunder-ally.ThetRNA-appropriateaminoacidisattachedtostood,buttheyarenecessaryforribosomalassemblythe3′-OHgroupoftheAmoietyoftheacceptorarm.andseemtoplaykeyrolesinthebindingofmRNAto
309NUCLEICACIDSTRUCTURE&FUNCTION/311OHOHCCHHHCCHOH2NNNHC5′O–NH22OPONHNONNO–OPO5′OCH3O–PCH2NNOOOCAPHCCHHH2′3′CCOOCH3NH5′OOCH2NOPOOmRNAO–HCCHHH3′CCOHOPOO–Figure35–10.Thecapstructureattachedtothe5′terminalofmosteukaryoticmessen-gerRNAmolecules.A7-methylguanosinetriphosphate(black)isattachedatthe5′terminalofthemRNA(showninblue),whichusuallycontainsa2′-O-methylpurinenucleotide.Thesemodifications(thecapandmethylgroup)areaddedafterthemRNAistranscribedfromDNA.ribosomesanditstranslation.Recentstudiessuggestsizefrom90to300nucleotidesandarepresentinthatanrRNAcomponentperformsthepeptidyltrans-100,000–1,000,000copiespercell.feraseactivityandthusisanenzyme(aribozyme).SmallnuclearRNAs(snRNAs),asubsetoftheseRNAs,aresignificantlyinvolvedinmRNAprocessingandgeneregulation.OftheseveralsnRNAs,U1,U2,D.SMALLSTABLERNAU4,U5,andU6areinvolvedinintronremovalandtheAlargenumberofdiscrete,highlyconserved,andsmallprocessingofhnRNAintomRNA(Chapter37).ThestableRNAspeciesarefoundineukaryoticcells.TheU7snRNAmaybeinvolvedinproductionofthecor-majorityofthesemoleculesarecomplexedwithpro-rect3′endsofhistonemRNA—whichlacksapoly(A)teinstoformribonucleoproteinsandaredistributedintail.TheU4andU6snRNAsmayalsoberequiredforthenucleus,inthecytoplasm,orinboth.Theyrangeinpoly(A)processing.
310312/CHAPTER35aaSPECIFICNUCLEASESDIGESTNUCLEICACIDSEnzymescapableofdegradingnucleicacidshavebeen3′Arecognizedformanyyears.Thesenucleasescanbeclas-CAcceptorsifiedinseveralways.ThosewhichexhibitspecificityarmCfordeoxyribonucleicacidarereferredtoasdeoxyri-bonucleases.Thosewhichspecificallyhydrolyzeri-5′Pbonucleicacidsareribonucleases.WithinbothoftheseclassesareenzymescapableofcleavinginternalRegionofhydrogenphosphodiesterbondstoproduceeither3′-hydroxylbondingbetweenbasepairsand5′-phosphorylterminalsor5′-hydroxyland3′-phosphorylterminals.Thesearereferredtoasendonu-TψCarmcleases.Somearecapableofhydrolyzingbothstrandsofadouble-strandedmolecule,whereasotherscanonlycleavesinglestrandsofnucleicacids.Somenucle-Gasescanhydrolyzeonlyunpairedsinglestrands,whileGothersarecapableofhydrolyzingsinglestrandspartici-TCpatingintheformationofadouble-strandedmolecule.DarmψThereexistclassesofendonucleasesthatrecognizespe-ExtraarmcificsequencesinDNA;themajorityofthesearetherestrictionendonucleases,whichhaveinrecentyearsbecomeimportanttoolsinmoleculargeneticsandmed-UAlkylatedpurineicalsciences.Alistofsomecurrentlyrecognizedrestric-tionendonucleasesispresentedinTable40–2.AnticodonarmSomenucleasesarecapableofhydrolyzinganu-cleotideonlywhenitispresentataterminalofamole-Figure35–11.TypicalaminoacyltRNAinwhichthecule;thesearereferredtoasexonucleases.Exonucle-aminoacid(aa)isattachedtothe3′CCAterminal.Theasesactinonedirection(3′→5′or5′→3′)only.Inanticodon,TΨC,anddihydrouracil(D)armsareindi-bacteria,a3′→5′exonucleaseisanintegralpartofthecated,asarethepositionsoftheintramolecularhydro-DNAreplicationmachineryandthereservestoedit—genbondingbetweenthesebasepairs.(FromWatsonorproofread—themostrecentlyaddeddeoxynucleo-JD:MolecularBiologyoftheGene,3rded.Copyright©tideforbase-pairingerrors.1976,1970,1965,byW.A.Benjamin,Inc.,MenloPark,Cali-fornia.)1Table35–2.Componentsofmammalianribosomes.MassProteinRNAComponent(mw)NumberMassSizeMassBases65540Ssubunit1.4×10~357×1018S7×1019006660Ssubunit2.8×10~501×105S35,0001205.8S45,000160628S1.6×1047001Theribosomalsubunitsaredefinedaccordingtotheirsedimentationve-locityinSvedbergunits(40Sor60S).Thistableillustratesthetotalmass(MW)ofeach.Thenumberofuniqueproteinsandtheirtotalmass(MW)andtheRNAcomponentsofeachsubunitinsize(Svedbergunits),mass,andnumberofbasesarelisted.
311NUCLEICACIDSTRUCTURE&FUNCTION/313SUMMARYsis.ThelineararrayofnucleotidesinRNAconsistsofA,G,C,andU,andthesugarmoietyisribose.•DNAconsistsoffourbases—A,G,C,andT—•ThemajorformsofRNAincludemessengerRNAwhichareheldinlineararraybyphosphodiester(mRNA),ribosomalRNA(rRNA),andtransferbondsthroughthe3′and5′positionsofadjacentde-RNA(tRNA).CertainRNAmoleculesactascata-oxyribosemoieties.lysts(ribozymes).•DNAisorganizedintotwostrandsbythepairingofbasesAtoTandGtoConcomplementarystrands.Thesestrandsformadoublehelixaroundacentralaxis.REFERENCES9•The3×10basepairsofDNAinhumansareorga-nizedintothehaploidcomplementof23chromo-GreenR,NollerHF:Ribosomesandtranslation.AnnuRevBio-chem1997;66:689.somes.Theexactsequenceofthese3billionnu-cleotidesdefinestheuniquenessofeachindividual.GuthrieC,PattersonB:SpliceosomalsnRNAs.AnnRevGenet1988;22:387.•DNAprovidesatemplateforitsownreplicationandHuntT:DNAMakesRNAMakesProtein.Elsevier,1983.thusmaintenanceofthegenotypeandforthetran-WatsonJD,CrickFHC:Molecularstructureofnucleicacids.Na-scriptionofthe30,000–50,000genesintoavarietyture1953;171:737.ofRNAmolecules.WatsonJD:TheDoubleHelix.Atheneum,1968.•RNAexistsinseveraldifferentsingle-strandedstruc-WatsonJDetal:MolecularBiologyoftheGene,5thed.Benjamin-tures,mostofwhichareinvolvedinproteinsynthe-Cummings,2000.
312DNAOrganization,Replication,&Repair36DarylK.Granner,MD,&P.AnthonyWeil,PhDBIOMEDICALIMPORTANCE*largerthanhistones)andasmallquantityofRNA.ThenonhistoneproteinsincludeenzymesinvolvedinDNAThegeneticinformationintheDNAofachromosomereplication,suchasDNAtopoisomerases.Alsoin-canbetransmittedbyexactreplicationoritcanbeex-cludedareproteinsinvolvedintranscription,suchaschangedbyanumberofprocesses,includingcrossingtheRNApolymerasecomplex.Thedouble-strandedover,recombination,transposition,andconversion.DNAhelixineachchromosomehasalengththatisTheseprovideameansofensuringadaptabilityanddi-thousandsoftimesthediameterofthecellnucleus.versityfortheorganismbut,whentheseprocessesgoOnepurposeofthemoleculesthatcomprisechro-awry,canalsoresultindisease.Anumberofenzymematin,particularlythehistones,istocondensethesystemsareinvolvedinDNAreplication,alteration,DNA.Electronmicroscopicstudiesofchromatinhaveandrepair.Mutationsareduetoachangeinthebasedemonstrateddensesphericalparticlescallednucleo-sequenceofDNAandmayresultfromthefaultyrepli-somes,whichareapproximately10nmindiametercation,movement,orrepairofDNAandoccurwitha6andconnectedbyDNAfilaments(Figure36–1).Nu-frequencyofaboutoneinevery10celldivisions.Ab-cleosomesarecomposedofDNAwoundaroundacol-normalitiesingeneproducts(eitherinproteinfunctionlectionofhistonemolecules.oramount)canbetheresultofmutationsthatoccurincodingorregulatory-regionDNA.AmutationinaHistonesAretheMostAbundantgermcellwillbetransmittedtooffspring(so-calledver-ticaltransmissionofhereditarydisease).AnumberofChromatinProteinsfactors,includingviruses,chemicals,ultravioletlight,Thehistonesareasmallfamilyofcloselyrelatedbasicandionizingradiation,increasetherateofmutation.proteins.H1histonesaretheonesleasttightlyboundMutationsoftenaffectsomaticcellsandsoarepassedtochromatinFigure36–1)andare,therefore,easilyre-ontosuccessivegenerationsofcells,butonlywithinanmovedwithasaltsolution,afterwhichchromatinbe-organism.Itisbecomingapparentthatanumberofcomessoluble.Theorganizationalunitofthissolublediseases—andperhapsmostcancers—areduetothechromatinisthenucleosome.Nucleosomescontaincombinedeffectsofverticaltransmissionofmutationsfourclassesofhistones:H2A,H2B,H3,andH4.Theaswellashorizontaltransmissionofinducedmutations.structuresofallfourhistones—H2A,H2B,H3,andH4,theso-calledcorehistonesformingthenucleo-CHROMATINISTHECHROMOSOMALsome—havebeenhighlyconservedbetweenspecies.MATERIALEXTRACTEDFROMNUCLEIThisextremeconservationimpliesthatthefunctionofOFCELLSOFEUKARYOTICORGANISMShistonesisidenticalinalleukaryotesandthattheentiremoleculeisinvolvedquitespecificallyincarryingoutChromatinconsistsofverylongdouble-strandedDNAthisfunction.Thecarboxylterminaltwo-thirdsofthemoleculesandanearlyequalmassofrathersmallbasicmoleculeshaveatypicalrandomaminoacidcomposi-proteinstermedhistonesaswellasasmalleramountoftion,whiletheiraminoterminalthirdsareparticularlynonhistoneproteins(mostofwhichareacidicandrichinbasicaminoacids.Thesefourcorehistonesaresubjecttoatleastfivetypesofcovalentmodifica-tion:acetylation,methylation,phosphorylation,ADP-ribosylation,andcovalentlinkage(H2Aonly)toubiq-*Sofarasispossible,thediscussioninthischapterandinChaptersuitin.Thesehistonemodificationsprobablyplayan37,38,and39willpertaintomammalianorganisms,whichare,ofimportantroleinchromatinstructureandfunctionascourse,amongthehighereukaryotes.AttimesitwillbenecessaryillustratedinTable36–1.torefertoobservationsinprokaryoticorganismssuchasbacteriaandviruses,butinsuchcasestheinformationwillbeofakindthatThehistonesinteractwitheachotherinveryspecificcanbeextrapolatedtomammalianorganisms.ways.H3andH4formatetramercontainingtwomol-314
313DNAORGANIZATION,REPLICATION,&REPAIR/315Inthenucleosome,theDNAissupercoiledinaleft-handedhelixoverthesurfaceofthedisk-shapedhistoneoctamer(Figure36–2).ThemajorityofcorehistoneproteinsinteractwiththeDNAontheinsideofthesu-percoilwithoutprotruding,thoughtheaminoterminaltailsofallthehistonesprobablyprotrudeoutsideofthisstructureandareavailableforregulatorycovalentmod-ifications(seeTable36–1).The(H3/H4)2tetrameritselfcanconfernucleo-some-likepropertiesonDNAandthushasacentralroleintheformationofthenucleosome.TheadditionoftwoH2A-H2Bdimersstabilizestheprimaryparticleandfirmlybindstwoadditionalhalf-turnsofDNApre-viouslyboundonlylooselytothe(H3/H4)2.Thus,1.75superhelicalturnsofDNAarewrappedaroundthesurfaceofthehistoneoctamer,protecting146basepairsofDNAandformingthenucleosomecoreparticleFigure36–1.Electronmicrographofnucleosomes(Figure36–2).Thecoreparticlesareseparatedbyanattachedbystrandsofnucleicacid.(Thebarrepresentsabout30-bplinkerregionofDNA.MostoftheDNA2.5μm.)(Reproduced,withpermission,fromOudetP,isinarepeatingseriesofthesestructures,givingtheso-Gross-BellardM,ChambonP:Electronmicroscopicandcalled“beads-on-a-string”appearancewhenexaminedbiochemicalevidencethatchromatinstructureisare-byelectronmicroscopy(seeFigure36–1).peatingunit.Cell1975;4:281.)Theassemblyofnucleosomesismediatedbyoneofseveralchromatinassemblyfactorsfacilitatedbyhistonechaperones,proteinssuchastheanionicnuclearproteineculesofeach(H3/H4)2,whileH2AandH2Bformnucleoplasmin.Asthenucleosomeisassembled,his-dimers(H2A-H2B).Underphysiologicconditions,tonesarereleasedfromthehistonechaperones.Nucleo-thesehistoneoligomersassociatetoformthehistoneoc-somesappeartoexhibitpreferenceforcertainregionsontamerofthecomposition(H3/H4)2-(H2A-H2B)2.specificDNAmolecules,butthebasisforthisnonran-domdistribution,termedphasing,isnotcompletelyTheNucleosomeContainsHistone&DNAWhenthehistoneoctamerismixedwithpurified,dou-ble-strandedDNA,thesamex-raydiffractionpatternisformedasthatobservedinfreshlyisolatedchromatin.ElectronmicroscopicstudiesconfirmtheexistenceofHistoneoctamerreconstitutednucleosomes.Furthermore,thereconsti-tutionofnucleosomesfromDNAandhistonesH2A,H2B,H3,andH4isindependentoftheorganismalorcellularoriginofthevariouscomponents.ThehistoneH1andthenonhistoneproteinsarenotnecessaryforthereconstitutionofthenucleosomecore.HistoneDNATable36–1.Possiblerolesofmodifiedhistones.H1Figure36–2.Modelforthestructureofthenucleo-1.AcetylationofhistonesH3andH4isassociatedwiththeac-some,inwhichDNAiswrappedaroundthesurfaceofativationorinactivationofgenetranscription(Chapter37).flatproteincylinderconsistingoftwoeachofhistones2.Acetylationofcorehistonesisassociatedwithchromoso-H2A,H2B,H3,andH4thatformthehistoneoctamer.malassemblyduringDNAreplication.3.PhosphorylationofhistoneH1isassociatedwiththecon-The146basepairsofDNA,consistingof1.75superheli-densationofchromosomesduringthereplicationcycle.calturns,areincontactwiththehistoneoctamer.This4.ADP-ribosylationofhistonesisassociatedwithDNArepair.protectstheDNAfromdigestionbyanuclease.Thepo-5.MethylationofhistonesiscorrelatedwithactivationandsitionofhistoneH1,whenitispresent,isindicatedbyrepressionofgenetranscription.thedashedoutlineatthebottomofthefigure.
314316/CHAPTER36understood.ItisprobablyrelatedtotherelativephysicaltionbyanucleasesuchasDNaseI.DNaseImakessin-flexibilityofcertainnucleotidesequencesthatareabletogle-strandcutsinanysegmentofDNA(nosequenceaccommodatetheregionsofkinkingwithinthesuper-specificity).ItwilldigestDNAnotprotectedbyproteincoilaswellasthepresenceofotherDNA-boundfactorsintoitscomponentdeoxynucleotides.Thesensitivitytothatlimitthesitesofnucleosomedeposition.DNaseIofchromatinregionsbeingactivelytranscribedThesuper-packingofnucleosomesinnucleiisseem-reflectsonlyapotentialfortranscriptionratherthaninglydependentupontheinteractionoftheH1his-transcriptionitselfandinseveralsystemscanbecorre-toneswithadjacentnucleosomes.latedwitharelativelackof5-methyldeoxycytidineintheDNAandparticularhistonecovalentmodificationsHIGHER-ORDERSTRUCTURESPROVIDE(phosphorylation,acetylation,etc;seeTable36–1).FORTHECOMPACTIONOFCHROMATINWithinthelargeregionsofactivechromatinthereexistshorterstretchesof100–300nucleotidesthatex-Electronmicroscopyofchromatinrevealstwohigherhibitanevengreater(anothertenfold)sensitivitytoordersofstructure—the10-nmfibrilandthe30-nmDNaseI.Thesehypersensitivesitesprobablyresultchromatinfiber—beyondthatofthenucleosomeitself.fromastructuralconformationthatfavorsaccessoftheThedisk-likenucleosomestructurehasa10-nmdiame-nucleasetotheDNA.Theseregionsareoftenlocatedterandaheightof5nm.The10-nmfibrilconsistsofimmediatelyupstreamfromtheactivegeneandarethenucleosomesarrangedwiththeiredgesseparatedbyalocationofinterruptednucleosomalstructurecausedbysmalldistance(30bpofDNA)withtheirflatfacespar-thebindingofnonhistoneregulatorytranscriptionfactorallelwiththefibrilaxis(Figure36–3).The10-nmfibrilproteins.(SeeChapters37and39.)Inmanycases,itisprobablyfurthersupercoiledwithsixorsevennucleo-seemsthatifageneiscapableofbeingtranscribed,itsomesperturntoformthe30-nmchromatinfiberveryoftenhasaDNase-hypersensitivesite(s)inthechro-(Figure36–3).Eachturnofthesupercoilisrelativelymatinimmediatelyupstream.Asnotedabove,nonhis-flat,andthefacesofthenucleosomesofsuccessivetoneregulatoryproteinsinvolvedintranscriptioncontrolturnswouldbenearlyparalleltoeachother.H1his-andthoseinvolvedinmaintainingaccesstothetemplatetonesappeartostabilizethe30-nmfiber,buttheirposi-strandleadtotheformationofhypersensitivesites.Hy-tionandthatofthevariablelengthspacerDNAarenotpersensitivesitesoftenprovidethefirstclueabouttheclear.Itisprobablethatnucleosomescanformavarietypresenceandlocationofatranscriptioncontrolelement.ofpackedstructures.Inordertoformamitoticchro-Transcriptionallyinactivechromatinisdenselymosome,the30-nmfibermustbecompactedinlengthpackedduringinterphaseasobservedbyelectronmi-another100-fold(seebelow).croscopicstudiesandisreferredtoasheterochro-Ininterphasechromosomes,chromatinfibersap-matin;transcriptionallyactivechromatinstainslesspeartobeorganizedinto30,000–100,000bploopsordenselyandisreferredtoaseuchromatin.Generally,domainsanchoredinascaffolding(orsupportingma-euchromatinisreplicatedearlierthanheterochromatintrix)withinthenucleus.Withinthesedomains,someinthemammaliancellcycle(seebelow).DNAsequencesmaybelocatednonrandomly.IthasTherearetwotypesofheterochromatin:constitutivebeensuggestedthateachloopeddomainofchromatinandfacultative.Constitutiveheterochromatinisal-correspondstooneormoreseparategeneticfunctions,wayscondensedandthusinactive.Itisfoundinthecontainingbothcodingandnoncodingregionsoftheregionsnearthechromosomalcentromereandatchro-cognategeneorgenes.mosomalends(telomeres).Facultativeheterochro-matinisattimescondensed,butatothertimesitisac-SOMEREGIONSOFCHROMATINAREtivelytranscribedand,thus,uncondensedandappears“ACTIVE”&OTHERSARE“INACTIVE”aseuchromatin.OfthetwomembersoftheXchromo-somepairinmammalianfemales,oneXchromosomeisGenerally,everycellofanindividualmetazoanorganismalmostcompletelyinactivetranscriptionallyandishete-containsthesamegeneticinformation.Thus,thediffer-rochromatic.However,theheterochromaticXchromo-encesbetweencelltypeswithinanorganismmustbeex-somedecondensesduringgametogenesisandbecomesplainedbydifferentialexpressionofthecommongenetictranscriptionallyactiveduringearlyembryogenesis—information.Chromatincontainingactivegenes(ie,thus,itisfacultativeheterochromatin.transcriptionallyactivechromatin)hasbeenshowntoCertaincellsofinsects,eg,Chironomus,containdifferinseveralwaysfromthatofinactiveregions.Thegiantchromosomesthathavebeenreplicatedfortennucleosomestructureofactivechromatinappearstobecycleswithoutseparationofdaughterchromatids.altered,sometimesquiteextensively,inhighlyactivere-ThesecopiesofDNAlineupsidebysideinprecisereg-gions.DNAinactivechromatincontainslargeregionsisterandproduceabandedchromosomecontainingre-(about100,000baseslong)thataresensitivetodiges-gionsofcondensedchromatinandlighterbandsof
315Metaphasechromosome1400nmCondensedloops700nmNuclear-scaffoldassociatedformChromosomescaffoldNon-condensedloops300nm30-nmchromatinfibril30nmcomposedofnucleosomesH1H1Oct“Beads-on-a-string”OctOct10nm10-nmchromatinH1fibrilNakeddouble-helical2nmDNAFigure36–3.ShownistheextentofDNApackaginginmetaphasechromosomes(top)tonotedduplexDNA(bot-tom).ChromosomalDNAispackagedandorganizedatseverallevelsasshown(seeTable36–2).Eachphaseofcon-densationorcompactionandorganization(bottomtotop)decreasesoverallDNAaccessibilitytoanextentthattheDNAsequencesinmetaphasechromosomesarealmosttotallytranscriptionallyinert.Intoto,thesefivelevelsof4DNAcompactionresultinnearlya10-foldlineardecreaseinend-to-endDNAlength.CompletecondensationanddecondensationofthelinearDNAinchromosomesoccurinthespaceofhoursduringthenormalreplicativecellcycle(seeFigure36–20).317
316318/CHAPTER36moreextendedchromatin.Transcriptionallyactivere-sitionofwhichischaracteristicforagivenchromosomegionsofthesepolytenechromosomesareespecially(Figure36–5).Thecentromereisanadenine-thymine2decondensedinto“puffs”thatcanbeshowntocontain(A–T)richregionranginginsizefrom10(brewers’6theenzymesresponsiblefortranscriptionandtobetheyeast)to10(mammals)basepairs.Itbindsseveralpro-sitesofRNAsynthesis(Figure36–4).teinswithhighaffinity.Thiscomplex,calledthekine-tochore,providestheanchorforthemitoticspindle.ItDNAISORGANIZEDthusisanessentialstructureforchromosomalsegrega-INTOCHROMOSOMEStionduringmitosis.TheendsofeachchromosomecontainstructuresAtmetaphase,mammalianchromosomespossessacalledtelomeres.Telomeresconsistofshort,repeattwofoldsymmetry,withtheidenticalduplicatedsisterTG-richsequences.Humantelomereshaveavariablechromatidsconnectedatacentromere,therelativepo-numberofrepeatsofthesequence5′-TTAGGG-3′,whichcanextendforseveralkilobases.Telomerase,amultisubunitRNA-containingcomplexrelatedtoviralRNA-dependentDNApolymerases(reversetranscrip-tases),istheenzymeresponsiblefortelomeresynthesisandthusformaintainingthelengthofthetelomere.Sincetelomereshorteninghasbeenassociatedwithbothmalignanttransformationandaging,telomerasehasbecomeanattractivetargetforcancerchemother-apyanddrugdevelopment.Eachsisterchromatidcon-tainsonedouble-strandedDNAmolecule.Duringin-terphase,thepackingoftheDNAmoleculeislessdensethanitisinthecondensedchromosomeduringmetaphase.Metaphasechromosomesarenearlycom-pletelytranscriptionallyinactive.Thehumanhaploidgenomeconsistsofabout973×10bpandabout1.7×10nucleosomes.Thus,eachofthe23chromatidsinthehumanhaploidgenome8wouldcontainontheaverage1.3×10nucleotidesinonedouble-strandedDNAmolecule.ThelengthofeachDNAmoleculemustbecompressedabout8000-foldtogeneratethestructureofacondensedmetaphase5Cchromosome!Inmetaphasechromosomes,the30-nm5CchromatinfibersarealsofoldedintoaseriesofloopedBR3BR3domains,theproximalportionsofwhichareanchoredABtoanonhistoneproteinaceousscaffoldingwithintheFigure36–4.Illustrationofthetightcorrelationbe-nucleus(Figure36–3).ThepackingratiosofeachoftweenthepresenceofRNApolymeraseIIandRNAsyn-theordersofDNAstructurearesummarizedinTable36–2.thesis.AnumberofgenesareactivatedwhenChirono-Thepackagingofnucleoproteinswithinchromatidsmustentanslarvaearesubjectedtoheatshock(39°Cisnotrandom,asevidencedbythecharacteristicpat-for30minutes).A:DistributionofRNApolymeraseIIternsobservedwhenchromosomesarestainedwithspe-(alsocalledtypeB)inisolatedchromosomeIVfromthecificdyessuchasquinacrineorGiemsa’sstain(Figuresalivarygland(atarrows).Theenzymewasdetectedby36–6).immunofluorescenceusinganantibodydirectedFromindividualtoindividualwithinasingleagainstthepolymerase.The5CandBR3arespecificspecies,thepatternofstaining(banding)oftheentirebandsofchromosomeIV,andthearrowsindicatepuffs.chromosomecomplementishighlyreproducible;none-B:AutoradiogramofachromosomeIVthatwasincu-theless,itdifferssignificantlyfromotherspecies,even3batedinH-uridinetolabeltheRNA.Notethecorre-thosecloselyrelated.Thus,thepackagingofthenucleo-spondenceoftheimmunofluorescenceandpresenceproteinsinchromosomesofhighereukaryotesmustinoftheradioactiveRNA(blackdots).Bar=7μm.(Repro-somewaybedependentuponspecies-specificcharacter-duced,withpermission,fromSassH:RNApolymeraseBinisticsoftheDNAmolecules.polytenechromosomes.Cell1982;28:274.Copyright©Acombinationofspecializedstainingtechniques1982bytheMassachusettsInstituteofTechnology.)andhigh-resolutionmicroscopyhasallowedgeneticists
317DNAORGANIZATION,REPLICATION,&REPAIR/319SisterchromatidNo.2SisterchromatidNo.1CentromereFigure36–5.Thetwosisterchromatidsofhumanchromosome12(×27,850).ThelocationoftheA+T-richcentromericregionconnectingsisterchromatidsisindicated,asaretwoofthefourtelomeresresidingattheveryendsofthechromatidsthatareattachedonetotheotheratthecentromere.(Modifiedandreproduced,withTelomerespermission,fromDuPrawEJ:DNAandChromo-(TTAGG)nsomes.Holt,Rinehart,andWinston,1970.)toquitepreciselymapthousandsofgenestospecificre-nonproteincodingDNA.Accordingly,theprimarygionsofmouseandhumanchromosomes.Withthere-transcriptsofDNA(mRNAprecursors,originallycentelucidationofthehumanandmousegenomese-termedhnRNAbecausethisspeciesofRNAwasquitequences,ithasbecomeclearthatmanyofthesevisualheterogeneousinsize[length]andmostlyrestrictedtomappingmethodswereremarkablyaccurate.thenucleus),containnoncodinginterveningsequencesofRNAthatmustberemovedinaprocesswhichalsojoinstogethertheappropriatecodingsegmentstoformCodingRegionsAreOftenInterruptedthematuremRNA.MostcodingsequencesforasinglebyInterveningSequencesmRNAareinterruptedinthegenome(andthusintheprimarytranscript)byatleastone—andinsomecasesTheproteincodingregionsofDNA,thetranscriptsasmanyas50—noncodinginterveningsequences(in-ofwhichultimatelyappearinthecytoplasmassingletrons).Inmostcases,theintronsaremuchlongerthanmRNAmolecules,areusuallyinterruptedintheeu-thecontinuouscodingregions(exons).Theprocessingkaryoticgenomebylargeinterveningsequencesofoftheprimarytranscript,whichinvolvesremovalofin-tronsandsplicingofadjacentexons,isdescribedinde-tailinChapter37.Thefunctionoftheinterveningsequences,orin-Table36–2.Thepackingratiosofeachofthetrons,isnotclear.Theymayservetoseparatefunc-ordersofDNAstructure.tionaldomains(exons)ofcodinginformationinaformthatpermitsgeneticrearrangementbyrecombinationtooccurmorerapidlythanifallcodingregionsforaChromatinFormPackingRatiogivengeneticfunctionwerecontiguous.Suchanen-Nakeddouble-helicalDNA~1.0hancedrateofgeneticrearrangementoffunctionaldo-10-nmfibrilofnucleosomes7–10mainsmightallowmorerapidevolutionofbiologic25-to30-nmchromatinfiberofsuperheli-40–60function.TherelationshipsamongchromosomalcalnucleosomesDNA,geneclustersonthechromosome,theexon-Condensedmetaphasechromosomeof8000intronstructureofgenes,andthefinalmRNAproductloopsareillustratedinFigure36–7.
318320/CHAPTER3612345678910111218131415161719202122XYFigure36–6.Ahumankaryotype(ofamanwithanormal46,XYconstitution),inwhichthemetaphasechromosomeshavebeenstainedbytheGiemsamethodandalignedaccordingtotheParisConvention.(CourtesyofHLawceandFConte.)MUCHOFTHEMAMMALIANGENOMETheDNAinaeukaryoticgenomecanbedividedISREDUNDANT&MUCHISintodifferent“sequenceclasses.”Theseareunique-NOTTRANSCRIBEDsequence,ornonrepetitive,DNAandrepetitive-sequenceDNA.Inthehaploidgenome,unique-se-ThehaploidgenomeofeachhumancellconsistsofquenceDNAgenerallyincludesthesinglecopygenes93×10basepairsofDNAsubdividedinto23chromo-thatcodeforproteins.TherepetitiveDNAinthehap-somes.Theentirehaploidgenomecontainssufficientloidgenomeincludessequencesthatvaryincopynum-7DNAtocodefornearly1.5millionaverage-sizedberfromtwotoasmanyas10copiespercell.genes.However,studiesofmutationratesandofthecomplexitiesofthegenomesofhigherorganismsstronglysuggestthathumanshave<100,000proteinsMoreThanHalftheDNAinEukaryoticencodedbythe~1.1%ofthehumangenomethatisOrganismsIsinUniqueorcomposedofexonicDNA.ThisimpliesthatmostofNonrepetitiveSequencestheDNAisnoncoding—ie,itsinformationisnevertranslatedintoanaminoacidsequenceofaproteinThisestimation(andthedistributionofrepetitive-molecule.Certainly,someoftheexcessDNAsequencessequenceDNA)isbasedonavarietyofDNA-RNAhy-servetoregulatetheexpressionofgenesduringdevel-bridizationtechniquesand,morerecently,ondirectopment,differentiation,andadaptationtotheenviron-DNAsequencing.Similartechniquesareusedtoesti-ment.Someexcessclearlymakesuptheinterveningse-matethenumberofactivegenesinapopulationofquencesorintrons(24%ofthetotalhumangenome)unique-sequenceDNA.Inbrewers’yeast(Saccha-thatsplitthecodingregionsofgenes,butmuchoftheromycescerevisiae,alowereukaryote),abouttwothirdsexcessappearstobecomposedofmanyfamiliesofre-ofits6200genesareexpressed.Intypicaltissuesinapeatedsequencesforwhichnofunctionshavebeenhighereukaryote(eg,mammalianliverandkidney),be-clearlydefined.Asummaryofthesalientfeaturesofthetween10,000and15,000genesareexpressed.Differ-humangenomeispresentedinChapter40.entcombinationsofgenesareexpressedineachtissue,
319DNAORGANIZATION,REPLICATION,&REPAIR/321Chromosome8(1–2×103genes)1.5×10bpGenecluster1.5×106bp(~20genes)Gene2×104bpPrimarytranscript8×103ntmRNA3nt2×10Figure36–7.TherelationshipbetweenchromosomalDNAandmRNA.Thehuman9haploidDNAcomplementof3×10basepairs(bp)isdistributedbetween23chromo-4somes.Genesareclusteredonthesechromosomes.Anaveragegeneis2×10bpinlength,includingtheregulatoryregion(hatchedarea),whichisusuallylocatedatthe5′endofthegene.Theregulatoryregionisshownhereasbeingadjacenttothetranscrip-tioninitiationsite(arrow).Mosteukaryoticgeneshavealternatingexonsandintrons.Inthisexample,therearenineexons(darkblueareas)andeightintrons(lightblueareas).Theintronsareremovedfromtheprimarytranscriptbytheprocessingreaction,andtheexonsareligatedtogetherinsequencetoformthematuremRNA.(nt,nucleotides.)ofcourse,andhowthisisaccomplishedisoneoftheDependingontheirlength,moderatelyrepetitivemajorunansweredquestionsinbiology.sequencesareclassifiedaslonginterspersedrepeatsequences(LINEs)orshortinterspersedrepeatInHumanDNA,atLeast30%ofthesequences(SINEs).BothtypesappeartobeGenomeConsistsofRepetitiveSequencesretroposons,ie,theyarosefrommovementfromonelocationtoanother(transposition)throughanRNARepetitive-sequenceDNAcanbebroadlyclassifiedasintermediatebytheactionofreversetranscriptasethatmoderatelyrepetitiveorashighlyrepetitive.ThehighlytranscribesanRNAtemplateintoDNA.Mammalianrepetitivesequencesconsistof5–500basepairlengthsgenomescontain20–50thousandcopiesofthe6–7kbrepeatedmanytimesintandem.ThesesequencesareLINEs.Theserepresentspecies-specificfamiliesofre-usuallyclusteredincentromeresandtelomeresofthepeatelements.SINEsareshorter(70–300bp),andchromosomeandarepresentinabout1–10milliontheremaybemorethan100,000copiespergenome.copiesperhaploidgenome.Thesesequencesaretran-OftheSINEsinthehumangenome,onefamily,thescriptionallyinactiveandmayplayastructuralroleinAlufamily,ispresentinabout500,000copiesperhap-thechromosome(seeChapter40).loidgenomeandaccountsforatleast5–6%oftheThemoderatelyrepetitivesequences,whicharede-humangenome.MembersofthehumanAlufamily6finedasbeingpresentinnumbersoflessthan10andtheircloselyrelatedanalogsinotheranimalsarecopiesperhaploidgenome,arenotclusteredbutarein-transcribedasintegralcomponentsofhnRNAorasdis-terspersedwithuniquesequences.Inmanycases,thesecreteRNAmolecules,includingthewell-studied4.5SlonginterspersedrepeatsaretranscribedbyRNApoly-RNAand7SRNA.TheseparticularfamilymembersmeraseIIandcontaincapsindistinguishablefromthosearehighlyconservedwithinaspeciesaswellasbetweenonmRNA.mammalianspecies.Componentsoftheshortinter-
320322/CHAPTER36spersedrepeats,includingthemembersoftheAlufam-withageneinaffectedfamilymembers—andthelackily,maybemobileelements,capableofjumpingintoofthisassociationinunaffectedmembers—maybetheandoutofvarioussiteswithinthegenome(seebelow).firstclueaboutthegeneticbasisofadisease.Thiscanhavedisastrousresults,asexemplifiedbytheTrinucleotidesequencesthatincreaseinnumberinsertionofAlusequencesintoagene,which,whenso(microsatelliteinstability)cancausedisease.Theunsta-mutated,causesneurofibromatosis.blep(CGG)nrepeatsequenceisassociatedwiththefragileXsyndrome.OthertrinucleotiderepeatsthatMicrosatelliteRepeatSequencesundergodynamicmutation(usuallyanincrease)areassociatedwithHuntington’schorea(CAG),myotonicOnecategoryofrepeatsequencesexistsasbothdis-dystrophy(CTG),spinobulbarmuscularatrophy(CAG),persedandgroupedtandemarrays.Thesequencescon-andKennedy’sdisease(CAG).sistof2–6bprepeatedupto50times.Thesemi-crosatellitesequencesmostcommonlyarefoundasONEPERCENTOFCELLULARDNAdinucleotiderepeatsofACononestrandandTGonISINMITOCHONDRIAtheoppositestrand,butseveralotherformsoccur,in-cludingCG,AT,andCA.TheACrepeatsequencesareThemajorityofthepeptidesinmitochondria(aboutestimatedtooccurat50,000–100,000locationsinthe54outof67)arecodedbynucleargenes.Therestaregenome.Atanylocus,thenumberoftheserepeatsmaycodedbygenesfoundinmitochondrial(mt)DNA.varyonthetwochromosomes,thusprovidingheterozy-Humanmitochondriacontaintwototencopiesofagosityofthenumberofcopiesofaparticularmi-smallcirculardouble-strandedDNAmoleculethatcrosatellitenumberinanindividual.Thisisaheritablemakesupapproximately1%oftotalcellularDNA.trait,and,becauseoftheirnumberandtheeaseofde-ThismtDNAcodesformtribosomalandtransfertectingthemusingthepolymerasechainreactionRNAsandfor13proteinsthatplaykeyrolesintheres-(PCR)(Chapter40),ACrepeatsareveryusefulincon-piratorychain.Thelinearizedstructuralmapofthestructinggeneticlinkagemaps.Mostgenesareassoci-humanmitochondrialgenesisshowninFigure36–8.atedwithoneormoremicrosatellitemarkers,sotherel-SomeofthefeaturesofmtDNAareshowninTableativepositionofgenesonchromosomescanbe36–3.assessed,ascantheassociationofagenewithadisease.AnimportantfeatureofhumanmitochondrialUsingPCR,alargenumberoffamilymemberscanbemtDNAisthat—becauseallmitochondriaarecon-rapidlyscreenedforacertainmicrosatellitepolymor-tributedbytheovumduringzygoteformation—itisphism.Theassociationofaspecificpolymorphismtransmittedbymaternalnonmendelianinheritance.kb246810121416PH1OHOHPH2ATPase86ND3ND4L12S16SND1ND2CO1CO2CO3ND4ND5CYTBND6PLOLFigure36–8.Mapsofhumanmitochondrialgenes.Themapsrepresenttheheavy(upperstrand)andlight(lowermap)strandsoflinearizedmitochondrial(mt)DNA,showingthegenesforthesubunitsofNADH-coenzymeQoxidoreductase(ND1throughND6),cytochromecoxidase(CO1throughCO3),cytochromeb(CYTB),andATPsynthase(ATPase8and6)andforthe12Sand16SribosomalmtrRNAs.ThetransferRNAsarede-notedbysmallopenboxes.Theoriginofheavy-strand(OH)andlight-strand(OL)replicationandthepromotersfortheinitiationofheavy-strand(PH1andPH2)andlight-strand(PL)transcriptionareindicatedbyarrows.(Reproduced,withpermission,fromMoraesCTetal:MitochondrialDNAdeletionsinprogressiveexternalophthal-moplegiaandKearns-Sayresyndrome.NEnglJMed1989;320:1293.)
321DNAORGANIZATION,REPLICATION,&REPAIR/323Table36–3.SomemajorfeaturesofthestructurecrossingoveroccursasshowninFigure36–9.ThisandfunctionofhumanmitochondrialDNA.1usuallyresultsinanequalandreciprocalexchangeofgeneticinformationbetweenhomologouschromo-somes.Ifthehomologouschromosomespossessdiffer-•Iscircular,double-stranded,andcomposedofheavy(H)entallelesofthesamegenes,thecrossovermayproduceandalight(L)chainsorstrands.noticeableandheritablegeneticlinkagedifferences.In•Contains16,569bp.•Encodes13proteinsubunitsoftherespiratorychain(ofatherarecasewherethealignmentofhomologouschro-totalofabout67):mosomesisnotexact,thecrossingoverorrecombina-SevensubunitsofNADHdehydrogenase(complexI)tioneventmayresultinanunequalexchangeofinfor-CytochromebofcomplexIIImation.OnechromosomemayreceivelessgeneticThreesubunitsofcytochromeoxidase(complexIV)materialandthusadeletion,whiletheotherpartnerofTwosubunitsofATPsynthasethechromosomepairreceivesmoregeneticmaterial•Encodeslarge(16s)andsmall(12s)mtribosomalRNAs.andthusaninsertionorduplication(Figure36–9).•Encodes22mttRNAmolecules.Unequalcrossingoverdoesoccurinhumans,asevi-•Geneticcodediffersslightlyfromthestandardcode:dencedbytheexistenceofhemoglobinsdesignatedUGA(standardstopcodon)isreadasTrp.Leporeandanti-Lepore(Figure36–10).ThefartherAGAandAGG(standardcodonsforArg)arereadasstopaparttwosequencesareonanindividualchromosome,codons.thegreaterthelikelihoodofacrossoverrecombination•Containsveryfewuntranslatedsequences.•Highmutationrate(fivetotentimesthatofnuclearDNA).•ComparisonsofmtDNAsequencesprovideevidenceaboutevolutionaryoriginsofprimatesandotherspecies.1AdaptedfromHardingAE:Neurologicaldiseaseandmitochon-drialgenes.TrendsNeurolSci1991;14:132.Thus,indiseasesresultingfrommutationsofmtDNA,anaffectedmotherwouldintheorypassthediseasetoallofherchildrenbutonlyherdaughterswouldtrans-mitthetrait.However,insomecases,deletionsinmtDNAoccurduringoogenesisandthusarenotinher-itedfromthemother.AnumberofdiseaseshavenowbeenshowntobeduetomutationsofmtDNA.Theseincludeavarietyofmyopathies,neurologicdisorders,andsomecasesofdiabetesmellitus.GENETICMATERIALCANBEALTERED&REARRANGEDAnalterationinthesequenceofpurineandpyrimidinebasesinageneduetoachange—aremovaloraninser-tion—ofoneormorebasesmayresultinanalteredgeneproduct.Suchalterationinthegeneticmaterialre-sultsinamutationwhoseconsequencesarediscussedindetailinChapter38.ChromosomalRecombinationIsOneWayofRearrangingGeneticMaterialGeneticinformationcanbeexchangedbetweensimilarorhomologouschromosomes.Theexchangeorrecom-binationeventoccursprimarilyduringmeiosisinmammaliancellsandrequiresalignmentofhomolo-Figure36–9.Theprocessofcrossing-overbetweengousmetaphasechromosomes,analignmentthatal-homologousmetaphasechromosomestogeneratere-mostalwaysoccurswithgreatexactness.Aprocessofcombinantchromosomes.SeealsoFigure36–12.
322324/CHAPTER36GγAγδβδβFigure36–10.Theprocessofunequalcross-Anti-Leporeoverintheregionofthemammaliangenomethatharborsthestructuralgenesencodinghe-GγAγδβmoglobinsandthegenerationoftheunequalrecombinantproductshemoglobindelta-betaLeporeandbeta-deltaanti-Lepore.Theexam-GγAγplesgivenshowthelocationsofthecrossoverregionsbetweenaminoacidresidues.(Redrawnandreproduced,withpermission,fromCleggJB,0GγAγδβWeatherallDJ:βThalassemia:Timeforareap-Leporepraisal?Lancet1974;2:133.)event.Thisisthebasisforgeneticmappingmethods.DNApolymerase,orreversetranscriptase—canbeinte-Unequalcrossoveraffectstandemarraysofrepeatedgratedintochromosomesofthemammaliancell.TheDNAswhethertheyarerelatedglobingenes,asinFig-integrationoftheanimalvirusDNAintotheanimalure36–10,ormoreabundantrepetitiveDNA.Un-genomegenerallyisnot“site-specific”butdoesdisplayequalcrossoverthroughslippageinthepairingcanre-sitepreferences.sultinexpansionorcontractioninthecopynumberoftherepeatfamilyandmaycontributetotheexpansionTranspositionCanProduceandfixationofvariantmembersthroughoutthearray.ProcessedGenesChromosomalIntegrationOccursIneukaryoticcells,smallDNAelementsthatclearlyarenotvirusesarecapableoftransposingthemselvesinandWithSomeVirusesSomebacterialviruses(bacteriophages)arecapableofrecombiningwiththeDNAofabacterialhostinsuchawaythatthegeneticinformationofthebacteriophageisBincorporatedinalinearfashionintothegeneticinfor-mationofthehost.Thisintegration,whichisaformofrecombination,occursbythemechanismillustratedinFigure36–11.Thebackboneofthecircularbacterio-ACphagegenomeisbroken,asisthatoftheDNAmole-culeofthehost;theappropriateendsareresealedwith12theproperpolarity.ThebacteriophageDNAisfigura-Btivelystraightenedout(“linearized”)asitisintegratedintothebacterialDNAmolecule—frequentlyaclosedcircleaswell.ThesiteatwhichthebacteriophageACgenomeintegratesorrecombineswiththebacterialgenomeischosenbyoneoftwomechanisms.Ifthe1B2bacteriophagecontainsaDNAsequencehomologoustoasequenceinthehostDNAmolecule,thenarecom-CAbinationeventanalogoustothatoccurringbetweenho-mologouschromosomescanoccur.However,some12bacteriophagessynthesizeproteinsthatbindspecificsitesonbacterialchromosomestoanonhomologousCBAsitecharacteristicofthebacteriophageDNAmolecule.Integrationoccursatthesiteandissaidtobe“site-12specific.”Manyanimalviruses,particularlytheoncogenicFigure36–11.Theintegrationofacirculargenomeviruses—eitherdirectlyor,inthecaseofRNAvirusesfromavirus(withgenesA,B,andC)intotheDNAmole-suchasHIVthatcausesAIDS,theirDNAtranscriptsculeofahost(withgenes1and2)andtheconsequentgeneratedbytheactionoftheviralRNA-dependentorderingofthegenes.
323DNAORGANIZATION,REPLICATION,&REPAIR/325outofthehostgenomeinwaysthataffectthefunctionchromatidscontainsidenticalgeneticinformationsinceofneighboringDNAsequences.Thesemobileele-eachisaproductofthesemiconservativereplicationofments,sometimescalled“jumpingDNA,”cancarrytheoriginalparentDNAmoleculeofthatchromosome.flankingregionsofDNAand,therefore,profoundlyaf-Crossingoveroccursbetweenthesegeneticallyidenticalfectevolution.Asmentionedabove,theAlufamilyofsisterchromatids.Ofcourse,thesesisterchromatidex-moderatelyrepeatedDNAsequenceshasstructuralchanges(Figure36–12)havenogeneticconsequenceascharacteristicssimilartotheterminiofretroviruses,longastheexchangeistheresultofanequalcrossover.whichwouldaccountfortheabilityofthelattertomoveintoandoutofthemammaliangenome.ImmunoglobulinGenesRearrangeDirectevidenceforthetranspositionofothersmallDNAelementsintothehumangenomehasbeenpro-Inmammaliancells,someinterestinggenerearrange-videdbythediscoveryof“processedgenes”forim-mentsoccurnormallyduringdevelopmentanddifferen-tiation.Forexample,inmicetheVandCgenesforamunoglobulinmolecules,α-globinmolecules,andsev-LLeralothers.TheseprocessedgenesconsistofDNAsingleimmunoglobulinmolecule(seeChapter39)aresequencesidenticalornearlyidenticaltothoseofthewidelyseparatedinthegermlineDNA.IntheDNAofamessengerRNAfortheappropriategeneproduct.Thatdifferentiatedimmunoglobulin-producing(plasma)cell,thesameVandCgeneshavebeenmovedphysicallyis,the5′nontranscribedregion,thecodingregionLLwithoutintronrepresentation,andthe3′poly(A)tailclosertogetherinthegenomeandintothesametran-areallpresentcontiguously.ThisparticularDNAse-scriptionunit.However,eventhen,thisrearrangementofDNAduringdifferentiationdoesnotbringtheVandquencearrangementmusthaveresultedfromthere-LCgenesintocontiguityintheDNA.Instead,theDNAversetranscriptionofanappropriatelyprocessedmes-LsengerRNAmoleculefromwhichtheintronregionshadbeenremovedandthepoly(A)tailadded.Theonlyrecognizedmechanismthisreversetranscriptcouldhaveusedtointegrateintothegenomewouldhavebeenatranspositionevent.Infact,these“processedgenes”haveshortterminalrepeatsateachend,asdoknowntransposedsequencesinlowerorganisms.Intheabsenceoftheirtranscriptionandthusgeneticselectionforfunction,manyoftheprocessedgeneshavebeenrandomlyalteredthroughevolutionsothattheynowcontainnonsensecodonswhichprecludetheirabilitytoencodeafunctional,intactprotein(seeChapter38).Thus,theyarereferredtoas“pseudogenes.”GeneConversionProducesRearrangementsBesidesunequalcrossoverandtransposition,athirdmechanismcaneffectrapidchangesinthegeneticma-terial.Similarsequencesonhomologousornonhomol-ogouschromosomesmayoccasionallypairupandelim-inateanymismatchedsequencesbetweenthem.Thismayleadtotheaccidentalfixationofonevariantoran-otherthroughoutafamilyofrepeatedsequencesandtherebyhomogenizethesequencesofthemembersofrepetitiveDNAfamilies.Thislatterprocessisreferredtoasgeneconversion.Figure36–12.SisterchromatidexchangesbetweenSisterChromatidsExchangehumanchromosomes.ThesearedetectablebyGiemsaIndiploideukaryoticorganismssuchashumans,afterstainingofthechromosomesofcellsreplicatedfortwocellsprogressthroughtheSphasetheycontainacyclesinthepresenceofbromodeoxyuridine.Thear-tetraploidcontentofDNA.Thisisintheformofsisterrowsindicatesomeregionsofexchange.(Courtesyofchromatidsofchromosomepairs.EachofthesesisterSWolffandJBodycote.)
324326/CHAPTER36containsaninterspersedorinterruptionsequenceofTable36–4.StepsinvolvedinDNAreplicationabout1200basepairsatornearthejunctionoftheVineukaryotes.andCregions.TheinterspersedsequenceistranscribedintoRNAalongwiththeVandCgenes,andtheinter-LL1.Identificationoftheoriginsofreplication.spersedinformationisremovedfromtheRNAduringits2.Unwinding(denaturation)ofdsDNAtoprovideanssDNAnuclearprocessing(Chapters37and39).template.3.Formationofthereplicationfork.DNASYNTHESIS&REPLICATION4.InitiationofDNAsynthesisandelongation.ARERIGIDLYCONTROLLED5.FormationofreplicationbubbleswithligationofthenewlysynthesizedDNAsegments.TheprimaryfunctionofDNAreplicationisunder-6.Reconstitutionofchromatinstructure.stoodtobetheprovisionofprogenywiththegeneticinformationpossessedbytheparent.Thus,thereplica-tionofDNAmustbecompleteandcarriedoutinsuchaseriesofdirectrepeatDNAsequences.Inbacterio-awayastomaintaingeneticstabilitywithintheorgan-phageλ,theoriλisboundbytheλ-encodedOproteinismandthespecies.TheprocessofDNAreplicationistofouradjacentsites.InEcoli,theoriCisboundbycomplexandinvolvesmanycellularfunctionsandsev-theproteindnaA.Inbothcases,acomplexisformederalverificationprocedurestoensurefidelityinreplica-consistingof150–250bpofDNAandmultimersoftion.About30proteinsareinvolvedinthereplicationtheDNA-bindingprotein.Thisleadstothelocalde-oftheEcolichromosome,andthisprocessisalmostnaturationandunwindingofanadjacentA+T-richre-certainlymorecomplexineukaryoticorganisms.ThegionofDNA.FunctionallysimilarautonomouslyfirstenzymologicobservationsonDNAreplicationreplicatingsequences(ARS)havebeenidentifiedinweremadeinEcolibyKornberg,whodescribedinthatyeastcells.TheARScontainsasomewhatdegenerateorganismtheexistenceofanenzymenowcalledDNA11-bpsequencecalledtheoriginreplicationelementpolymeraseI.Thisenzymehasmultiplecatalyticactivi-(ORE).TheOREbindsasetofproteins,analogoustoties,acomplexstructure,andarequirementforthethednaAproteinofEcoli,whichiscollectivelycalledtriphosphatesofthefourdeoxyribonucleosidesofade-theoriginrecognitioncomplex(ORC).TheOREisnine,guanine,cytosine,andthymine.Thepolymeriza-locatedadjacenttoanapproximately80-bpA+T-richtionreactioncatalyzedbyDNApolymeraseIofEcolisequencethatiseasytounwind.ThisiscalledtheDNAhasservedasaprototypeforallDNApolymerasesofunwindingelement(DUE).TheDUEistheoriginofbothprokaryotesandeukaryotes,eventhoughitisnowreplicationinyeast.recognizedthatthemajorroleofthispolymeraseistoConsensussequencessimilartooriorARSinstruc-completereplicationonthelaggingstrand.tureorfunctionhavenotbeenpreciselydefinedinInallcells,replicationcanoccuronlyfromasingle-mammaliancells,thoughseveraloftheproteinsthatstrandedDNA(ssDNA)template.Mechanismsmustparticipateinorirecognitionandfunctionhavebeenexisttotargetthesiteofinitiationofreplicationandtoidentifiedandappearquitesimilartotheiryeastcoun-unwindthedouble-strandedDNA(dsDNA)inthatre-terpartsinbothaminoacidsequenceandfunction.gion.Thereplicationcomplexmustthenform.Afterreplicationiscompleteinanarea,theparentanddaughterUnwindingofDNAstrandsmustre-formdsDNA.Ineukaryoticcells,anad-ditionalstepmustoccur.ThedsDNAmustpreciselyre-Theinteractionofproteinswithoridefinesthestartsiteformthechromatinstructure,includingnucleosomes,ofreplicationandprovidesashortregionofssDNAes-thatexistedpriortotheonsetofreplication.AlthoughthissentialforinitiationofsynthesisofthenascentDNAentireprocessisnotwellunderstoodineukaryoticcells,strand.Thisprocessrequirestheformationofanumberreplicationhasbeenquitepreciselydescribedinprokary-ofprotein-proteinandprotein-DNAinteractions.Aoticcells,andthegeneralprinciplesarethoughttobethecriticalstepisprovidedbyaDNAhelicasethatallowssameinboth.ThemajorstepsarelistedinTable36–4,il-forprocessiveunwindingofDNA.InuninfectedEcoli,lustratedinFigure36–13,anddiscussed,insequence,thisfunctionisprovidedbyacomplexofdnaBhelicasebelow.Anumberofproteins,mostwithspecificenzy-andthednaCprotein.Single-strandedDNA-bindingmaticaction,areinvolvedinthisprocess(Table36–5).proteins(SSBs)stabilizethiscomplex.Inλphage-infectedEcoli,thephageproteinPbindstodnaBandtheP/dnaBcomplexbindstooriλbyinteractingwithTheOriginofReplicationtheOprotein.dnaBisnotanactivehelicasewhenintheAttheoriginofreplication(ori),thereisanassocia-P/dnaB/Ocomplex.ThreeEcoliheatshockproteinstionofsequence-specificdsDNA-bindingproteinswith(dnaK,dnaJ,andGrpE)cooperatetoremovetheP
325DNAORGANIZATION,REPLICATION,&REPAIR/327OriA+TregionA+T-richOri-bindingDenaturationregionproteinBinding()ofSSB()Bindingoffactors,formationof3′replicationfork,5′initiationofreplicationLeadingstrandPolymeraseHelicase3′Primase5′SSB5′3′3′Lagging=Ori–bindingproteinstrand5′=Polymerase=NascentDNA=RNAprimerReplicationfork=Helicase=Primase=SSBFigure36–13.StepsinvolvedinDNAreplication.ThisfiguredescribesDNAreplicationinanEcolicell,butthegeneralstepsaresimilarineukaryotes.Aspecificinteractionofaprotein(theOprotein)totheoriginofreplication(ori)resultsinlocalunwindingofDNAatanadjacentA+T-richregion.TheDNAinthisareaismaintainedinthesingle-strandconformation(ssDNA)bysingle-strand-bindingproteins(SSBs).Thisallowsavarietyofproteins,includ-inghelicase,primase,andDNApolymerase,tobindandtoinitiateDNAsynthesis.ThereplicationforkproceedsasDNAsynthesisoccurscontinuously(longarrow)ontheleadingstrandanddiscontinuously(shortarrows)onthelag-gingstrand.ThenascentDNAisalwayssynthesizedinthe5′to3′direction,asDNApolymerasescanaddanucleotideonlytothe3′endofaDNAstrand.proteinandactivatethednaBhelicase.Incooperation(2)aprimaseinitiatessynthesisofanRNAmoleculewithSSB,thisleadstoDNAunwindingandactivethatisessentialforprimingDNAsynthesis;(3)thereplication.Inthisway,thereplicationoftheλphageisDNApolymeraseinitiatesnascent,daughterstrandaccomplishedattheexpenseofreplicationofthehostsynthesis;and(4)SSBsbindtossDNAandpreventEcolicell.prematurereannealingofssDNAtodsDNA.Thesere-actionsareillustratedinFigure36–13.ThepolymeraseIIIholoenzyme(thednaEgeneFormationoftheReplicationForkproductinEcoli)bindstotemplateDNAaspartofaAreplicationforkconsistsoffourcomponentsthatmultiproteincomplexthatconsistsofseveralpolym-forminthefollowingsequence:(1)theDNAhelicaseeraseaccessoryfactors(β,γ,δ,δ′,andτ).DNApolym-unwindsashortsegmentoftheparentalduplexDNA;erasesonlysynthesizeDNAinthe5′to3′direction,
326328/CHAPTER36Table36–5.Classesofproteinsinvolvedreplicationfork.Ofallpolymerases,itcatalyzestheinreplication.highestrateofchainelongationandisthemostproces-sive.Itiscapableofpolymerizing0.5MbofDNAdur-ingonecycleontheleadingstrand.PolIIIisalargeProteinFunction(>1MDa),ten-subunitproteincomplexinEcoli.TheDNApolymerasesDeoxynucleotidepolymerizationtwoidenticalβsubunitsofpolIIIencircletheDNAHelicasesProcessiveunwindingofDNAtemplateinasliding“clamp,”whichaccountsforthestabilityofthecomplexandforthehighdegreeofpro-TopoisomerasesRelievetorsionalstrainthatresultscessivitytheenzymeexhibits.fromhelicase-inducedunwindingPolymeraseII(polII)ismostlyinvolvedinproof-DNAprimaseInitiatessynthesisofRNAprimersreadingandDNArepair.PolymeraseI(polI)com-pleteschainsynthesisbetweenOkazakifragmentsonSingle-strandbindingPreventprematurereannealingofthelaggingstrand.EukaryoticcellshavecounterpartsproteinsdsDNAforeachoftheseenzymesplussomeadditionalones.ADNAligaseSealsthesinglestrandnickbetweencomparisonisshowninTable36–6.thenascentchainandOkazakifrag-Inmammaliancells,thepolymeraseiscapableofmentsonlaggingstrandpolymerizingabout100nucleotidespersecond,arateatleasttenfoldslowerthantherateofpolymerizationofdeoxynucleotidesbythebacterialDNApolymeraseandonlyoneoftheseveraldifferenttypesofpolym-complex.Thisreducedratemayresultfrominterfer-erasesisinvolvedatthereplicationfork.Becausetheencebynucleosomes.Itisnotknownhowthereplica-DNAstrandsareantiparallel(Chapter35),thepolym-tioncomplexnegotiatesnucleosomes.erasefunctionsasymmetrically.Ontheleading(for-ward)strand,theDNAissynthesizedcontinuously.Initiation&ElongationofDNASynthesisOnthelagging(retrograde)strand,theDNAissyn-TheinitiationofDNAsynthesis(Figure36–14)re-thesizedinshort(1–5kb;seeFigure36–16)fragments,quiresprimingbyashortlengthofRNA,abouttheso-calledOkazakifragments.SeveralOkazakifrag-10–200nucleotideslong.Thisprimingprocessinvolvesments(upto250)mustbesynthesized,insequence,forthenucleophilicattackbythe3′-hydroxylgroupoftheeachreplicationfork.Toensurethatthishappens,theRNAprimerontheαphosphateofthefirstenteringhelicaseactsonthelaggingstrandtounwinddsDNAindeoxynucleosidetriphosphate(NinFigure36–14)a5′to3′direction.Thehelicaseassociateswiththepri-withthesplittingoffofpyrophosphate.The3′-hy-masetoaffordthelatterproperaccesstothetemplate.droxylgroupoftherecentlyattacheddeoxyribonu-ThisallowstheRNAprimertobemadeand,inturn,cleosidemonophosphateisthenfreetocarryoutathepolymerasetobeginreplicatingtheDNA.Thisisnucleophilicattackonthenextenteringdeoxyribonu-animportantreactionsequencesinceDNApoly-cleosidetriphosphate(N+1inFigure36–14),againatmerasescannotinitiateDNAsynthesisdenovo.Theitsαphosphatemoiety,withthesplittingoffofpy-mobilecomplexbetweenhelicaseandprimasehasbeenrophosphate.Ofcourse,selectionoftheproperde-calledaprimosome.AsthesynthesisofanOkazakioxyribonucleotidewhoseterminal3′-hydroxylgroupisfragmentiscompletedandthepolymeraseisreleased,atobeattackedisdependentuponproperbasepairingnewprimerhasbeensynthesized.ThesamepolymerasemoleculeremainsassociatedwiththereplicationforkandproceedstosynthesizethenextOkazakifragment.Table36–6.AcomparisonofprokaryoticandTheDNAPolymeraseComplexeukaryoticDNApolymerases.AnumberofdifferentDNApolymerasemoleculesen-EcoliMammalianFunctiongageinDNAreplication.Thesesharethreeimportantproperties:(1)chainelongation,(2)processivity,andIαGapfillingandsynthesisoflagging(3)proofreading.Chainelongationaccountsforthestrandrate(innucleotidespersecond)atwhichpolymeriza-IIεDNAproofreadingandrepairtionoccurs.ProcessivityisanexpressionofthenumberofnucleotidesaddedtothenascentchainbeforetheβDNArepairpolymerasedisengagesfromthetemplate.Theproof-γMitochondrialDNAsynthesisreadingfunctionidentifiescopyingerrorsandcorrectsIIIδProcessive,leadingstrandsynthesisthem.InEcoli,polymeraseIII(polIII)functionsatthe
327X1COHHHHX2HOCOORNAprimerPOHHHHX3HOCOOPOHHHHX4HOCOOPOHHHHOHNOHCOOOPFirstenteringdNTPOOO–HH–PHHOOO–POHHOO–X4COOPOHHHHNHOCOOPOHHHHHN+1OHCOOOPSecondenteringdNTPOOO–HH–PHHOOO–POHHOO–Figure36–14.TheinitiationofDNAsynthesisuponaprimerofRNAandthesub-sequentattachmentoftheseconddeoxyribonucleosidetriphosphate.329
328330/CHAPTER36withtheotherstrandoftheDNAmoleculeaccordingofnewlysynthesizedDNAbyenzymesreferredtoastotherulesproposedoriginallybyWatsonandCrickDNAligases.(Figure36–15).Whenanadeninedeoxyribonucleosidemonophosphorylmoietyisinthetemplateposition,aReplicationExhibitsPolaritythymidinetriphosphatewillenteranditsαphosphatewillbeattackedbythe3′-hydroxylgroupoftheAshasalreadybeennoted,DNAmoleculesaredouble-deoxyribonucleosidemonophosphorylmostrecentlystrandedandthetwostrandsareantiparallel,ie,run-addedtothepolymer.Bythisstepwiseprocess,theninginoppositedirections.ThereplicationofDNAintemplatedictateswhichdeoxyribonucleosidetriphos-prokaryotesandeukaryotesoccursonbothstrandssi-phateiscomplementaryandbyhydrogenbondingmultaneously.However,anenzymecapableofpoly-holdsitinplacewhilethe3′-hydroxylgroupofthemerizingDNAinthe3′to5′directiondoesnotexistingrowingstrandattacksandincorporatesthenewnu-anyorganism,sothatbothofthenewlyreplicatedcleotideintothepolymer.ThesesegmentsofDNADNAstrandscannotgrowinthesamedirectionsimul-attachedtoanRNAinitiatorcomponentarethetaneously.Nevertheless,thesameenzymedoesreplicateOkazakifragments(Figure36–16).Inmammals,afterbothstrandsatthesametime.Thesingleenzymerepli-manyOkazakifragmentsaregenerated,thereplicationcatesonestrand(“leadingstrand”)inacontinuouscomplexbeginstoremovetheRNAprimers,tofillinmannerinthe5′to3′direction,withthesameoverallthegapsleftbytheirremovalwiththeproperbase-forwarddirection.Itreplicatestheotherstrand(“lag-paireddeoxynucleotide,andthentosealthefragmentsgingstrand”)discontinuouslywhilepolymerizingthe′3TPCA′5AGOHGUOHACOHARNAprimerUOHCTDNAtemplateOHAGTTTAGACCGrowingDNApolymerAGGTOHTPGPOH3′APACEnteringTTP′5Figure36–15.TheRNA-primedsynthesisofDNAdemonstratingthetemplatefunctionofthecomplementarystrandofparentalDNA.
329DNAORGANIZATION,REPLICATION,&REPAIR/331DNAtemplate3′5′5′3′NewlysynthesizedRNA10bp10bpDNAstrandprimer100bpOkazakifragmentsFigure36–16.Thediscontinuouspolymerizationofdeoxyribonucleotidesonthelaggingstrand;formationofOkazakifragmentsduringlaggingstrandDNAsynthesisisillustrated.Okazakifragmentsare100–250ntlongineukaryotes,1000–2000bpinprokaryotes.nucleotidesinshortspurtsof150–250nucleotides,hours!Metazoanorganismsgetaroundthisproblemagaininthe5′to3′direction,butatthesametimeitusingtwostrategies.First,replicationisbidirectional.facestowardthebackendoftheprecedingRNASecond,replicationproceedsfrommultipleoriginsinprimerratherthantowardtheunreplicatedportion.eachchromosome(atotalofasmanyas100inhu-ThisprocessofsemidiscontinuousDNAsynthesisismans).Thus,replicationoccursinbothdirectionsshowndiagrammaticallyinFigures36–13and36–16.alongallofthechromosomes,andbothstrandsareInthemammaliannucleargenome,mostofthereplicatedsimultaneously.Thisreplicationprocessgen-RNAprimersareeventuallyremovedaspartoftheerates“replicationbubbles”(Figure36–17).replicationprocess,whereasafterreplicationofthemi-ThemultiplesitesthatserveasoriginsforDNAtochondrialgenomethesmallpieceofRNAremainsasreplicationineukaryotesarepoorlydefinedexceptinaanintegralpartoftheclosedcircularDNAstructure.fewanimalvirusesandinyeast.However,itisclearthatinitiationisregulatedbothspatiallyandtemporally,sinceclustersofadjacentsitesinitiatereplicationsyn-FormationofReplicationBubbleschronously.Therearesuggestionsthatfunctionaldo-Replicationproceedsfromasingleoriinthecircularmainsofchromatinreplicateasintactunits,implying6thattheoriginsofreplicationarespecificallylocatedbacterialchromosome,composedofroughly6×10bpofDNA.Thisprocessiscompletedinabout30min-withrespecttotranscriptionunits.5DuringthereplicationofDNA,theremustbeasep-utes,areplicationrateof3×10bp/min.Theentiremammaliangenomereplicatesinapproximately9arationofthetwostrandstoalloweachtoserveasahours,theaverageperiodrequiredforformationofatemplatebyhydrogenbondingitsnucleotidebasestotetraploidgenomefromadiploidgenomeinareplicat-theincomingdeoxynucleosidetriphosphate.Thesepara-9tionoftheDNAdoublehelixispromotedbySSBs,spe-ingcell.Ifamammaliangenome(3×10bp)repli-5cificproteinmoleculesthatstabilizethesingle-strandedcatedatthesamerateasbacteria(ie,3×10bp/min)frombutasingleori,replicationwouldtakeover150structureasthereplicationforkprogresses.Thesestabi-Originofreplication“Replicationbubble”3′5′5′3′UnwindingproteinsatreplicationforksDirectionsofreplicationFigure36–17.Thegenerationof“replicationbubbles”duringtheprocessofDNAsynthesis.Thebidirectionalreplicationandtheproposedpositionsofunwindingproteinsatthereplicationforksaredepicted.
330332/CHAPTER36lizingproteinsbindcooperativelyandstoichiometricallywithoutrequiringenergyinput,becauseoftheforma-tothesinglestrandswithoutinterferingwiththeabili-tionofahigh-energycovalentbondbetweenthenickedtiesofthenucleotidestoserveastemplates(Figurephosphodiesterbackboneandthenicking-sealingen-36–13).Inadditiontoseparatingthetwostrandsofthezyme.Thenicking-resealingenzymesarecalledDNAdoublehelix,theremustbeanunwindingofthemole-topoisomerases.Thisprocessisdepicteddiagrammati-cule(onceevery10nucleotidepairs)toallowstrandsep-callyinFigure36–18andtherecomparedwiththearation.Thismusthappeninsegments,giventhetimeATP-dependentresealingcarriedoutbytheDNAli-duringwhichDNAreplicationoccurs.Therearemulti-gases.Topoisomerasesarealsocapableofunwindingsu-ple“swivels”interspersedintheDNAmoleculesofallpercoiledDNA.SupercoiledDNAisahigher-orderedorganisms.TheswivelfunctionisprovidedbyspecificstructureoccurringincircularDNAmoleculeswrappedenzymesthatintroduce“nicks”inonestrandofthearoundacore,asdepictedinFigure36–19.unwindingdoublehelix,therebyallowingtheunwind-Thereexistsinonespeciesofanimalviruses(retro-ingprocesstoproceed.Thenicksarequicklyresealedviruses)aclassofenzymescapableofsynthesizingasin-Step1DNAtopoisomeraseI=EDNAligase=EE+ATPEPRA(AMP-Enzyme)5′5′P-EPEnzyme(E)-generatedSingle-strandnickO3′single-strandnickO3′presentHH3′5′3′5′EPRAStep2E5′5′P-EPPRAOFormationofhigh-OHenergybondHStep3EPRA(AMP)NickrepairedNickrepairedFigure36–18.Comparisonoftwotypesofnick-sealingreactionsonDNA.TheseriesofreactionsatleftiscatalyzedbyDNAtopoisomeraseI,thatatrightbyDNAligase;P=phosphate,R=ribose,A=ademine.(Slightlymodifiedandrepro-duced,withpermission,fromLehningerAL:Biochemistry,2nded.Worth,1975.)
331DNAORGANIZATION,REPLICATION,&REPAIR/333preexistingandnewlyassembledhistoneoctamersarerandomlydistributedtoeacharmofthereplicationfork.DNASynthesisOccursDuringtheSPhaseoftheCellCycleInanimalcells,includinghumancells,thereplicationoftheDNAgenomeoccursonlyataspecifiedtimeduringthelifespanofthecell.ThisperiodisreferredtoasthesyntheticorSphase.Thisisusuallytemporallyseparatedfromthemitoticphasebynonsyntheticperi-odsreferredtoasgap1(G1)andgap2(G2),occurringbeforeandaftertheSphase,respectively(Figure36–20).Amongotherthings,thecellpreparesforDNAsynthesisinG1andformitosisinG2.Thecellregu-latesitsDNAsynthesisgrosslybyallowingittooccuronlyatspecifictimesandmostlyincellspreparingtodividebyamitoticprocess.Itappearsthatalleukaryoticcellshavegeneprod-uctsthatgovernthetransitionfromonephaseofthecellcycletoanother.Thecyclinsareafamilyofpro-teinswhoseconcentrationincreasesanddecreasesthroughoutthecellcycle—thustheirname.ThecyclinsFigure36–19.SupercoilingofDNA.Aleft-handedturnon,attheappropriatetime,differentcyclin-toroidal(solenoidal)supercoil,atleft,willconverttoadependentproteinkinases(CDKs)thatphosphory-right-handedinterwoundsupercoil,atright,whenthelatesubstratesessentialforprogressionthroughthecellcylindriccoreisremoved.Suchatransitionisanalogouscycle(Figure36–21).Forexample,cyclinDlevelsrisetothatwhichoccurswhennucleosomesaredisruptedinlateG1phaseandallowprogressionbeyondthestartbythehighsaltextractionofhistonesfromchromatin.(yeast)orrestrictionpoint(mammals),thepointbe-yondwhichcellsirrevocablyproceedintotheSorDNAsynthesisphase.gle-strandedandthenadouble-strandedDNAmole-TheDcyclinsactivateCDK4andCDK6.Theseculefromasingle-strandedRNAtemplate.Thispoly-twokinasesarealsosynthesizedduringG1incellsun-merase,RNA-dependentDNApolymerase,or“reversedergoingactivedivision.TheDcyclinsandCDK4andtranscriptase,”firstsynthesizesaDNA-RNAhybridCDK6arenuclearproteinsthatassembleasacomplexmoleculeutilizingtheRNAgenomeasatemplate.AinlateG1phase.Thecomplexisanactiveserine-specificnuclease,RNaseH,degradestheRNAstrand,threonineproteinkinase.OnesubstrateforthiskinaseandtheremainingDNAstrandinturnservesasatem-istheretinoblastoma(Rb)protein.Rbisacellcycleplatetoformadouble-strandedDNAmoleculecon-regulatorbecauseitbindstoandinactivatesatranscrip-tainingtheinformationoriginallypresentintheRNAtionfactor(E2F)necessaryforthetranscriptionofcer-genomeoftheanimalvirus.taingenes(histonegenes,DNAreplicationproteins,etc)neededforprogressionfromG1toSphase.ThephosphorylationofRbbyCDK4orCDK6resultsinthereleaseofE2FfromRb-mediatedtranscriptionre-ReconstitutionofChromatinStructurepression—thus,geneactivationensuesandcellcycleThereisevidencethatnuclearorganizationandchro-progressiontakesplace.matinstructureareinvolvedindeterminingtheregu-OthercyclinsandCDKsareinvolvedindifferentlationandinitiationofDNAsynthesis.Asnotedaspectsofcellcycleprogression(Table36–7).CyclinEabove,therateofpolymerizationineukaryoticcells,andCDK2formacomplexinlateG1.CyclinEiswhichhavechromatinandnucleosomes,istenfoldrapidlydegraded,andthereleasedCDK2thenformsaslowerthanthatinprokaryoticcells,whichhavecomplexwithcyclinA.ThissequenceisnecessaryfornakedDNA.ItisalsoclearthatchromatinstructuretheinitiationofDNAsynthesisinSphase.Acomplexmustbere-formedafterreplication.NewlyreplicatedbetweencyclinBandCDK1israte-limitingfortheDNAisrapidlyassembledintonucleosomes,andtheG2/Mtransitionineukaryoticcells.
332334/CHAPTER36ImproperspindledetectedFigure36–20.MammaliancellcycleandcellMcyclecheckpoints.DNA,chromosome,andchro-mosomesegregationintegrityiscontinuouslyGmonitoredthroughoutthecellcycle.IfDNAdam-2DamagedDNAGlageisdetectedineithertheG1ortheG2phaseofdetectedthecellcycle,ifthegenomeisincompletelyrepli-Scated,orifnormalchromosomesegregationma-DamagedDNAdetectedchineryisincomplete(ie,adefectivespindle),cellswillnotprogressthroughthephaseofthecycleinIncompletewhichdefectsaredetected.Insomecases,ifthereplicationdamagecannotberepaired,suchcellsundergodetectedprogrammedcelldeath(apoptosis).Manyofthecancer-causingviruses(oncoviruses)ducedbyseveralDNAvirusestargettheRbtranscrip-andcancer-inducinggenes(oncogenes)arecapableoftionrepressorforinactivation,inducingcelldivisionin-alleviatingordisruptingtheapparentrestrictionthatappropriately.normallycontrolstheentryofmammaliancellsfromDuringtheSphase,mammaliancellscontainG1intotheSphase.Fromtheforegoing,onemightgreaterquantitiesofDNApolymerasethanduringthehavesurmisedthatexcessiveproductionofacyclin—ornonsyntheticphasesofthecellcycle.Furthermore,productionataninappropriatetime—mightresultinthoseenzymesresponsibleforformationofthesub-abnormalorunrestrainedcelldivision.InthiscontextitstratesforDNAsynthesis—ie,deoxyribonucleosideisnoteworthythatthebcloncogeneassociatedwithBtriphosphates—arealsoincreasedinactivity,andtheircelllymphomaappearstobethecyclinD1gene.Simi-activitywilldiminishfollowingthesyntheticphaselarly,theoncoproteins(ortransformingproteins)pro-untilthereappearanceofthesignalforrenewedDNACdk1-cyclinBCdk1-cyclinAG2MCdk4-cyclinDCdk6-cyclinDG1SRestrictionpointCdk2-cyclinAFigure36–21.Schematicillustrationofthepointsduringthemammaliancellcycleduringwhichtheindicatedcyclinsandcyclin-dependentkinasesareactivated.ThethicknessofthevariousCdk2-cyclinEcoloredlinesisindicativeoftheextentofactivity.
333DNAORGANIZATION,REPLICATION,&REPAIR/335Table36–7.Cyclinsandcyclin-dependentkinasesringmorefrequentlythanonceevery108–1010baseinvolvedincellcycleprogression.pairsofDNAsynthesized.ThemechanismsresponsibleforthismonitoringmechanisminEcoliincludethe3′CyclinKinaseFunctionto5′exonucleaseactivitiesofoneofthesubunitsofthepolIIIcomplexandofthepolImolecule.Theanalo-DCDK4,CDK6Progressionpastrestrictionpointatgousmammalianenzymes(δandα)donotseemtoG1/Sboundarypossesssuchanucleaseproofreadingfunction.OtherE,ACDK2InitiationofDNAsynthesisinearlySenzymesprovidethisrepairfunction.phaseReplicationerrors,evenwithaveryefficientrepairsystem,leadtotheaccumulationofmutations.ABCDK1TransitionfromG2toM149humanhas10nucleatedcellseachwith3×10base16pairsofDNA.Ifabout10celldivisionsoccurina−10lifetimeand10mutationsperbasepairpercellgen-erationescaperepair,theremayeventuallybeasmanysynthesis.DuringtheSphase,thenuclearDNAisasonemutationper106bpinthegenome.Fortunately,completelyreplicatedonceandonlyonce.ItseemsmostofthesewillprobablyoccurinDNAthatdoesnotthatoncechromatinhasbeenreplicated,itismarkedsoencodeproteinsorwillnotaffectthefunctionofen-astopreventitsfurtherreplicationuntilitagainpassescodedproteinsandsoareofnoconsequence.Inaddi-throughmitosis.Themolecularmechanismsforthistion,spontaneousandchemicallyinduceddamagetophenomenonhaveyettobeelucidated.DNAmustberepaired.Ingeneral,agivenpairofchromosomeswillrepli-DamagetoDNAbyenvironmental,physical,andcatesimultaneouslyandwithinafixedportionoftheSchemicalagentsmaybeclassifiedintofourtypesphaseuponeveryreplication.Onachromosome,clus-(Table36–8).AbnormalregionsofDNA,eitherfromtersofreplicationunitsreplicatecoordinately.Thena-copyingerrorsorDNAdamage,arereplacedbyfourtureofthesignalsthatregulateDNAsynthesisatthesemechanisms:(1)mismatchrepair,(2)baseexcision-levelsisunknown,buttheregulationdoesappeartoberepair,(3)nucleotideexcision-repair,and(4)double-anintrinsicpropertyofeachindividualchromosome.strandbreakrepair(Table36–9).ThesemechanismsexploittheredundancyofinformationinherentintheEnzymesRepairDamagedDNAdoublehelicalDNAstructure.Thedefectiveregioninonestrandcanbereturnedtoitsoriginalformbyrely-ThemaintenanceoftheintegrityoftheinformationiningonthecomplementaryinformationstoredintheDNAmoleculesisofutmostimportancetothesurvivalunaffectedstrand.ofaparticularorganismaswellastosurvivalofthespecies.Thus,itcanbeconcludedthatsurvivingspecieshaveevolvedmechanismsforrepairingDNAdamageoccurringasaresultofeitherreplicationerrorsorenvi-ronmentalinsults.Table36–8.TypesofdamagetoDNA.AsdescribedinChapter35,themajorresponsibilityforthefidelityofreplicationresidesinthespecificpair-I.Single-basealterationingofnucleotidebases.ProperpairingisdependentA.DepurinationuponthepresenceofthefavoredtautomersoftheB.Deaminationofcytosinetouracilpurineandpyrimidinenucleotides,buttheequilibriumC.DeaminationofadeninetohypoxanthinewherebyonetautomerismorestablethananotherisD.Alkylationofbase45onlyabout10or10infavorofthatwiththegreaterE.Insertionordeletionofnucleotidestability.Althoughthisisnotfavorableenoughtoen-F.Base-analogincorporationsurethehighfidelitythatisnecessary,favoringoftheII.Two-basealterationpreferredtautomers—andthusoftheproperbasepair-A.UVlight–inducedthymine-thymine(pyrimidine)dimering—couldbeensuredbymonitoringthebasepairingB.Bifunctionalalkylatingagentcross-linkagetwice.SuchdoublemonitoringdoesappeartooccurinIII.Chainbreaksbothbacterialandmammaliansystems:onceattheA.Ionizingradiationtimeofinsertionofthedeoxyribonucleosidetriphos-B.Radioactivedisintegrationofbackboneelementphates,andlaterbyafollow-upenergy-requiringmech-C.OxidativefreeradicalformationanismthatremovesallimproperbaseswhichmayoccurIV.Cross-linkageinthenewlyformedstrand.This“proofreading”pre-A.Betweenbasesinsameoroppositestrandsventstautomer-inducedmisincorporationfromoccur-B.BetweenDNAandproteinmolecules(eg,histones)
334336/CHAPTER36Table36–9.MechanismofDNArepairCH3CH33′5′MechanismProblemSolution5′3′MismatchCopyingerrors(singleMethyl-directedSINGLE-SITESTRANDCUTrepairbaseortwo-tofive-strandcutting,exo-BYGATCENDONUCLEASEbaseunpairedloops)nucleasedigestion,andreplacementCH3CH33′5′BaseSpontaneous,chem-BaseremovalbyN-excision-ical,orradiationdam-glycosylase,abasic5′3′repairagetoasinglebasesugarremoval,re-DEFECTREMOVEDplacementBYEXONUCLEASENucleotideSpontaneous,chem-Removalofanap-CH3CH3excision-ical,orradiationdam-proximately30-3′5′repairagetoaDNAsegmentnucleotideoligomerandreplacement5′3′DEFECTREPAIREDDouble-Ionizingradiation,Synapsis,unwind-BYPOLYMERASEstrandchemotherapy,ing,alignment,breakrepairoxidativefreeligationCH3CH3radicals3′5′RELIGATEDBYLIGASEMismatchRepairCH3CH3MismatchrepaircorrectserrorsmadewhenDNAis3′5′copied.Forexample,aCcouldbeinsertedoppositeanA,orthepolymerasecouldsliporstutterandinserttwo5′3′tofiveextraunpairedbases.SpecificproteinsscantheFigure36–22.MismatchrepairofDNA.Thismecha-newlysynthesizedDNA,usingadeninemethylationnismcorrectsasinglemismatchbasepair(eg,CtoAwithinaGATCsequenceasthepointofreference(Fig-ratherthanTtoA)orashortregionofunpairedDNA.ure36–22).Thetemplatestrandismethylated,andthenewlysynthesizedstrandisnot.ThisdifferenceallowsThedefectiveregionisrecognizedbyanendonucleasetherepairenzymestoidentifythestrandthatcontainsthatmakesasingle-strandcutatanadjacentmethy-theerrantnucleotidewhichrequiresreplacement.IfalatedGATCsequence.TheDNAstrandisremovedmismatchorsmallloopisfound,aGATCendonucle-throughthemutation,replaced,andreligated.asecutsthestrandbearingthemutationatasitecorre-spondingtotheGATC.AnexonucleasethendigeststhisstrandfromtheGATCthroughthemutation,thusEcoliMutSproteinthatisinvolvedinmismatchrepairremovingthefaultyDNA.Thiscanoccurfromeither(seeabove).MutationsofhMSH2accountfor50–60%endifthedefectisbracketedbytwoGATCsites.ThisofHNPCCcases.Anothergene,hMLH1,isassociateddefectisthenfilledinbynormalcellularenzymesac-withmostoftheothercases.hMLH1isthehumanana-cordingtobasepairingrules.InEcoli,threeproteinslogofthebacterialmismatchrepairgeneMutL.How(MutS,MutC,andMutH)arerequiredforrecogni-doesfaultymismatchrepairresultincoloncancer?Thetionofthemutationandnickingofthestrand.Otherhumangeneswerelocalizedbecausemicrosatellitein-cellularenzymes,includingligase,polymerase,andstabilitywasdetected.Thatis,thecancercellshadami-SSBs,removeandreplacethestrand.Theprocessiscrosatelliteofalengthdifferentfromthatfoundinthesomewhatmorecomplicatedinmammaliancells,asnormalcellsoftheindividual.Itappearsthattheaf-aboutsixproteinsareinvolvedinthefirststeps.fectedcells,whichharboramutatedhMSH2orFaultymismatchrepairhasbeenlinkedtoheredi-hMLH1mismatchrepairenzyme,areunabletoremovetarynonpolyposiscoloncancer(HNPCC),oneofthesmallloopsofunpairedDNA,andthemicrosatellitemostcommoninheritedcancers.Geneticstudieslinkedthusincreasesinsize.Ultimately,microsatelliteDNAHNPCCinsomefamiliestoaregionofchromosomeexpansionmustaffecteithertheexpressionorthefunc-2.Thegenelocated,designatedhMSH2,wassub-tionofaproteincriticalinsurveillanceofthecellcyclesequentlyshowntoencodethehumananalogoftheinthesecoloncells.
335DNAORGANIZATION,REPLICATION,&REPAIR/337BaseExcision-Repair3′5′ATCGGCTCATCCGATThedepurinationofDNA,whichhappenssponta-neouslyowingtothethermallabilityofthepurineN-TAGCCGAGTAGGCTA5′3′glycosidicbond,occursatarateof5000–10,000/cell/dat37°C.SpecificenzymesrecognizeadepurinatedsiteHeatenergyandreplacetheappropriatepurinedirectly,withoutin-ATCGGCTUATCCGATterruptionofthephosphodiesterbackbone.Cytosine,adenine,andguaninebasesinDNAspon-TAGCCGAGTAGGCTAtaneouslyformuracil,hypoxanthine,orxanthine,re-spectively.SincenoneofthesenormallyexistinDNA,UURACILDNAGLYCOSYLASEitisnotsurprisingthatspecificN-glycosylasescanrec-ognizetheseabnormalbasesandremovethebaseitselfATCGGCT*ATCCGATfromtheDNA.Thisremovalmarksthesiteofthede-fectandallowsanapurinicorapyrimidinicendonu-TAGCCGAGTAGGCTAcleasetoexcisetheabasicsugar.TheproperbaseisthenreplacedbyarepairDNApolymerase,andaligaseNUCLEASESreturnstheDNAtoitsoriginalstate(Figure36–23).ATCGGCTCCGATThisseriesofeventsiscalledbaseexcision-repair.ByasimilarseriesofstepsinvolvinginitiallytherecognitionTAGCCGAGTAGGCTAofthedefect,alkylatedbasesandbaseanalogscanbere-movedfromDNAandtheDNAreturnedtoitsorigi-DNAPOLYMERASE+DNALIGASEnalinformationalcontent.ThismechanismissuitableforreplacementofasinglebasebutisnoteffectiveatATCGGCTCATCCGATreplacingregionsofdamagedDNA.TAGCCGAGTAGGCTANucleotideExcision-RepairFigure36–23.Baseexcision-repairofDNA.Theen-ThismechanismisusedtoreplaceregionsofdamagedzymeuracilDNAglycosylaseremovestheuracilcreatedDNAupto30basesinlength.CommonexamplesofbyspontaneousdeaminationofcytosineintheDNA.AnDNAdamageincludeultraviolet(UV)light,whichin-endonucleasecutsthebackbonenearthedefect;then,ducestheformationofcyclobutanepyrimidine-pyrimi-afteranendonucleaseremovesafewbases,thedefectdinedimers,andsmoking,whichcausesformationofisfilledinbytheactionofarepairpolymeraseandthebenzo[a]pyrene-guanineadducts.Ionizingradiation,strandisrejoinedbyaligase.(CourtesyofBAlberts.)cancerchemotherapeuticagents,andavarietyofchemi-calsfoundintheenvironmentcausebasemodification,strandbreaks,cross-linkagebetweenbasesonoppositestrandsorbetweenDNAandprotein,andnumerousmarkedsensitivitytosunlight(ultraviolet)withsubse-otherdefects.Thesearerepairedbyaprocesscallednu-quentformationofmultipleskincancersandprema-cleotideexcision-repair(Figure36–24).Thiscomplexturedeath.Theriskofdevelopingskincancerisin-process,whichinvolvesmoregeneproductsthanthetwocreased1000-to2000-fold.Theinheriteddefectseemsothertypesofrepair,essentiallyinvolvesthehydrolysisoftoinvolvetherepairofdamagedDNA,particularlytwophosphodiesterbondsonthestrandcontainingthethyminedimers.Cellsculturedfrompatientswithxero-defect.Aspecialexcisionnuclease(exinuclease),consist-dermapigmentosumexhibitlowactivityforthenu-ingofatleastthreesubunitsinEcoliand16polypep-cleotideexcision-repairprocess.Sevencomplementa-tidesinhumans,accomplishesthistask.Ineukaryotictiongroupshavebeenidentifiedusinghybridcellcellstheenzymescutbetweenthethirdtofifthphospho-analyses,soatleastsevengeneproducts(XPA–XPG)diesterbond3′fromthelesion,andonthe5′sidethecutareinvolved.Twoofthese(XPAandXPC)arein-issomewherebetweenthetwenty-firstandtwenty-fifthvolvedinrecognitionandexcision.XPBandXPDarebonds.Thus,afragmentofDNA27–29nucleotideshelicasesand,interestingly,aresubunitsofthetran-longisexcised.Afterthestrandisremoveditisreplaced,scriptionfactorTFIIH(seeChapter37).againbyexactbasepairing,throughtheactionofyetan-otherpolymerase(δ/εinhumans),andtheendsareDouble-StrandBreakRepairjoinedtotheexistingstrandsbyDNAligase.Xerodermapigmentosum(XP)isanautosomalre-Therepairofdouble-strandbreaksispartofthephysio-cessivegeneticdisease.Theclinicalsyndromeincludeslogicprocessofimmunoglobulingenerearrangement.It
336338/CHAPTER363′5′ase;andthegapsarefilledandclosedbyDNAligase.ThisrepairmechanismisillustratedinFigure36–25.5′3′RECOGNITIONANDUNWINDINGSomeRepairEnzymesAreMultifunctionalSomewhatsurprisingistherecentobservationthat3′5′DNArepairproteinscanserveotherpurposes.Forex-ample,somerepairenzymesarealsofoundascompo-5′3′nentsofthelargeTFIIHcomplexthatplaysacentralroleingenetranscription(Chapter37).Anothercom-OLIGONUCLEOTIDEEXCISIONponentofTFIIHisinvolvedincellcycleregulation.BYCUTTINGATTWOSITESThus,threecriticalcellularprocessesmaybelinked3′5′throughuseofcommonproteins.Thereisalsogoodevidencethatsomerepairenzymesareinvolvedingene5′3′rearrangementsthatoccurnormally.Inpatientswithataxia-telangiectasia,anautosomalDEGRADATIONOFMUTATEDDNArecessivediseaseinhumansresultinginthedevelopmentRESYNTHESISANDRELIGATIONofcerebellarataxiaandlymphoreticularneoplasms,3′5′thereappearstoexistanincreasedsensitivitytodamagebyx-ray.PatientswithFanconi’sanemia,anautosomal5′3′recessiveanemiacharacterizedalsobyanincreasedfre-Figure36–24.Nucleotideexcision-repair.Thisquencyofcancerandbychromosomalinstability,prob-mechanismisemployedtocorrectlargerdefectsinablyhavedefectiverepairofcross-linkingdamage.DNAandgenerallyinvolvesmoreproteinsthaneithermismatchorbaseexcision-repair.Afterdefectrecogni-tion(indicatedbyXXXX)andunwindingoftheDNAen-compassingthedefect,anexcisionnuclease(exinucle-KuandDNA-PKbindase)cutstheDNAupstreamanddownstreamofthedefectiveregion.Thisgapisthenfilledinbyapoly-merase(δ/εinhumans)andreligated.ApproximationisalsoanimportantmechanismforrepairingdamagedPDNA,suchasoccursasaresultofionizingradiationorPoxidativefreeradicalgeneration.Somechemotherapeu-Unwindingticagentsdestroycellsbycausingdsbreaksorprevent-ingtheirrepair.Twoproteinsareinitiallyinvolvedinthenonho-mologousrejoiningofadsbreak.Ku,aheterodimerof70kDaand86kDasubunits,bindstofreeDNAendsAlignmentandbasepairingandhaslatentATP-dependenthelicaseactivity.TheDNA-boundKuheterodimerrecruitsauniqueproteinkinase,DNA-dependentproteinkinase(DNA-PK).DNA-PKhasabindingsiteforDNAfreeendsandan-otherfordsDNAjustinsidetheseends.Itthereforeal-Ligationlowsfortheapproximationofthetwoseparatedends.ThefreeendDNA-Ku-DNA-PKcomplexactivatesthekinaseactivityinthelatter.DNA-PKreciprocallyphos-Figure36–25.Double-strandbreakrepairofDNA.phorylatesKuandtheotherDNA-PKmolecule,ontheTheproteinsKuandDNA-dependentproteinkinaseopposingstrand,intrans.DNA-PKthendissociatescombinetoapproximatethetwostrandsandunwindfromtheDNAandKu,resultinginactivationofthethem.Thealignedfragmentsformbasepairs;theextraKuhelicase.Thisresultsinunwindingofthetwoends.endsareremoved,probablybyaDNA-PK-associatedTheunwound,approximatedDNAformsbasepairs;endo-orexonuclease,andthegapsarefilledin;andtheextranucleotidetailsareremovedbyanexonucle-continuityisrestoredbyligation.
337DNAORGANIZATION,REPLICATION,&REPAIR/339Allthreeoftheseclinicalsyndromesareassociated•MuchoftheDNAisassociatedwithhistoneproteinswithanincreasedfrequencyofcancer.Itislikelythattoformastructurecalledthenucleosome.Nucleo-otherhumandiseasesresultingfromdisorderedDNAsomesarecomposedofanoctamerofhistonesandrepaircapabilitieswillbefoundinthefuture.150bpofDNA.•Nucleosomesandhigher-orderstructuresformedDNA&ChromosomeIntegrityIsfromthemservetocompacttheDNA.MonitoredThroughouttheCellCycle•Asmuchas90%ofDNAmaybetranscriptionallyinactiveasaresultofbeingnuclease-resistant,highlyGiventheimportanceofnormalDNAandchromosomecompacted,andnucleosome-associated.functiontosurvival,itisnotsurprisingthateukaryoticcellshavedevelopedelaboratemechanismstomonitor•DNAintranscriptionallyactiveregionsissensitivetotheintegrityofthegeneticmaterial.Asdetailedabove,anucleaseattack;someregionsareexceptionallysensi-numberofcomplexmulti-subunitenzymesystemshavetiveandareoftenfoundtocontaintranscriptionevolvedtorepairdamagedDNAatthenucleotidese-controlsites.quencelevel.Similarly,DNAmishapsatthechromo-•TranscriptionallyactiveDNA(thegenes)isoftensomelevelarealsomonitoredandrepaired.Asshowninclusteredinregionsofeachchromosome.WithinFigure36–20,DNAintegrityandchromosomalin-theseregions,genesmaybeseparatedbyinactivetegrityarecontinuouslymonitoredthroughoutthecellDNAinnucleosomalstructures.Thetranscriptioncycle.Thefourspecificstepsatwhichthismonitoringunit—thatportionofagenethatiscopiedbyRNAoccurshavebeentermedcheckpointcontrols.Ifprob-polymerase—consistsofcodingregionsofDNAlemsaredetectedatanyofthesecheckpoints,progression(exons)interruptedbyinterveningsequencesofnon-throughthecycleisinterruptedandtransitthroughthecodingDNA(introns).cellcycleishalteduntilthedamageisrepaired.Themol-•Aftertranscription,duringRNAprocessing,intronsecularmechanismsunderlyingdetectionofDNAdam-areremovedandtheexonsareligatedtogethertoageduringtheG1andG2phasesofthecycleareunder-formthematuremRNAthatappearsinthecyto-stoodbetterthanthoseoperativeduringSandMphases.plasm.Thetumorsuppressorp53,aproteinofMW53•DNAineachchromosomeisexactlyreplicatedac-kDa,playsakeyroleinbothG1andG2checkpointcon-cordingtotherulesofbasepairingduringtheStrol.Normallyaveryunstableprotein,p53isaDNAphaseofthecellcycle.bindingtranscriptionfactor,oneofafamilyofrelated•Eachstrandofthedoublehelixisreplicatedsimulta-proteins,thatissomehowstabilizedinresponsetoDNAneouslybutbysomewhatdifferentmechanisms.Adamage,perhapsbydirectp53-DNAinteractions.In-complexofproteins,includingDNApolymerase,creasedlevelsofp53activatetranscriptionofanensemblereplicatestheleadingstrandcontinuouslyinthe5′toofgenesthatcollectivelyservetodelaytransitthroughtheCIP3′direction.Thelaggingstrandisreplicateddiscon-cycle.Oneoftheseinducedproteins,p21,isapotenttinuously,inshortpiecesof150–250nucleotides,inCDK-cyclininhibitor(CKI)thatiscapableofefficientlythe3′to5′direction.inhibitingtheactionofallCDKs.Clearly,inhibitionofCDKswillhaltprogressionthroughthecellcycle(see•DNAreplicationoccursatseveralsites—calledrepli-Figures36–19and36–20).IfDNAdamageistooexten-cationbubbles—ineachchromosome.Theentiresivetorepair,theaffectedcellsundergoapoptosis(pro-processtakesabout9hoursinatypicalcell.grammedcelldeath)inap53-dependentfashion.Inthis•Avarietyofmechanismsemployingdifferenten-case,p53inducestheactivationofacollectionofgeneszymesrepairdamagedDNA,asafterexposuretothatinduceapoptosis.Cellslackingfunctionalp53failtochemicalmutagensorultravioletradiation.undergoapoptosisinresponsetohighlevelsofradiationorDNA-activechemotherapeuticagents.ItmaycomeasREFERENCESnosurprise,then,thatp53isoneofthemostfrequentlymutatedgenesinhumancancers.AdditionalresearchintoDePamphilisML:OriginsofDNAreplicationinmetazoanchro-themechanismsofcheckpointcontrolwillproveinvalu-mosomes.JBiolChem1993;268:1.ableforthedevelopmentofeffectiveanticancertherapeu-HartwellLH,KastanMB:Cellcyclecontrolandcancer.Science1994;266:1821.ticoptions.JenuweinT,AllisCD:Translatingthehistonecode.Science2001;293:1074.SUMMARYLanderESetal:Initialsequencingandanalysisofthehumangenome.Nature2001;409:860.•DNAineukaryoticcellsisassociatedwithavarietyLugerLetal:Crystalstructureofthenucleosomecoreparticleatofproteins,resultinginastructurecalledchromatin.2.8Åresolution.Nature1997;398:251.
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339RNASynthesis,Processing,&Modification37DarylK.Granner,MD,&P.AnthonyWeil,PhDBIOMEDICALIMPORTANCEfollowing:(1)ribonucleotidesareusedinRNAsynthe-sisratherthandeoxyribonucleotides;(2)UreplacesTThesynthesisofanRNAmoleculefromDNAisaasthecomplementarybasepairforAinRNA;(3)acomplexprocessinvolvingoneofthegroupofRNAprimerisnotinvolvedinRNAsynthesis;(4)onlyaverypolymeraseenzymesandanumberofassociatedpro-smallportionofthegenomeistranscribedorcopiedteins.Thegeneralstepsrequiredtosynthesizethepri-intoRNA,whereastheentiregenomemustbecopiedmarytranscriptareinitiation,elongation,andtermina-duringDNAreplication;and(5)thereisnoproofread-tion.Mostisknownaboutinitiation.AnumberofingfunctionduringRNAtranscription.DNAregions(generallylocatedupstreamfromtheini-TheprocessofsynthesizingRNAfromaDNAtem-tiationsite)andproteinfactorsthatbindtothesese-platehasbeencharacterizedbestinprokaryotes.Al-quencestoregulatetheinitiationoftranscriptionhavethoughinmammaliancellstheregulationofRNAsyn-beenidentified.CertainRNAs—mRNAsinparticu-thesisandtheprocessingoftheRNAtranscriptsarelar—haveverydifferentlifespansinacell.Itisimpor-differentfromthoseinprokaryotes,theprocessofRNAtanttounderstandthebasicprinciplesofmessengersynthesisperseisquitesimilarinthesetwoclassesofRNAsynthesisandmetabolism,formodulationofthisorganisms.Therefore,thedescriptionofRNAsynthesisprocessresultsinalteredratesofproteinsynthesisandinprokaryotes,whereitisbetterunderstood,isapplica-thusavarietyofmetabolicchanges.Thisishowallor-bletoeukaryoteseventhoughtheenzymesinvolvedganismsadapttochangesofenvironment.Itisalsohowandtheregulatorysignalsaredifferent.differentiatedcellstructuresandfunctionsareestab-lishedandmaintained.TheRNAmoleculessynthe-sizedinmammaliancellsaremadeasprecursormole-culesthathavetobeprocessedintomature,activeTheTemplateStrandofDNARNA.Errorsorchangesinsynthesis,processing,andIsTranscribedsplicingofmRNAtranscriptsareacauseofdisease.ThesequenceofribonucleotidesinanRNAmoleculeiscomplementarytothesequenceofdeoxyribonu-RNAEXISTSINFOURMAJORCLASSEScleotidesinonestrandofthedouble-strandedDNAAlleukaryoticcellshavefourmajorclassesofRNA:ri-molecule(Figure35–8).ThestrandthatistranscribedbosomalRNA(rRNA),messengerRNA(mRNA),trans-orcopiedintoanRNAmoleculeisreferredtoastheferRNA(tRNA),andsmallnuclearRNA(snRNA).templatestrandoftheDNA.TheotherDNAstrandisThefirstthreeareinvolvedinproteinsynthesis,andfrequentlyreferredtoasthecodingstrandofthatgene.snRNAisinvolvedinmRNAsplicing.AsshowninItiscalledthisbecause,withtheexceptionofTforUTable37–1,thesevariousclassesofRNAaredifferentchanges,itcorrespondsexactlytothesequenceoftheintheirdiversity,stability,andabundanceincells.primarytranscript,whichencodestheproteinproductofthegene.Inthecaseofadouble-strandedDNAmol-RNAISSYNTHESIZEDFROMADNAeculecontainingmanygenes,thetemplatestrandforeachgenewillnotnecessarilybethesamestrandoftheTEMPLATEBYANRNAPOLYMERASEDNAdoublehelix(Figure37–1).Thus,agivenstrandTheprocessesofDNAandRNAsynthesisaresimilarofadouble-strandedDNAmoleculewillserveastheinthattheyinvolve(1)thegeneralstepsofinitiation,templatestrandforsomegenesandthecodingstrandelongation,andterminationwith5′to3′polarity;(2)ofothergenes.Notethatthenucleotidesequenceofanlarge,multicomponentinitiationcomplexes;and(3)RNAtranscriptwillbethesame(exceptforUreplacingadherencetoWatson-Crickbase-pairingrules.TheseT)asthatofthecodingstrand.Theinformationintheprocessesdifferinseveralimportantways,includingthetemplatestrandisreadoutinthe3′to5′direction.341
340342/CHAPTER37Table37–1.ClassesofeukaryoticRNA.RNAtranscript5′P-P-Pβ′RNATypesAbundanceStabilityβRibosomal28S,18S,5.8S,5S80%oftotalVerystable3′3OH′Transcription(rRNA)5′5Messenger~10different2–5%oftotalUnstableto(mRNA)speciesveryσαα5′stable3′Transfer~60different~15%oftotalVerystable(tRNA)speciesRNAPcomplexSmallnuclear~30different≤1%oftotalVerystableFigure37–2.RNApolymerase(RNAP)catalyzesthe(snRNA)speciespolymerizationofribonucleotidesintoanRNAse-quencethatiscomplementarytothetemplatestrandDNA-DependentRNAPolymeraseofthegene.TheRNAtranscripthasthesamepolarity(5′to3′)asthecodingstrandbutcontainsUratherInitiatesTranscriptionataDistinctthanT.EcoliRNAPconsistsofacorecomplexoftwoSite,thePromoterαsubunitsandtwoβsubunits(βandβ′).Theholoen-DNA-dependentRNApolymeraseistheenzymere-zymecontainstheσsubunitboundtotheα2ββ′coresponsibleforthepolymerizationofribonucleotidesintoassembly.Theωsubunitisnotshown.Thetranscriptionasequencecomplementarytothetemplatestrandof“bubble”isanapproximately20-bpareaofmeltedthegene(seeFigures37–2and37–3).Theenzymeat-DNA,andtheentirecomplexcovers30–75bp,depend-tachesataspecificsite—thepromoter—onthetem-ingontheconformationofRNAP.platestrand.ThisisfollowedbyinitiationofRNAsyn-thesisatthestartingpoint,andtheprocesscontinuesuntilaterminationsequenceisreached(Figure37–3).(1)TemplatebindingAtranscriptionunitisdefinedasthatregionofDNApthatincludesthesignalsfortranscriptioninitiation,ATP+NTPelongation,andtermination.TheRNAproduct,whichRNAPpissynthesizedinthe5′to3′direction,istheprimary(2)Chaininitiationtranscript.Inprokaryotes,thiscanrepresenttheprod-pppApNuctofseveralcontiguousgenes;inmammaliancells,itpppApNusuallyrepresentstheproductofasinglegene.The5′pppApNNTPsterminalsoftheprimaryRNAtranscriptandthema-(5)ChainterminationturecytoplasmicRNAareidentical.Thus,thestartingandRNAPrelease(3)Promoterpointoftranscriptioncorrespondstothe5nu-pppApNclearanceNTPscleotideofthemRNA.Thisisdesignatedposition+1,asisthecorrespondingnucleotideintheDNA.The(4)ChainelongationFigure37–3.Thetranscriptioncycleinbacteria.Bac-GeneAGeneBGeneCGeneDterialRNAtranscriptionisdescribedinfoursteps:5′3′(1)Templatebinding:RNApolymerase(RNAP)binds3′5′toDNAandlocatesapromoter(P)meltsthetwoDNAstrandstoformapreinitiationcomplex(PIC).(2)ChainTemplatestrandsinitiation:RNAPholoenzyme(core+oneofmultipleFigure37–1.Thisfigureillustratesthatgenescanbesigmafactors)catalyzesthecouplingofthefirstbasetranscribedoffbothstrandsofDNA.Thearrowheadsin-(usuallyATPorGTP)toasecondribonucleosidedicatethedirectionoftranscription(polarity).Notethattriphosphatetoformadinucleotide.(3)Chainelonga-thetemplatestrandisalwaysreadinthe3′to5′direc-tion:Successiveresiduesareaddedtothe3′-OHtermi-tion.Theoppositestrandiscalledthecodingstrandbe-nusofthenascentRNAmolecule.(4)Chaintermina-causeitisidentical(exceptforTforUchanges)tothetionandrelease:ThecompletedRNAchainandRNAPmRNAtranscript(theprimarytranscriptineukaryoticarereleasedfromthetemplate.TheRNAPholoenzymecells)thatencodestheproteinproductofthegene.re-forms,findsapromoter,andthecycleisrepeated.
341RNASYNTHESIS,PROCESSING,&MODIFICATION/343numbersincreaseasthesequenceproceedsdownstream.Table37–2.NomenclatureandpropertiesofThisconventionmakesiteasytolocateparticularre-mammaliannuclearDNA-dependentRNAgions,suchasintronandexonboundaries.Thenu-polymerases.cleotideinthepromoteradjacenttothetranscriptioninitiationsiteisdesignated−1,andthesenegativenum-FormofRNASensitivitytobersincreaseasthesequenceproceedsupstream,awayPolymerase-AmanitinMajorProductsfromtheinitiationsite.ThisprovidesaconventionalwayofdefiningthelocationofregulatoryelementsinI(A)InsensitiverRNAthepromoter.II(B)HighsensitivitymRNATheprimarytranscriptsgeneratedbyRNApolym-III(C)IntermediatesensitivitytRNA/5SrRNAeraseII—oneofthreedistinctnuclearDNA-depen-dentRNApolymerasesineukaryotes—arepromptlycappedby7-methylguanosinetriphosphatecaps(Fig-ferentsetsofgenes.ThesizesoftheRNApolymerasesure35–10)thatpersistandeventuallyappearonthe5′rangefromMW500,000toMW600,000.Theseen-endofmaturecytoplasmicmRNA.Thesecapsarenec-zymesaremuchmorecomplexthanprokaryoticRNAessaryforthesubsequentprocessingoftheprimarypolymerases.TheyallhavetwolargesubunitsandatranscripttomRNA,forthetranslationofthemRNA,numberofsmallersubunits—asmanyas14inthecaseandforprotectionofthemRNAagainstexonucleolyticofRNApolIII.TheeukaryoticRNApolymeraseshaveattack.extensiveaminoacidhomologieswithprokaryoticRNApolymerases.Thishomologyhasbeenshownre-BacterialDNA-DependentRNAcentlytoextendtothelevelofthree-dimensionalstruc-PolymeraseIsaMultisubunitEnzymetures.Thefunctionsofeachofthesubunitsarenotyetfullyunderstood.Manycouldhaveregulatoryfunc-TheDNA-dependentRNApolymerase(RNAP)ofthetions,suchasservingtoassistthepolymeraseinthebacteriumEscherichiacoliexistsasanapproximatelyrecognitionofspecificsequenceslikepromotersand400kDacorecomplexconsistingoftwoidenticalαterminationsignals.subunits,similarbutnotidenticalβandβ′subunits,OnepeptidetoxinfromthemushroomAmanitaandanωsubunit.Betaisthoughttobethecatalyticphalloides,α-amanitin,isaspecificdifferentialinhibitorsubunit(Figure37–2).RNAP,ametalloenzyme,alsooftheeukaryoticnuclearDNA-dependentRNApolym-containstwozincmolecules.ThecoreRNApolymeraseerasesandassuchhasprovedtobeapowerfulresearchassociateswithaspecificproteinfactor(thesigma[σ]tool(Table37–2).α-Amanitinblocksthetranslocationfactor)thathelpsthecoreenzymerecognizeandbindofRNApolymeraseduringtranscription.tothespecificdeoxynucleotidesequenceofthepro-moterregion(Figure37–5)toformthepreinitiationcomplex(PIC).Sigmafactorshaveadualroleintheprocessofpromoterrecognition;σassociationwithRNASYNTHESISISACYCLICALPROCESScoreRNApolymerasedecreasesitsaffinityfornonpro-&INVOLVESINITIATION,ELONGATION,moterDNAwhilesimultaneouslyincreasingholoen-&TERMINATIONzymeaffinityforpromoterDNA.Bacteriacontainmul-tipleσfactors,eachofwhichactsasaregulatoryTheprocessofRNAsynthesisinbacteria—depictedinproteinthatmodifiesthepromoterrecognitionspeci-Figure37–3—involvesfirstthebindingoftheRNAficityoftheRNApolymerase.Theappearanceofdif-holopolymerasemoleculetothetemplateatthepro-ferentσfactorscanbecorrelatedtemporallywithvari-motersitetoformaPIC.Bindingisfollowedbyacon-ousprogramsofgeneexpressioninprokaryoticsystemsformationalchangeoftheRNAP,andthefirstnu-suchasbacteriophagedevelopment,sporulation,andcleotide(almostalwaysapurine)thenassociateswiththeresponsetoheatshock.theinitiationsiteontheβsubunitoftheenzyme.Inthepresenceoftheappropriatenucleotide,theRNAPMammalianCellsPossessThreecatalyzestheformationofaphosphodiesterbond,andDistinctNuclearDNA-DependentthenascentchainisnowattachedtothepolymerizationsiteontheβsubunitofRNAP.(TheanalogytotheARNAPolymerasesandPsitesontheribosomeshouldbenoted;seeFigureThepropertiesofmammalianpolymerasesarede-38–9.)scribedinTable37–2.EachoftheseDNA-dependentInitiationofformationoftheRNAmoleculeatitsRNApolymerasesisresponsiblefortranscriptionofdif-5′endthenfollows,whileelongationoftheRNAmole-
342344/CHAPTER37culefromthe5′toits3′endcontinuescyclically,an-tiparalleltoitstemplate.TheenzymepolymerizestheribonucleotidesinaspecificsequencedictatedbythetemplatestrandandinterpretedbyWatson-Crickbase-pairingrules.Pyrophosphateisreleasedinthepolymer-izationreaction.Thispyrophosphate(PPi)israpidlydegradedto2molofinorganicphosphate(Pi)byubiq-uitouspyrophosphatases,therebyprovidingirreversibil-ityontheoverallsyntheticreaction.Inbothprokary-otesandeukaryotes,apurineribonucleotideisusuallythefirsttobepolymerizedintotheRNAmolecule.Aswitheukaryotes,5′triphosphateofthisfirstnucleotideismaintainedinprokaryoticmRNA.AstheelongationcomplexcontainingthecoreRNApolymeraseprogressesalongtheDNAmolecule,DNAunwindingmustoccurinordertoprovideaccessfortheappropriatebasepairingtothenucleotidesofthecodingstrand.Theextentofthistranscriptionbub-ble(ie,DNAunwinding)isconstantthroughouttran-scriptionandhasbeenestimatedtobeabout20basepairsperpolymerasemolecule.Thus,itappearsthatthesizeoftheunwoundDNAregionisdictatedbythepolymeraseandisindependentoftheDNAsequenceinthecomplex.ThissuggeststhatRNApolymerasehasassociatedwithitan“unwindase”activitythatopensFigure37–4.ElectronphotomicrographofmultipletheDNAhelix.ThefactthattheDNAdoublehelixcopiesofamphibianribosomalRNAgenesinthemustunwindandthestrandspartatleasttransientlyprocessofbeingtranscribed.Themagnificationisfortranscriptionimpliessomedisruptionofthenucleo-somestructureofeukaryoticcells.Topoisomerasebothabout6000×.Notethatthelengthofthetranscriptsin-precedesandfollowstheprogressingRNAPtopreventcreasesastheRNApolymerasemoleculesprogresstheformationofsuperhelicalcomplexes.alongtheindividualribosomalRNAgenes;transcrip-TerminationofthesynthesisoftheRNAmoleculetionstartsites(filledcircles)totranscriptiontermina-inbacteriaissignaledbyasequenceinthetemplatetionsites(opencircles).RNApolymeraseI(notvisual-strandoftheDNAmolecule—asignalthatisrecog-izedhere)isatthebaseofthenascentrRNAtranscripts.nizedbyaterminationprotein,therho(ρ)factor.RhoThus,theproximalendofthetranscribedgenehasisanATP-dependentRNA-stimulatedhelicasethatshorttranscriptsattachedtoit,whilemuchlongertran-disruptsthenascentRNA-DNAcomplex.Aftertermi-scriptsareattachedtothedistalendofthegene.ThenationofsynthesisoftheRNAmolecule,theenzymearrowsindicatethedirection(5′to3′)oftranscription.separatesfromtheDNAtemplateandprobablydisso-(Reproducedwithpermission,fromMillerOLJr,BeattyBR:ciatestofreecoreenzymeandfreeσfactor.WiththePortraitofagene.JCellPhysiol1969;74[Suppl1]:225.)assistanceofanotherσfactor,thecoreenzymethenrecognizesapromoteratwhichthesynthesisofanewRNAmoleculecommences.Ineukaryoticcells,termi-nationislesswelldefined.ItappearstobesomehowTHEFIDELITY&FREQUENCYOFlinkedbothtoinitiationandtoadditionofthe3′TRANSCRIPTIONISCONTROLLEDpolyAtailofmRNAandcouldinvolvedestabilizationBYPROTEINSBOUNDTOCERTAINoftheRNA-DNAcomplexataregionofA–UbaseDNASEQUENCESpairs.MorethanoneRNApolymerasemoleculemaytranscribethesametemplatestrandofagenesimulta-TheDNAsequenceanalysisofspecificgeneshasal-neously,buttheprocessisphasedandspacedinsuchalowedtherecognitionofanumberofsequencesimpor-waythatatanyonemomenteachistranscribingadif-tantingenetranscription.FromthelargenumberofferentportionoftheDNAsequence.Anelectronmi-bacterialgenesstudieditispossibletoconstructcon-crographofextremelyactiveRNAsynthesisisshownsensusmodelsoftranscriptioninitiationandtermina-inFigure37–4.tionsignals.
343RNASYNTHESIS,PROCESSING,&MODIFICATION/345Thequestion,“HowdoesRNAPfindthecorrecttionstartsitethereisaconsensussequenceofeightnu-sitetoinitiatetranscription?”isnottrivialwhenthecleotidepairs(5′-TGTTGACA-3′)towhichtheRNAPcomplexityofthegenomeisconsidered.Ecolihasbindstoformtheso-calledclosedcomplex.More364×10transcriptioninitiationsitesin4×10baseproximaltothetranscriptionstartsite—abouttennu-pairs(bp)ofDNA.Thesituationisevenmorecomplexcleotidesupstream—isasix-nucleotide-pairA+T-rich5inhumans,whereperhaps10transcriptioninitiationsequence(5′-TATAAT-3′).Theseconservedsequence9sitesaredistributedthroughoutin3×10bpofDNA.elementscomprisingthepromoterareshownschemati-RNAPcanbindtomanyregionsofDNA,butitscanscallyinFigure37–5.Thelattersequencehasalow3theDNAsequence—atarateof≥10bp/s—untilitmeltingtemperaturebecauseofitsdeficiencyofGCrecognizescertainspecificregionsofDNAtowhichitnucleotidepairs.Thus,theTATAboxisthoughttobindswithhigheraffinity.Thisregioniscalledthepro-easethedissociationbetweenthetwoDNAstrandssomoter,anditistheassociationofRNAPwiththepro-thatRNApolymeraseboundtothepromoterregionmoterthatensuresaccurateinitiationoftranscription.canhaveaccesstothenucleotidesequenceofitsimme-Thepromoterrecognition-utilizationprocessisthetar-diatelydownstreamtemplatestrand.Oncethisprocessgetforregulationinbothbacteriaandhumans.occurs,thecombinationofRNApolymerasepluspro-moteriscalledtheopencomplex.Otherbacteriahaveslightlydifferentconsensussequencesintheirpromot-BacterialPromotersAreRelativelySimpleers,butallgenerallyhavetwocomponentstothepro-Bacterialpromotersareapproximately40nucleotidesmoter;thesetendtobeinthesamepositionrelativeto(40bporfourturnsoftheDNAdoublehelix)inthetranscriptionstartsite,andinallcasesthesequenceslength,aregionsmallenoughtobecoveredbyanbetweentheboxeshavenosimilaritybutstillprovideEcoliRNAholopolymerasemolecule.Inthisconsensuscriticalspacingfunctionsfacilitatingrecognitionof−35promoterregionaretwoshort,conservedsequenceele-and−10sequencebyRNApolymeraseholoenzyme.ments.Approximately35bpupstreamofthetranscrip-Withinabacterialcell,differentsetsofgenesareoftenTRANSCRIPTIONUNITPromoterTranscribedregionTranscriptionstartsite+1Codingstrand5′Termination3′TGTTGACATATAATDNATemplatestrand3′signals5′−35−10regionregionOHPPPRNA3′5′5′Flanking3′FlankingsequencessequencesFigure37–5.Bacterialpromoters,suchasthatfromEcolishownhere,sharetworegionsofhighlyconservednucleotidesequence.Theseregionsarelocated35and10bpupstream(inthe5′directionofthecodingstrand)fromthestartsiteoftranscription,whichisindicatedas+1.Byconvention,allnucleotidesupstreamofthetranscriptioninitiationsite(at+1)arenum-beredinanegativesenseandarereferredtoas5′-flankingsequences.Alsobyconvention,theDNAregulatorysequenceelements(TATAbox,etc)aredescribedinthe5′to3′directionandasbeingonthecodingstrand.Theseelementsfunctiononlyindouble-strandedDNA,however.Notethatthetranscriptproducedfromthistranscriptionunithasthesamepolarityor“sense”(ie,5′to3′orientation)asthecodingstrand.Terminationcis-elementsresideattheendofthetranscriptionunit(seeFigure37–6formoredetail).Byconventionthesequencesdownstreamofthesiteatwhichtranscriptionterminationoccursaretermed3′-flankingsequences.
344346/CHAPTER37coordinatelyregulated.Oneimportantwaythatthisissimplexvirus,whichutilizestranscriptionfactorsofitsaccomplishedisthroughthefactthattheseco-regulatedmammalianhostforgeneexpression,thereisasinglegenesshareunique−35and−10promotersequences.uniquetranscriptionstartsite,andaccuratetranscriptionTheseuniquepromotersarerecognizedbydifferentσfromthisstartsitedependsuponanucleotidesequencefactorsboundtocoreRNApolymerase.located32nucleotidesupstreamfromthestartsite(ie,atRho-dependenttranscriptionterminationsignals−32)(Figure37–7).ThisregionhasthesequenceofinEcolialsoappeartohaveadistinctconsensusse-TATAAAAGandbearsremarkablesimilaritytothequence,asshowninFigure37–6.Theconservedcon-functionallyrelatedTATAboxthatislocatedabout10sensussequence,whichisabout40nucleotidepairsinbpupstreamfromtheprokaryoticmRNAstartsite(Fig-length,canbeseentocontainahyphenatedorinter-ure37–5).MutationorinactivationoftheTATAboxruptedinvertedrepeatfollowedbyaseriesofATbasemarkedlyreducestranscriptionofthisandmanyotherpairs.Astranscriptionproceedsthroughthehyphen-genesthatcontainthisconsensusciselement(seeFiguresated,invertedrepeat,thegeneratedtranscriptcanform37–7,37–8).MostmammaliangeneshaveaTATAboxtheintramolecularhairpinstructure,alsodepictedinthatisusuallylocated25–30bpupstreamfromthetran-Figure37–6.scriptionstartsite.TheconsensussequenceforaTATATranscriptioncontinuesintotheATregion,andboxisTATAAA,thoughnumerousvariationshavebeenwiththeaidoftheρterminationproteintheRNAcharacterized.TheTATAboxisboundby34kDapolymerasestops,dissociatesfromtheDNAtemplate,TATAbindingprotein(TBP),whichinturnbindssev-andreleasesthenascenttranscript.eralotherproteinscalledTBP-associatedfactors(TAFs).ThiscomplexofTBPandTAFsisreferredtoasEukaryoticPromotersAreMoreComplexTFIID.BindingofTFIIDtotheTATAboxsequenceisthoughttorepresentthefirststepintheformationoftheItisclearthatthesignalsinDNAwhichcontroltran-transcriptioncomplexonthepromoter.scriptionineukaryoticcellsareofseveraltypes.TwoAsmallnumberofgeneslackaTATAbox.Insuchtypesofsequenceelementsarepromoter-proximal.Oneinstances,twoadditionalciselements,aninitiatorse-ofthesedefineswheretranscriptionistocommencequence(Inr)andtheso-calleddownstreampromoteralongtheDNA,andtheothercontributestothemecha-element(DPE),directRNApolymeraseIItothepro-nismsthatcontrolhowfrequentlythiseventistooccur.moterandinsodoingprovidebasaltranscriptionstart-Forexample,inthethymidinekinasegeneoftheherpesingfromthecorrectsite.TheInrelementspansthestartDirectionoftranscriptionCodingstrand5′AGCCCGCGCGGGCTTTTTTTTT3′DNATemplatestrand3′TCGGGCGCGCCCGAAAAAAAAA5′TTTTTTTTCodingstrand5′3′DNATemplatestrand3′AAAAAAAA5′UUUUUUU-3′UAUGCCGCGCGGCCGRNAtranscript5′Figure37–6.Thepredominantbacterialtranscriptionterminationsignalcontainsaninverted,hyphenatedre-peat(thetwoboxedareas)followedbyastretchofATbasepairs(topfigure).Theinvertedrepeat,whentran-scribedintoRNA,cangeneratethesecondarystructureintheRNAtranscriptshownatthebottomofthefigure.FormationofthisRNAhairpincausesRNApolymerasetopauseandsubsequentlytheρterminationfactorinter-actswiththepausedpolymeraseandsomehowinduceschaintermination.
345RNASYNTHESIS,PROCESSING,&MODIFICATION/347PromoterproximalPromoterupstreamelementsTFIIDSp1+1GCCAATGCTATAboxtkcodingregionCTF−25Sp1Figure37–7.Transcriptionelementsandbindingfactorsintheherpessimplexvirusthymidineki-nase(tk)gene.DNA-dependentRNApolymeraseIIbindstotheregionoftheTATAbox(whichisboundbytranscriptionfactorTFIID)toformamulticomponentpreinitiationcomplexcapableofinitiatingtranscriptionatasinglenucleotide(+1).Thefrequencyofthiseventisincreasedbythepresenceofup-streamcis-actingelements(theGCandCAATboxes).Theseelementsbindtrans-actingtranscriptionfactors,inthisexampleSp1andCTF(alsocalledC/EBP,NF1,NFY).Theseciselementscanfunctioninde-pendentlyoforientation(arrows).Regulatedexpression“Basal”expressionDistalPromoterregulatoryproximalPromoterelementselements+1OtherEnhancer(+)Promoterregulatoryandproximalelementsrepressor(−)elementsTATAInrDPECodingregionelements(GC/CAAT,etc)Figure37–8.SchematicdiagramshowingthetranscriptioncontrolregionsinahypotheticalclassII(mRNA-producing)eukaryoticgene.Suchagenecanbedividedintoitscodingandregulatoryregions,asdefinedbythetranscriptionstartsite(arrow;+1).ThecodingregioncontainstheDNAsequencethatistranscribedintomRNA,whichisultimatelytranslatedintoprotein.Theregulatoryregionconsistsoftwoclassesofelements.Oneclassisresponsibleforensuringbasalexpression.Theseelementsgener-allyhavetwocomponents.Theproximalcomponent,generallytheTATAbox,orInrorDPEelementsdi-rectRNApolymeraseIItothecorrectsite(fidelity).InTATA-lesspromoters,aninitiator(Inr)elementthatspanstheinitiationsite(+1)maydirectthepolymerasetothissite.Anothercomponent,theupstreamelements,specifiesthefrequencyofinitiation.AmongthebeststudiedoftheseistheCAATbox,butseveralotherelements(Sp1,NF1,AP1,etc)maybeusedinvariousgenes.Asecondclassofregulatorycis-actingelementsisresponsibleforregulatedexpression.Thisclassconsistsofelementsthatenhanceorrepressexpressionandofothersthatmediatetheresponsetovarioussignals,includinghormones,heatshock,heavymetals,andchemicals.Tissue-specificexpressionalsoinvolvesspecificsequencesofthissort.Theorientationdependenceofalltheelementsisindicatedbythearrowswithintheboxes.Forexample,theproximalelement(theTATAbox)mustbeinthe5′to3′orientation.Theupstreamele-mentsworkbestinthe5′to3′orientation,butsomeofthemcanbereversed.Thelocationsofsomeel-ementsarenotfixedwithrespecttothetranscriptionstartsite.Indeed,someelementsresponsibleforregulatedexpressioncanbelocatedeitherinterspersedwiththeupstreamelements,ortheycanbelo-cateddownstreamfromthestartsite.
346348/CHAPTER37site(from−3to+5)andconsistsofthegeneralconsen-belowandFigures37–9and37–10).Theprotein-sussequenceTCA+1G/TTT/CwhichissimilartotheDNAinteractionattheTATAboxinvolvingRNAinitiationsitesequenceperse.(A+1indicatesthefirstpolymeraseIIandothercomponentsofthebasaltran-nucleotidetranscribed.)TheproteinsthatbindtoInrinscriptionmachineryensuresthefidelityofinitiation.ordertodirectpolIIbindingincludeTFIID.PromotersTogether,then,thepromoterandpromoter-proxi-thathavebothaTATAboxandanInrmaybestrongermalcis-activeupstreamelementsconferfidelityandfre-thanthosethathavejustoneoftheseelements.Thequencyofinitiationuponagene.TheTATAboxhasaDPEhastheconsensussequenceA/GGA/TCGTGandparticularlyrigidrequirementforbothpositionandori-islocalizedabout25bpdownstreamofthe+1startsite.entation.Single-basechangesinanyofthesecisele-LiketheInr,DPEsequencesarealsoboundbytheTAFmentshavedramaticeffectsonfunctionbyreducingsubunitsofTFIID.Inasurveyofover200eukaryoticthebindingaffinityofthecognatetransfactors(eithergenes,roughly30%containedaTATAboxandInr,TFIID/TBPorSp1,CTF,andsimilarfactors).The25%containedInrandDPE,15%containedallthreespacingoftheseelementswithrespecttothetranscrip-elements,while~30%containedjusttheInr.tionstartsitecanalsobecritical.ThisisparticularlySequencesfartherupstreamfromthestartsitedeter-truefortheTATAboxInrandDPE.minehowfrequentlythetranscriptioneventoccurs.AthirdclassofsequenceelementscaneitherincreaseMutationsintheseregionsreducethefrequencyofordecreasetherateoftranscriptioninitiationofeukary-transcriptionalstartstenfoldtotwentyfold.Typicalofoticgenes.TheseelementsarecalledeitherenhancersortheseDNAelementsaretheGCandCAATboxes,sorepressors(orsilencers),dependingonwhicheffectnamedbecauseoftheDNAsequencesinvolved.Asil-theyhave.TheyhavebeenfoundinavarietyoflocationslustratedinFigure37–7,eachoftheseboxesbindsabothupstreamanddownstreamofthetranscriptionstartprotein,Sp1inthecaseoftheGCboxandCTF(orsiteandevenwithinthetranscribedportionsofsomeC/EPB,NF1,NFY)bytheCAATbox;bothbindgenes.Incontrasttoproximalandupstreampromoterel-throughtheirdistinctDNAbindingdomains(DBDs).ements,enhancersandsilencerscanexerttheireffectsThefrequencyoftranscriptioninitiationisaconse-whenlocatedhundredsoreventhousandsofbasesawayquenceoftheseprotein-DNAinteractionsandcomplexfromtranscriptionunitslocatedonthesamechromo-interactionsbetweenparticulardomainsofthetran-some.Surprisingly,enhancersandsilencerscanfunctionscriptionfactors(distinctfromtheDBDdomains—so-inanorientation-independentfashion.Literallyhun-calledactivationdomains;ADs)oftheseproteinsanddredsoftheseelementshavebeendescribed.Insometherestofthetranscriptionmachinery(RNApolym-cases,thesequencerequirementsforbindingarerigidlyeraseIIandthebasalfactorsTFIIA,B,D,E,F).(Seeconstrained;inothers,considerablesequencevariationisHFAEDBTATApolII–50–30–10+10+30+50Figure37–9.Theeukaryoticbasaltranscriptioncomplex.FormationofthebasaltranscriptioncomplexbeginswhenTFIIDbindstotheTATAbox.Itdirectstheassemblyofseveralothercomponentsbyprotein-DNAandprotein-proteininteractions.TheentirecomplexspansDNAfromposition−30to+30relativetotheinitiationsite(+1,markedbybentarrow).Theatomiclevel,x-ray-derivedstructuresofRNApolymeraseIIaloneandofTBPboundtoTATApromoterDNAinthepresenceofeitherTFIIBorTFIIAhaveallbeensolvedat3Åresolution.ThestructureofTFIIDcomplexeshavebeendeterminedbyelectronmicroscopyat30Åresolution.Thus,themolecu-larstructuresofthetranscriptionmachineryarebeginningtobeelucidated.Muchofthisstructuralinformationisconsistentwiththemodelspresentedhere.
347RNASYNTHESIS,PROCESSING,&MODIFICATION/349RateofRateoftranscriptiontranscriptionBasalTBPcomplexTAFTAFCTFTAFCCAATTBPBasalcomplexCAATTATAnil+CTFBasalTBPcomplexTAFCCAATCTFCTFCAATTATAnilTAFTAFTAFTBPBasalCAATTAFcomplexBasalcomplexCTFTBPTATAABFigure37–10.Twomodelsforassemblyoftheactivetranscriptioncomplexandforhowactivatorsandcoacti-vatorsmightenhancetranscription.ShownhereasasmallovalisTBP,whichcontainsTFIID,alargeovalthatcon-tainsallthecomponentsofthebasaltranscriptioncomplexillustratedinFigure37–9(ie,RNAPIIandTFIIA,TFIIB,TFIIE,TFIIF,andTFIIH).PanelA:ThebasaltranscriptioncomplexisassembledonthepromoteraftertheTBPsub-unitofTFIIDisboundtotheTATAbox.SeveralTAFs(coactivators)areassociatedwithTBP.Inthisexample,atran-scriptionactivator,CTF,isshownboundtotheCAATbox,formingaloopcomplexbyinteractingwithaTAFboundtoTBP.PanelB:Therecruitmentmodel.ThetranscriptionactivatorCTFbindstotheCAATboxandinter-actswithacoactivator(TAFinthiscase).ThisallowsforaninteractionwiththepreformedTBP-basaltranscriptioncomplex.TBPcannowbindtotheTATAbox,andtheassembledcomplexisfullyactive.allowed.Somesequencesbindonlyasingleprotein,butstood.However,itappearsthattheterminationsignalsthemajoritybindseveraldifferentproteins.Similarly,aexistfardownstreamofthecodingsequenceofeukary-singleproteincanbindtomorethanoneelement.oticgenes.Forexample,thetranscriptionterminationHormoneresponseelements(forsteroids,T3,reti-signalformouseβ-globinoccursatseveralpositionsnoicacid,peptides,etc)actas—orinconjunctionwith—1000–2000basesbeyondthesiteatwhichthepoly(A)enhancersorsilencers(Chapter43).Otherprocessestailwilleventuallybeadded.Littleisknownaboutthethatenhanceorsilencegeneexpression—suchasthere-terminationprocessorwhetherspecifictermination2+2+sponsetoheatshock,heavymetals(CdandZn),factorssimilartothebacterialρfactorareinvolved.andsometoxicchemicals(eg,dioxin)—aremediatedHowever,itisknownthatthemRNA3′terminalisthroughspecificregulatoryelements.Tissue-specificex-generatedposttranscriptionally,issomehowcoupledtopressionofgenes(eg,thealbumingeneinliver,thehe-eventsorstructuresformedatthetimeandsiteofiniti-moglobingeneinreticulocytes)isalsomediatedbyspe-ation,dependsonaspecialstructureinoneofthesub-cificDNAsequences.unitsofRNApolymeraseII(theCTD;seebelow),andappearstoinvolveatleasttwosteps.AfterRNApolym-SpecificSignalsRegulateeraseIIhastraversedtheregionofthetranscriptionunitencodingthe3′endofthetranscript,anRNAen-TranscriptionTerminationdonucleasecleavestheprimarytranscriptatapositionThesignalsfortheterminationoftranscriptionbyabout15bases3′oftheconsensussequenceAAUAAAeukaryoticRNApolymeraseIIareverypoorlyunder-thatservesineukaryotictranscriptsasacleavagesignal.
348350/CHAPTER37Finally,thisnewlyformed3′terminalispolyadenylateddistinct,polymerase-specificsetsofTAFs,isalsoanim-inthenucleoplasm,asdescribedbelow.portantcomponentofclassIandclassIIIinitiationcomplexeseveniftheydonotcontainTATAboxes.THEEUKARYOTICThebindingofTBPmarksaspecificpromoterforTRANSCRIPTIONCOMPLEXtranscriptionandistheonlystepintheassemblyprocessthatisentirelydependentonspecific,high-affinitypro-Acomplexapparatusconsistingofasmanyas50tein-DNAinteraction.Ofseveralsubsequentinvitrouniqueproteinsprovidesaccurateandregulatabletran-steps,thefirstisthebindingofTFIIBtotheTFIID-scriptionofeukaryoticgenes.TheRNApolymeraseen-promotercomplex.Thisresultsinastableternarycom-zymes(polI,polII,andpolIIIforclassI,II,andIIIplexwhichisthenmorepreciselylocatedandmoregenes,respectively)transcribeinformationcontainedintightlyboundatthetranscriptioninitiationsite.ThisthetemplatestrandofDNAintoRNA.Thesepolym-complexthenattractsandtethersthepolII-TFIIFcom-erasesmustrecognizeaspecificsiteinthepromoterinplextothepromoter.TFIIFisstructurallyandfunc-ordertoinitiatetranscriptionatthepropernucleotide.tionallysimilartothebacterialσfactorandisrequiredIncontrasttothesituationinprokaryotes,eukaryoticforthedeliveryofpolIItothepromoter.TFIIAbindsRNApolymerasesalonearenotabletodiscriminatebe-tothisassemblyandmayallowthecomplextorespondtweenpromotersequencesandotherregionsofDNA;toactivators,perhapsbythedisplacementofrepressors.thus,otherproteinsknownasgeneraltranscriptionfac-AdditionofTFIIEandTFIIHisthefinalstepintheas-torsorGTFsfacilitatepromoter-specificbindingofsemblyofthePIC.TFIIEappearstojointhecomplextheseenzymesandformationofthepreinitiationcom-withpolII-TFIIF,andTFIIHisthenrecruited.Eachofplex(PIC).Thiscombinationofcomponentscancat-thesebindingeventsextendsthesizeofthecomplexsoalyzebasalor(non)-unregulatedtranscriptioninvitro.thatfinallyabout60bp(from−30to+30relativeto+1,Anothersetofproteins—coactivators—helpregulatethenucleotidefromwhichtranscriptioncommences)therateoftranscriptioninitiationbyinteractingwitharecovered(Figure37–9).ThePICisnowcompletetranscriptionactivatorsthatbindtoupstreamDNAel-andcapableofbasaltranscriptioninitiatedfromthecor-ements(seebelow).rectnucleotide.IngenesthatlackaTATAbox,thesamefactors,includingTBP,arerequired.Insuchcases,FormationoftheBasalanInrortheDPEs(seeFigure37–8)positionthecom-TranscriptionComplexplexforaccurateinitiationoftranscription.Inbacteria,aσfactor–polymerasecomplexselectivelyPhosphorylationActivatesPolIIbindstoDNAinthepromoterformingthePIC.Thesituationismorecomplexineukaryoticgenes.ClassIIEukaryoticpolIIconsistsof12subunits.Thetwogenes—thosetranscribedbypolIItomakemRNA—largestsubunits,bothabout200kDa,arehomologousaredescribedasanexample.InclassIIgenes,thefunc-tothebacterialβandβ′subunits.Inadditiontothein-tionofσfactorsisassumedbyanumberofproteins.creasednumberofsubunits,eukaryoticpolIIdiffersBasaltranscriptionrequires,inadditiontopolII,afromitsprokaryoticcounterpartinthatithasaseriesofnumberofGTFscalledTFIIA,TFIIB,TFIID,heptadrepeatswithconsensussequenceTyr-Ser-Pro-TFIIE,TFIIF,andTFIIH.TheseGTFsservetopro-Thr-Ser-Pro-SeratthecarboxylterminalofthelargestmoteRNApolymeraseIItranscriptiononessentiallyallpolIIsubunit.Thiscarboxylterminalrepeatdomaingenes.SomeoftheseGTFsarecomposedofmultiple(CTD)has26repeatedunitsinbrewers’yeastand52subunits.TFIID,whichbindstotheTATAboxpro-unitsinmammaliancells.TheCTDisbothasubstratemoterelement,istheonlyoneofthesefactorscapa-forseveralkinases,includingthekinasecomponentofbleofbindingtospecificsequencesofDNA.Asde-TFIIH,andabindingsiteforawidearrayofproteins.scribedabove,TFIIDconsistsofTATAbindingTheCTDhasbeenshowntointeractwithRNApro-protein(TBP)and14TBP-associatedfactors(TAFs).cessingenzymes;suchbindingmaybeinvolvedwithTBPbindstotheTATAboxintheminorgrooveofRNApolyadenylation.TheassociationofthefactorsDNA(mosttranscriptionfactorsbindinthemajorwiththeCTDofRNApolymeraseII(andothercom-groove)andcausesanapproximately100-degreebendponentsofthebasalmachinery)somehowservestoorkinkoftheDNAhelix.ThisbendingisthoughttocoupleinitiationwithmRNA3′endformation.PolIIfacilitatetheinteractionofTBP-associatedfactorswithisactivatedwhenphosphorylatedontheSerandThrothercomponentsofthetranscriptioninitiationcom-residuesanddisplaysreducedactivitywhentheCTDisplexandpossiblywithfactorsboundtoupstreamele-dephosphorylated.PolIIlackingtheCTDtailisinca-ments.AlthoughdefinedasacomponentofclassIIpableofactivatingtranscription,whichunderscoresthegenepromoters,TBP,byvirtueofitsassociationwithimportanceofthisdomain.
349RNASYNTHESIS,PROCESSING,&MODIFICATION/351PolIIassociateswithotherproteinstoformaTAFs.Itisconceivablethatdifferentcombinationsofholoenzymecomplex.Inyeast,atleastninegeneprod-TAFswithTBP—oroneofseveralrecentlydiscovereducts—calledSrb(forsuppressorofRNApolymer-TBP-likefactors(TLFs)—maybindtodifferentpro-aseB)—bindtotheCTD.TheSrbproteins—ormedi-moters,andrecentreportssuggestthatthismayac-ators,astheyarealsocalled—areessentialforpolIIcountforselectiveactivationnotedinvariouspromot-transcription,thoughtheirexactroleinthisprocesshasersandforthedifferentstrengthsofcertainpromoters.notbeendefined.RelatedproteinscomprisingevenTAFs,sincetheyarerequiredfortheactionofacti-morecomplexformsofRNApolymeraseIIhavebeenvators,areoftencalledcoactivators.Therearethusdescribedinhumancells.threeclassesoftranscriptionfactorsinvolvedinthereg-ulationofclassIIgenes:basalfactors,coactivators,andTheRoleofTranscriptionActivatorsactivator-repressors(Table37–4).Howtheseclassesof&Coactivatorsproteinsinteracttogovernboththesiteandfrequencyoftranscriptionisaquestionofcentralimportance.TFIIDwasoriginallyconsideredtobeasingleprotein.However,severalpiecesofevidenceledtotheimpor-TwoModelsExplaintheAssemblytantdiscoverythatTFIIDisactuallyacomplexconsist-ofthePreinitiationComplexingofTBPandthe14TAFs.ThefirstevidencethatTFIIDwasmorecomplexthanjusttheTBPmoleculesTheformationofthePICdescribedaboveisbasedoncamefromtheobservationthatTBPbindstoa10-bpthesequentialadditionofpurifiedcomponentsininsegmentofDNA,immediatelyovertheTATAboxofvitroexperiments.Anessentialfeatureofthismodelisthegene,whereasnativeholo-TFIIDcoversa35bporthattheassemblytakesplaceontheDNAtemplate.largerregion(Figure37–9).Second,TBPhasamolec-Accordingly,transcriptionactivators,whichhaveau-ularmassof20–40kDa(dependingonthespecies),tonomousDNAbindingandactivationdomains(seewhereastheTFIIDcomplexhasamassofabout1000Chapter39),arethoughttofunctionbystimulatingei-kDa.Finally,andperhapsmostimportantly,TBPsup-therPICformationorPICfunction.TheTAFcoacti-portsbasaltranscriptionbutnottheaugmentedtran-vatorsareviewedasbridgingfactorsthatcommunicatescriptionprovidedbycertainactivators,eg,Sp1boundbetweentheupstreamactivators,theproteinsassociatedtotheGCbox.TFIID,ontheotherhand,supportswithpolII,orthemanyothercomponentsofTFIID.bothbasalandenhancedtranscriptionbySp1,Oct1,Thisview,whichassumesthatthereisstepwiseassem-AP1,CTF,ATF,etc.(Table37–3).TheTAFsarees-blyofthePIC—promotedbyvariousinteractionsbe-sentialforthisactivator-enhancedtranscription.Itistweenactivators,coactivators,andPICcomponents—notyetclearwhetherthereareoneorseveralformsofisillustratedinpanelAofFigure37–10.ThismodelTFIIDthatmightdifferslightlyintheircomplementofwassupportedbyobservationsthatmanyofthesepro-teinscouldindeedbindtooneanotherinvitro.Recentevidencesuggeststhatthereisanotherpossi-Table37–3.SomeofthetranscriptioncontrolblemechanismofPICformationandtranscriptionreg-ulation.First,largepreassembledcomplexesofGTFselements,theirconsensussequences,andtheandpolIIarefoundincellextracts,andthiscomplexfactorsthatbindtothemwhicharefoundincanassociatewithapromoterinasinglestep.Second,mammaliangenestranscribedbyRNAtherateoftranscriptionachievedwhenactivatorsarepolymeraseII.AcompletelistwouldincludeaddedtolimitingconcentrationsofpolIIholoenzymedozensofexamples.Theasterisksmeanthatcanbematchedbyincreasingtheconcentrationofthethereareseveralmembersofthisfamily.polIIholoenzymeintheabsenceofactivators.Thus,ElementConsensusSequenceFactorTATAboxTATAAATBPTable37–4.ThreeclassesoftranscriptionfactorsCAATboxCCAATCC/EBP*,NF-Y*inclassIIgenes.GCboxGGGCGGSp1*CAACTGACMyoDGeneralMechanismsSpecificComponentsT/CGGA/CN5GCCAANF1*lgoctamerATGCAAATOct1,2,4,6*BasalcomponentsTBP,TFIIA,B,E,F,andHAP1TGAG/CTC/AAJun,Fos,ATF*CoactivatorsTAFs(TBP+TAFs)=TFIID;SrbsSerumresponseGATGCCCATASRFHeatshock(NGAAN)3HSFActivatorsSP1,ATF,CTF,AP1,etc
350352/CHAPTER37activatorsarenotinthemselvesabsolutelyessentialforprecursormoleculesisrequiredforthegenerationofPICformation.Theseobservationsledtothe“recruit-thematurefunctionalmolecules.ment”hypothesis,whichhasnowbeentestedexperi-NearlyalleukaryoticRNAprimarytranscriptsun-mentally.Simplystated,theroleofactivatorsanddergoextensiveprocessingbetweenthetimetheyarecoactivatorsmaybesolelytorecruitapreformedsynthesizedandthetimeatwhichtheyservetheirulti-holoenzyme-GTFcomplextothepromoter.There-matefunction,whetheritbeasmRNAorasacom-quirementforanactivationdomainiscircumventedponentofthetranslationmachinerysuchasrRNA,wheneitheracomponentofTFIIDorthepolII5SRNA,ortRNAorRNAprocessingmachinery,holoenzymeisartificiallytethered,usingrecombinantsnRNAs.Processingoccursprimarilywithinthenu-DNAtechniques,totheDNAbindingdomain(DBD)cleusandincludesnucleolyticcleavagetosmallermole-ofanactivator.Thisanchoring,throughtheDBDculesandcouplednucleolyticandligationreactionscomponentoftheactivatormolecule,leadstoatran-(splicingofexons).Inmammaliancells,50–75%ofscriptionallycompetentstructure,andthereisnofur-thenuclearRNAdoesnotcontributetothecytoplas-therrequirementfortheactivationdomainoftheacti-micmRNA.ThisnuclearRNAlossissignificantlyvator.Inthisview,theroleofactivationdomainsandgreaterthancanbereasonablyaccountedforbythelossTAFsistoformanassemblythatdirectsthepreformedofinterveningsequencesalone(seebelow).Thus,theholoenzyme-GTFcomplextothepromoter;theydoexactfunctionoftheseeminglyexcessivetranscriptsinnotassistinPICassembly(seepanelB,Figure37–10).thenucleusofamammaliancellisnotknown.Theefficiencyofthisrecruitmentprocessdeterminestherateoftranscriptionatagivenpromoter.Hormones—andothereffectorsthatservetotrans-TheCodingPortions(Exons)mitinformationrelatedtotheextracellularenviron-ofMostEukaryoticGenesment—modulategeneexpressionbyinfluencingtheas-AreInterruptedbyIntronssemblyandactivityoftheactivatorandcoactivatorInterspersedwithintheaminoacid-codingportionscomplexesandthesubsequentformationofthePICat(exons)ofmanygenesarelongsequencesofDNAthatthepromoteroftargetgenes(seeChapter43).Thenu-donotcontributetothegeneticinformationultimatelymerouscomponentsinvolvedprovideforanabundancetranslatedintotheaminoacidsequenceofaproteinofpossiblecombinationsandthereforearangeoftran-molecule(seeChapter36).Infact,thesesequencesac-scriptionalactivityofagivengene.Itisimportanttotuallyinterruptthecodingregionofstructuralgenes.notethatthetwomodelsarenotmutuallyexclusive—Theseinterveningsequences(introns)existwithinstepwiseversusholoenzyme-mediatedPICformation.mostbutnotallmRNAencodinggenesofhighereu-Indeed,onecanenvisionvariousmorecomplexmodelskaryotes.Theprimarytranscriptsofthestructuralgenesinvokingelementsofbothmodelsoperatingonagene.containRNAcomplementarytotheinterspersedse-quences.However,theintronRNAsequencesarecleavedoutofthetranscript,andtheexonsofthetran-RNAMOLECULESAREUSUALLYscriptareappropriatelysplicedtogetherinthenucleusPROCESSEDBEFORETHEYbeforetheresultingmRNAmoleculeappearsinthecy-BECOMEFUNCTIONALtoplasmfortranslation(Figures37–11and37–12).Onespeculationisthatexons,whichoftenencodeanInprokaryoticorganisms,theprimarytranscriptsofactivitydomainofaprotein,representaconvenientmRNA-encodinggenesbegintoserveastranslationmeansofshufflinggeneticinformation,permittingor-templatesevenbeforetheirtranscriptionhasbeencom-ganismstoquicklytesttheresultsofcombiningnovelpleted.Thisisbecausethesiteoftranscriptionisnotproteinfunctionaldomains.compartmentalizedintoanucleusasitisineukaryoticorganisms.Thus,transcriptionandtranslationarecou-pledinprokaryoticcells.Consequently,prokaryoticIntronsAreRemoved&ExonsmRNAsaresubjectedtolittleprocessingpriortocarry-AreSplicedTogetheringouttheirintendedfunctioninproteinsynthesis.In-deed,appropriateregulationofsomegenes(eg,theTrpThemechanismswherebyintronsareremovedfromoperon)reliesuponthiscouplingoftranscriptionandtheprimarytranscriptinthenucleus,exonsareligatedtranslation.ProkaryoticrRNAandtRNAmoleculesaretoformthemRNAmolecule,andthemRNAmoleculetranscribedinunitsconsiderablylongerthantheulti-istransportedtothecytoplasmarebeingelucidated.matemolecule.Infact,manyofthetRNAtranscriptionFourdifferentsplicingreactionmechanismshavebeenunitscontainmorethanonemolecule.Thus,indescribed.TheonemostfrequentlyusedineukaryoticprokaryotestheprocessingoftheserRNAandtRNAcellsisdescribedbelow.Althoughthesequencesofnu-
351RNASYNTHESIS,PROCESSING,&MODIFICATION/353Exon1Exon2Intron5′CapGGAGAn3′PrimarytranscriptCapGGAGAnNucleophilicattackat5′endofintronGCapGOHAGAnLariatformationCapGGGACutat3′endofintronOHnAGCapG—GAnandLigationof3′endofexonA1to5′endofexon2IntronisdigestedFigure37–11.TheprocessingoftheprimarytranscripttomRNA.Inthishy-potheticaltranscript,the5′(left)endoftheintroniscut(↓)andalariatformsbetweentheGatthe5′endoftheintronandanAnearthe3′end,inthecon-sensussequenceUACUAAC.Thissequenceiscalledthebranchsite,anditisthe3′mostAthatformsthe5′–2’bondwiththeG.The3′(right)endoftheintronisthencut(⇓).Thisreleasesthelariat,whichisdigested,andexon1isjoinedtoexon2atGresidues.cleotidesintheintronsofthevariouseukaryotictran-marytranscript,fivesmallnuclearRNAs(U1,U2,U5,scripts—andeventhosewithinasingletranscript—areU4,andU6)andmorethan60proteins.Collectively,quiteheterogeneous,therearereasonablyconservedse-theseformasmallnucleoprotein(snRNP)complex,quencesateachofthetwoexon-intron(splice)junc-sometimescalleda“snurp.”Itislikelythatthispenta-tionsandatthebranchsite,whichislocated20–40nu-snRNPspliceosomeformspriortointeractionwithcleotidesupstreamfromthe3′splicesite(seeconsensusmRNAprecursors.SnurpsarethoughttopositionthesequencesinFigure37–12).Aspecialstructure,theRNAsegmentsforthenecessarysplicingreactions.Thespliceosome,isinvolvedinconvertingtheprimarysplicingreactionstartswithacutatthejunctionofthetranscriptintomRNA.Spliceosomesconsistofthepri-5′exon(donororleft)andintron(Figure37–11).ThisConsensussequencesA5′AGGUAAGUUACUAAC28-37nucleotidesCAGG3′C5′ExonIntronExon3′Figure37–12.Consensussequencesatsplicejunctions.The5′(donororleft)and3′(ac-ceptororright)sequencesareshown.Alsoshownistheyeastconsensussequence(UACUAAC)forthebranchsite.Inmammaliancells,thisconsensussequenceisPyNPyPy-PuAPy,wherePyisapyrimidine,Puisapurine,andNisanynucleotide.Thebranchsiteislo-cated20–40nucleotidesupstreamfromthe3′site.
352354/CHAPTER37isaccomplishedbyanucleophilicattackbyanadenylylabove,thesequenceofexon-intronsplicingeventsgen-residueinthebranchpointsequencelocatedjustup-erallyfollowsahierarchicalorderforagivengene.Thestreamfromthe3′endofthisintron.Thefree5′termi-factthatverycomplexRNAstructuresareformeddur-nalthenformsalooporlariatstructurethatislinkedingsplicing—andthatanumberofsnRNAsandpro-byanunusual5′–2′phosphodiesterbondtothereac-teinsareinvolved—affordsnumerouspossibilitiesforativeAinthePyNPyPyPuAPybranchsitesequencechangeofthisorderandforthegenerationofdifferent(Figure37–12).Thisadenylylresidueistypicallylo-mRNAs.Similarly,theuseofalternativetermination-cated28–37nucleotidesupstreamfromthe3′endofcleavage-polyadenylationsitesalsoresultsinmRNAtheintronbeingremoved.Thebranchsiteidentifiesheterogeneity.Someschematicexamplesofthesethe3′splicesite.Asecondcutismadeatthejunctionprocesses,allofwhichoccurinnature,areshowninoftheintronwiththe3′exon(donoronright).InthisFigure37–13.secondtransesterificationreaction,the3′hydroxylofFaultysplicingcancausedisease.Atleastonetheupstreamexonattacksthe5′phosphateattheformofβ-thalassemia,adiseaseinwhichtheβ-globindownstreamexon-intronboundary,andthelariatgeneofhemoglobinisseverelyunderexpressed,appearsstructurecontainingtheintronisreleasedandhy-toresultfromanucleotidechangeatanexon-introndrolyzed.The5′and3′exonsareligatedtoformacon-junction,precludingremovaloftheintronandthere-tinuoussequence.foreleadingtodiminishedorabsentsynthesisoftheThesnRNAsandassociatedproteinsarerequiredβ-chainprotein.Thisisaconsequenceofthefactthatforformationofthevariousstructuresandintermedi-thenormaltranslationreadingframeofthemRNAisates.U1withinthesnRNPcomplexbindsfirstbybasedisrupted—adefectinthisfundamentalprocess(splic-pairingtothe5′exon-intronboundary.U2withintheing)thatunderscorestheaccuracywhichtheprocessofsnRNPcomplexthenbindsbybasepairingtotheRNA-RNAsplicingmustachieve.branchsite,andthisexposesthenucleophilicAresidue.U5/U4/U6withinthesnRNPcomplexmediatesanAlternativePromoterUtilizationATP-dependentprotein-mediatedunwindingthatre-ProvidesaFormofRegulationsultsindisruptionofthebase-pairedU4-U6complexwiththereleaseofU4.U6isthenabletointeractfirstTissue-specificregulationofgeneexpressioncanbewithU2,thenwithU1.Theseinteractionsservetoap-providedbycontrolelementsinthepromoterorbytheproximatethe5′splicesite,thebranchpointwithitsreactiveA,andthe3′splicesite.Thisalignmentisen-hancedbyU5.Thisprocessalsoresultsintheforma-mRNAprecursortionofthelooporlariatstructure.Thetwoendsare123AAUAAAAUAA(A)ncleaved,probablybytheU2-U6withinthesnRNPcomplex.U6iscertainlyessential,sinceyeastsdeficientinthissnRNAarenotviable.ItisimportanttonoteSelectivesplicingthatRNAservesasthecatalyticagent.Thissequenceisthenrepeatedingenescontainingmultipleintrons.In123AAUAAAAUAA(A)nsuchcases,adefinitepatternisfollowedforeachgene,andtheintronsarenotnecessarilyremovedinse-Alternative5′donorsitequence—1,then2,then3,etc.TherelationshipbetweenhnRNAandthecorre-1′23AAUAAAAUAA(A)nspondingmaturemRNAineukaryoticcellsisnowap-parent.ThehnRNAmoleculesaretheprimarytran-Alternative3′acceptorsitescriptsplustheirearlyprocessedproducts,which,aftertheadditionofcapsandpoly(A)tailsandremovalof12′3AAUAAAAUAA(A)ntheportioncorrespondingtotheintrons,aretrans-portedtothecytoplasmasmaturemRNAmolecules.AlternativepolyadenylationsiteAlternativeSplicingProvides123AAUAA(A)nforDifferentmRNAsFigure37–13.Mechanismsofalternativeprocess-TheprocessingofhnRNAmoleculesisasiteforreg-ingofmRNAprecursors.ThisformofRNAprocessingulationofgeneexpression.Alternativepatternsofinvolvestheselectiveinclusionorexclusionofexons,RNAsplicingresultfromtissue-specificadaptiveandtheuseofalternative5′donoror3′acceptorsites,anddevelopmentalcontrolmechanisms.Asmentionedtheuseofdifferentpolyadenylationsites.
353RNASYNTHESIS,PROCESSING,&MODIFICATION/355useofalternativepromoters.Theglucokinase(GK)maturetRNA.AsmallfractionoftRNAsevencontaingeneconsistsoftenexonsinterruptedbynineintrons.introns.Thesequenceofexons2–10isidenticalinliverandpancreaticBcells,theprimarytissuesinwhichGKpro-RNASCANBEEXTENSIVELYMODIFIEDteinisexpressed.ExpressionoftheGKgeneisregulatedverydifferently—bytwodifferentpromoters—intheseEssentiallyallRNAsarecovalentlymodifiedaftertran-twotissues.Theliverpromoterandexon1Larelocatedscription.Itisclearthatatleastsomeofthesemodifica-nearexons2–10;exon1Lisligateddirectlytoexon2.tionsareregulatory.Incontrast,thepancreaticBcellpromoterislocatedabout30kbpupstream.Inthiscase,the3′boundaryofMessengerRNA(mRNA)IsModifiedexon1Bisligatedtothe5′boundaryofexon2.Theatthe5&3Endsliverpromoterandexon1LareexcludedandremovedAsmentionedabove,mammalianmRNAmoleculesduringthesplicingreaction(seeFigure37–14).Theex-containa7-methylguanosinecapstructureattheir5′istenceofmultipledistinctpromotersallowsforcell-terminal,andmosthaveapoly(A)tailatthe3′termi-andtissue-specificexpressionpatternsofaparticularnal.Thecapstructureisaddedtothe5′endofthegene(mRNA).newlytranscribedmRNAprecursorinthenucleuspriortotransportofthemRNAmoleculetothecyto-BothRibosomalRNAs&Mostplasm.The5capoftheRNAtranscriptisrequiredTransferRNAsAreProcessedbothforefficienttranslationinitiationandprotectionofthe5′endofmRNAfromattackby5′→3′exonu-FromLargerPrecursorscleases.ThesecondarymethylationsofmRNAmole-6Inmammaliancells,thethreerRNAmoleculesarecules,thoseonthe2′-hydroxyandtheNofadenylyltranscribedaspartofasinglelargeprecursormolecule.residues,occurafterthemRNAmoleculehasappearedTheprecursorissubsequentlyprocessedinthenu-inthecytoplasm.cleolustoprovidetheRNAcomponentfortheribo-Poly(A)tailsareaddedtothe3′endofmRNAmol-somesubunitsfoundinthecytoplasm.TherRNAeculesinaposttranscriptionalprocessingstep.Thegenesarelocatedinthenucleoliofmammaliancells.mRNAisfirstcleavedabout20nucleotidesdown-HundredsofcopiesofthesegenesarepresentineverystreamfromanAAUAArecognitionsequence.Anothercell.Thislargenumberofgenesisrequiredtosynthe-enzyme,poly(A)polymerase,addsapoly(A)tailwhichsizesufficientcopiesofeachtypeofrRNAtoformtheissubsequentlyextendedtoasmanyas200Aresidues.710ribosomesrequiredforeachcellreplication.Thepoly(A)tailappearstoprotectthe3′endofWhereasasinglemRNAmoleculemaybecopiedintomRNAfrom3′→5′exonucleaseattack.Thepresence510proteinmolecules,providingalargeamplification,orabsenceofthepoly(A)taildoesnotdeterminetherRNAsareendproducts.Thislackofamplificationwhetheraprecursormoleculeinthenucleusappearsinrequiresalargenumberofgenes.Similarly,transferthecytoplasm,becauseallpoly(A)-tailedhnRNAmole-RNAsareoftensynthesizedasprecursors,withextrase-culesdonotcontributetocytoplasmicmRNA,nordoquencesboth5′and3′ofthesequencescomprisingtheallcytoplasmicmRNAmoleculescontainpoly(A)tails(˜30kb)Liver1B1L2A2345678910(˜30kb)Bcell/pituitary1B1L2A2345678910Figure37–14.AlternativepromoteruseintheliverandpancreaticBcellglucokinasegenes.Differentialregulationoftheglucokinase(GK)geneisaccomplishedbytheuseoftissue-specificpromoters.TheBcellGKgenepromoterandexon1Barelocatedabout30kbpupstreamfromtheliverpromoterandexon1L.Eachpromoterhasauniquestructureandisregulateddifferently.Exons2–10areidenticalinthetwogenes,andtheGKproteinsencodedbytheliverandBcellmRNAshaveidenticalkineticproperties.
354356/CHAPTER37(thehistonesaremostnotableinthisregard).Cytoplas-mRNAintoproteinsequences.ThetRNAscontainmicenzymesinmammaliancellscanbothaddandre-manymodificationsofthestandardbasesA,U,G,andmoveadenylylresiduesfromthepoly(A)tails;thisC,includingmethylation,reduction,deamination,andprocesshasbeenassociatedwithanalterationofmRNArearrangedglycosidicbonds.Furthermodificationofstabilityandtranslatability.thetRNAmoleculesincludesnucleotidealkylationsThesizeofsomecytoplasmicmRNAmolecules,andtheattachmentofthecharacteristicCpCpAOHter-evenafterthepoly(A)tailisremoved,isstillconsider-minalatthe3′endofthemoleculebytheenzymenu-ablygreaterthanthesizerequiredtocodeforthespe-cleotidyltransferase.The3′OHoftheAriboseisthecificproteinforwhichitisatemplate,oftenbyafactorpointofattachmentforthespecificaminoacidthatisof2or3.Theextranucleotidesoccurinuntrans-toenterintothepolymerizationreactionofproteinlated(non-proteincoding)regionsboth5′and3′ofsynthesis.ThemethylationofmammaliantRNApre-thecodingregion;thelongestuntranslatedsequencescursorsprobablyoccursinthenucleus,whereastheareusuallyatthe3′end.Theexactfunctionofthesese-cleavageandattachmentofCpCpAOHarecytoplasmicquencesisunknown,buttheyhavebeenimplicatedinfunctions,sincetheterminalsturnovermorerapidlyRNAprocessing,transport,degradation,andtransla-thandothetRNAmoleculesthemselves.Enzymestion;eachofthesereactionspotentiallycontributesad-withinthecytoplasmofmammaliancellsarerequiredditionallevelsofcontrolofgeneexpression.fortheattachmentofaminoacidstotheCpCpAOHresidues.(SeeChapter38.)RNAEditingChangesmRNAAfterTranscriptionRNACANACTASACATALYSTThecentraldogmastatesthatforagivengeneandgeneInadditiontothecatalyticactionservedbytheproductthereisalinearrelationshipbetweenthecod-snRNAsintheformationofmRNA,severalotheringsequenceinDNA,themRNAsequence,andtheenzymaticfunctionshavebeenattributedtoRNA.proteinsequence(Figure36–7).ChangesintheDNARibozymesareRNAmoleculeswithcatalyticactivity.sequenceshouldbereflectedinachangeinthemRNAThesegenerallyinvolvetransesterificationreactions,sequenceand,dependingoncodonusage,inproteinse-andmostareconcernedwithRNAmetabolism(splic-quence.However,exceptionstothisdogmahavebeeningandendoribonuclease).Recently,aribosomalRNArecentlydocumented.CodinginformationcanbecomponentwasnotedtohydrolyzeanaminoacylesterchangedatthemRNAlevelbyRNAediting.Insuchandthustoplayacentralroleinpeptidebondfunctioncases,thecodingsequenceofthemRNAdiffersfrom(peptidyltransferases;seeChapter38).Theseobserva-thatinthecognateDNA.Anexampleistheapolipo-tions,madeinorganellesfromplants,yeast,viruses,proteinB(apoB)geneandmRNA.Inliver,thesingleandhighereukaryoticcells,showthatRNAcanactasapoBgeneistranscribedintoanmRNAthatdirectstheanenzyme.Thishasrevolutionizedthinkingabouten-synthesisofa100-kDaprotein,apoB100.Intheintes-zymeactionandtheoriginoflifeitself.tine,thesamegenedirectsthesynthesisoftheprimarytranscript;however,acytidinedeaminaseconvertsaCAAcodoninthemRNAtoUAAatasinglespecificSUMMARYsite.Ratherthanencodingglutamine,thiscodonbe-•RNAissynthesizedfromaDNAtemplatebytheen-comesaterminationsignal,anda48-kDaproteinzymeRNApolymerase.(apoB48)istheresult.ApoB100andapoB48havedif-ferentfunctionsinthetwoorgans.Agrowingnumber•TherearethreedistinctnuclearDNA-dependentofotherexamplesincludeaglutaminetoarginineRNApolymerasesinmammals:RNApolymerasesI,changeintheglutamatereceptorandseveralchangesII,andIII.TheseenzymescontrolthetranscriptionalintrypanosomemitochondrialmRNAs,generallyin-function—thetranscriptionofrRNA,mRNA,andvolvingtheadditionordeletionofuridine.TheexactsmallRNA(tRNA/5SrRNA,snRNA)genes,respec-extentofRNAeditingisunknown,butcurrentesti-tively.matessuggestthat<0.01%ofmRNAsareeditedin•RNApolymerasesinteractwithuniquecis-activere-thisfashion.gionsofgenes,termedpromoters,inordertoformpreinitiationcomplexes(PICs)capableofinitiation.IneukaryotestheprocessofPICformationisfacili-TransferRNA(tRNA)IsExtensivelytatedbymultiplegeneraltranscriptionfactorsProcessed&Modified(GTFs),TFIIA,B,D,E,F,andH.AsdescribedinChapters35and38,thetRNAmole-•EukaryoticPICformationcanoccureitherstep-culesserveasadaptermoleculesforthetranslationofwise—bythesequential,orderedinteractionsof
355RNASYNTHESIS,PROCESSING,&MODIFICATION/357GTFsandRNApolymerasewithpromoters—orinREFERENCESonestepbytherecognitionofthepromoterbyapre-formedGTF-RNApolymeraseholoenzymecomplex.BusbyS,EbrightRH:Promoterstructure,promoterrecognition,andtranscriptionactivationinprokaryotes.Cell1994;79:•Transcriptionexhibitsthreephases:initiation,elon-743.gation,andtermination.Allaredependentupondis-CramerP,BushnellDA,KornbergR:Structuralbasisoftranscrip-tinctDNAcis-elementsandcanbemodulatedbytion:RNApolymeraseIIat2.8angstromresolution.Sciencedistincttrans-actingproteinfactors.2001;292:1863.•MosteukaryoticRNAsaresynthesizedasprecursorsFedorMJ:Ribozymes.CurrBiol1998;8:R441.thatcontainexcesssequenceswhichareremovedGottJM,EmesonRB:FunctionsandmechanismsofRNAediting.priortothegenerationofmature,functionalRNA.AnnRevGenet2000;34:499.HiroseY,ManleyJL:RNApolymeraseIIandtheintegrationof•EukaryoticmRNAsynthesisresultsinapre-mRNAnuclearevents.GenesDev2000;14:1415.precursorthatcontainsextensiveamountsofexcessKeaveneyM,StruhlK:Activator-mediatedrecruitmentoftheRNA(introns)thatmustbepreciselyremovedbyRNApolymerasemachineryisthepredominantmechanismRNAsplicingtogeneratefunctional,translatablefortranscriptionalactivationinyeast.MolCell1998;1:917.mRNAcomposedofexoniccodingandnoncodingLemonB,TjianR:Orchestratedresponse:asymphonyoftran-sequences.scriptionfactorsforgenecontrol.GenesDev2000;14:2551.•Allsteps—fromchangesinDNAtemplate,sequence,ManiatisT,ReedR:AnextensivenetworkofcouplingamonggeneandaccessibilityinchromatintoRNAstability—areexpressionmachines.Nature2002;416:499.subjecttomodulationandhencearepotentialcon-OrphanidesG,ReinbergD:Aunifiedtheoryofgeneexpression.trolsitesforeukaryoticgeneregulation.Cell2002;108:439.ShatkinAJ,ManleyJL:Theendsoftheaffair:cappingandpoly-adenylation.NatStructBiol2000;7:838.StevensSWetal:Compositionandfunctionalcharacterizationoftheyeastspliceosomalpenta-snRNP.MolCell2002;9:31.TuckerM,ParkerR:MechanismsandcontrolofmRNAdecap-pinginSaccharomycescerevisiae.AnnRevBiochem2000;69:571.WoychikNA,HampseyM:TheRNApolymeraseIImachinery:structureilluminatesfunction.Cell2002;108:453.
356ProteinSynthesis&theGeneticCode38DarylK.Granner,MDBIOMEDICALIMPORTANCEThecellmustpossessthemachinerynecessarytotranslateinformationaccuratelyandefficientlyfromThelettersA,G,T,andCcorrespondtothenu-thenucleotidesequenceofanmRNAintothesequencecleotidesfoundinDNA.Theyareorganizedintothree-ofaminoacidsofthecorrespondingspecificprotein.lettercodewordscalledcodons,andthecollectionofClarificationofourunderstandingofthisprocess,thesecodonsmakesupthegeneticcode.Itwasimpos-whichistermedtranslation,awaiteddecipheringofthesibletounderstandproteinsynthesis—ortoexplaingeneticcode.ItwasrealizedearlythatmRNAmole-mutations—beforethegeneticcodewaselucidated.culesthemselveshavenoaffinityforaminoacidsand,Thecodeprovidesafoundationforexplainingthewaytherefore,thatthetranslationoftheinformationintheinwhichproteindefectsmaycausegeneticdiseaseandmRNAnucleotidesequenceintotheaminoacidse-forthediagnosisandperhapsthetreatmentofthesequenceofaproteinrequiresanintermediateadapterdisorders.Inaddition,thepathophysiologyofmanymolecule.Thisadaptermoleculemustrecognizeaspe-viralinfectionsisrelatedtotheabilityoftheseagentstocificnucleotidesequenceontheonehandaswellasadisrupthostcellproteinsynthesis.Manyantibacterialspecificaminoacidontheother.Withsuchanadapteragentsareeffectivebecausetheyselectivelydisruptpro-molecule,thecellcandirectaspecificaminoacidintoteinsynthesisintheinvadingbacterialcellbutdonotthepropersequentialpositionofaproteinduringitsaffectproteinsynthesisineukaryoticcells.synthesisasdictatedbythenucleotidesequenceofthespecificmRNA.Infact,thefunctionalgroupsoftheGENETICINFORMATIONFLOWSaminoacidsdonotthemselvesactuallycomeintocon-tactwiththemRNAtemplate.FROMDNATORNATOPROTEINThegeneticinformationwithinthenucleotidese-quenceofDNAistranscribedinthenucleusintotheTHENUCLEOTIDESEQUENCEspecificnucleotidesequenceofanRNAmolecule.TheOFANmRNAMOLECULECONSISTSsequenceofnucleotidesintheRNAtranscriptiscom-OFASERIESOFCODONSTHATSPECIFYplementarytothenucleotidesequenceofthetemplateTHEAMINOACIDSEQUENCEOFTHEstrandofitsgeneinaccordancewiththebase-pairingENCODEDPROTEINrules.SeveraldifferentclassesofRNAcombinetodi-rectthesynthesisofproteins.Twentydifferentaminoacidsarerequiredforthesyn-Inprokaryotesthereisalinearcorrespondencebe-thesisofthecellularcomplementofproteins;thus,tweenthegene,themessengerRNA(mRNA)tran-theremustbeatleast20distinctcodonsthatmakeupscribedfromthegene,andthepolypeptideproduct.thegeneticcode.Sincethereareonlyfourdifferentnu-ThesituationismorecomplicatedinhighereukaryoticcleotidesinmRNA,eachcodonmustconsistofmorecells,inwhichtheprimarytranscriptismuchlargerthanasinglepurineorpyrimidinenucleotide.CodonsthanthematuremRNA.ThelargemRNAprecursorsconsistingoftwonucleotideseachcouldprovidefor2containcodingregions(exons)thatwillformthema-only16(4)specificcodons,whereascodonsofthree3turemRNAandlonginterveningsequences(introns)nucleotidescouldprovide64(4)specificcodons.thatseparatetheexons.ThehnRNAisprocessedItisnowknownthateachcodonconsistsofase-withinthenucleus,andtheintrons,whichoftenmakequenceofthreenucleotides;ie,itisatripletcodeupmuchmoreofthisRNAthantheexons,arere-(seeTable38–1).Thedecipheringofthegeneticcodemoved.Exonsaresplicedtogethertoformmaturedependedheavilyonthechemicalsynthesisofnu-mRNA,whichistransportedtothecytoplasm,whereitcleotidepolymers,particularlytripletsinrepeatedse-istranslatedintoprotein.quence.358
357PROTEINSYNTHESIS&THEGENETICCODE/359Table38–1.Thegeneticcode(codonthecode.However,foranyspecificcodon,onlyasingleassignmentsinmammalianmessengerRNA).1aminoacidisindicated;withrareexceptions,thege-neticcodeisunambiguous—ie,givenaspecificcodon,onlyasingleaminoacidisindicated.ThedistinctionFirstSecondThirdbetweenambiguityanddegeneracyisanimportantNucleotideNucleotideNucleotideconcept.UCAGTheunambiguousbutdegeneratecodecanbeex-PheSerTyrCysUplainedinmolecularterms.TherecognitionofspecificPheSerTyrCysCcodonsinthemRNAbythetRNAadaptermoleculesisULeuSerTermTerm2AdependentupontheiranticodonregionandspecificLeuSerTermTrpGbase-pairingrules.EachtRNAmoleculecontainsaspe-cificsequence,complementarytoacodon,whichisLeuProHisArgUtermeditsanticodon.ForagivencodoninthemRNA,LeuProHisArgConlyasinglespeciesoftRNAmoleculepossessestheCLeuProGlnArgAproperanticodon.SinceeachtRNAmoleculecanbeLeuProGlnArgGchargedwithonlyonespecificaminoacid,eachcodonIleThrAsnSerUthereforespecifiesonlyoneaminoacid.However,someIleThrAsnSerCtRNAmoleculescanutilizetheanticodontorecognize22AIleThrLysArgAmorethanonecodon.Withfewexceptions,givena2MetThrLysArgGspecificcodon,onlyaspecificaminoacidwillbein-ValAlaAspGlyUcorporated—although,givenaspecificaminoacid,ValAlaAspGlyCmorethanonecodonmaybeused.GValAlaGluGlyAAsdiscussedbelow,thereadingofthegeneticcodeValAlaGluGlyGduringtheprocessofproteinsynthesisdoesnotinvolve1Thetermsfirst,second,andthirdnucleotiderefertotheindi-anyoverlapofcodons.Thus,thegeneticcodeisvidualnucleotidesofatripletcodon.U,uridinenucleotide;nonoverlapping.Furthermore,oncethereadingisC,cytosinenucleotide;A,adeninenucleotide;G,guaninenu-commencedataspecificcodon,thereisnopunctua-cleotide;Term,chainterminatorcodon.AUG,whichcodesfortionbetweencodons,andthemessageisreadinacon-Met,servesastheinitiatorcodoninmammaliancellsanden-tinuingsequenceofnucleotidetripletsuntilatransla-codesforinternalmethioninesinaprotein.(Abbreviationsoftionstopcodonisreached.aminoacidsareexplainedinChapter3.)Untilrecently,thegeneticcodewasthoughttobe2Inmammalianmitochondria,AUAcodesforMetandUGAforuniversal.IthasnowbeenshownthatthesetoftRNATrp,andAGAandAGGserveaschainterminators.moleculesinmitochondria(whichcontaintheirownseparateanddistinctsetoftranslationmachinery)fromlowerandhighereukaryotes,includinghumans,readsTHEGENETICCODEISDEGENERATE,fourcodonsdifferentlyfromthetRNAmoleculesinUNAMBIGUOUS,NONOVERLAPPING,thecytoplasmofeventhesamecells.AsnotedinTable38–1,thecodonAUAisreadasMet,andUGAcodesWITHOUTPUNCTUATION,&UNIVERSALforTrpinmammalianmitochondria.Inaddition,inThreeofthe64possiblecodonsdonotcodeforspecificmitochondria,thecodonsAGAandAGGarereadasaminoacids;thesehavebeentermednonsensecodons.stoporchainterminatorcodonsratherthanasArg.AsThesenonsensecodonsareutilizedinthecellastermi-aresult,mitochondriarequireonly22tRNAmoleculesnationsignals;theyspecifywherethepolymerizationtoreadtheirgeneticcode,whereasthecytoplasmicofaminoacidsintoaproteinmoleculeistostop.Thetranslationsystempossessesafullcomplementof31remaining61codonscodefor20aminoacids(TabletRNAspecies.Theseexceptionsnoted,thegenetic38–1).Thus,theremustbe“degeneracy”inthege-codeisuniversal.Thefrequencyofuseofeachaminoneticcode—ie,multiplecodonsmustdecodethesameacidcodonvariesconsiderablybetweenspeciesandaminoacid.Someaminoacidsareencodedbyseveralamongdifferenttissueswithinaspecies.Thespecificcodons;forexample,sixdifferentcodonsspecifyserine.tRNAlevelsgenerallymirrorthesecodonusagebiases.Otheraminoacids,suchasmethionineandtrypto-Thus,aparticularabundantlyusedcodonisdecodedphan,haveasinglecodon.Ingeneral,thethirdnu-byasimilarlyabundantspecifictRNAwhichrecognizescleotideinacodonislessimportantthanthefirsttwothatparticularcodon.Tablesofcodonusagearebe-indeterminingthespecificaminoacidtobeincorpo-comingmoreaccurateasmoregenesaresequenced.rated,andthisaccountsformostofthedegeneracyofThisisofconsiderableimportancebecauseinvestigators
358360/CHAPTER38Table38–2.Featuresofthegeneticcode.tRNAsynthetases.Theyformanactivatedintermedi-ateofaminoacyl-AMP-enzymecomplex(Figure38–1).•DegenerateThespecificaminoacyl-AMP-enzymecomplexthen•UnambiguousrecognizesaspecifictRNAtowhichitattachesthe•Nonoverlappingaminoacylmoietyatthe3′-hydroxyladenosylterminal.•NotpunctuatedThechargingreactionshaveanerrorrateoflessthan−4•Universal10andsoareextremelyaccurate.Theaminoacidre-mainsattachedtoitsspecifictRNAinanesterlinkageuntilitispolymerizedataspecificpositioninthefabri-cationofapolypeptideprecursorofaproteinmolecule.oftenneedtodeducemRNAstructurefromthepri-TheregionsofthetRNAmoleculereferredtoinmarysequenceofaportionofproteininordertosyn-Chapter35(andillustratedinFigure35–11)nowbe-thesizeanoligonucleotideprobeandinitiatearecombi-comeimportant.Thethymidine-pseudouridine-cyti-nantDNAcloningproject.Themainfeaturesofthedine(TΨC)armisinvolvedinbindingoftheamino-geneticcodearelistedinTable38–2.acyl-tRNAtotheribosomalsurfaceatthesiteofproteinsynthesis.TheDarmisoneofthesitesimpor-tantfortheproperrecognitionofagiventRNAspeciesATLEASTONESPECIESOFTRANSFERbyitsproperaminoacyl-tRNAsynthetase.TheacceptorRNA(tRNA)EXISTSFOREACHOFTHEarm,locatedatthe3′-hydroxyladenosylterminal,isthe20AMINOACIDSsiteofattachmentofthespecificaminoacid.Theanticodonregionconsistsofsevennucleotides,tRNAmoleculeshaveextraordinarilysimilarfunctionsanditrecognizesthethree-lettercodoninmRNA(Fig-andthree-dimensionalstructures.Theadapterfunctionure38–2).Thesequencereadfromthe3′to5′direc-ofthetRNAmoleculesrequiresthechargingofeachtioninthatanticodonloopconsistsofavariablespecifictRNAwithitsspecificaminoacid.Sincetherebase–modifiedpurine–XYZ–pyrimidine–pyrimidine-isnoaffinityofnucleicacidsforspecificfunctional5′.Notethatthisdirectionofreadingtheanticodonisgroupsofaminoacids,thisrecognitionmustbecarried3′to5′,whereasthegeneticcodeinTable38–1isreadoutbyaproteinmoleculecapableofrecognizingbotha5′to3′,sincethecodonandtheanticodonloopofthespecifictRNAmoleculeandaspecificaminoacid.AtmRNAandtRNAmolecules,respectively,areantipar-least20specificenzymesarerequiredforthesespecificallelintheircomplementarityjustlikeallotherinter-recognitionfunctionsandfortheproperattachmentofmolecularinteractionsbetweennucleicacidstrands.the20aminoacidstospecifictRNAmolecules.TheThedegeneracyofthegeneticcoderesidesmostlyinprocessofrecognitionandattachment(charging)thelastnucleotideofthecodontriplet,suggestingthatproceedsintwostepsbyoneenzymeforeachofthe20thebasepairingbetweenthislastnucleotideandtheaminoacids.Theseenzymesaretermedaminoacyl-correspondingnucleotideoftheanticodonisnotstrictlyATPPPiAMP+EnzOOHOOCHCREnz•AdenosineOPOCCHRH2NOHNH2Enzyme(Enz)Enz•AMP-aatRNAtRNA-aa(Activatedaminoacid)AMINOACYL-Aminoacid(aa)tRNASYNTHETASEAminoacyl-AMP-enzymeAminoacyl-tRNAcomplexFigure38–1.Formationofaminoacyl-tRNA.Atwo-stepreaction,involvingtheenzymeaminoacyl-tRNAsynthetase,resultsintheformationofaminoacyl-tRNA.Thefirstreactionin-volvestheformationofanAMP-aminoacid-enzymecomplex.ThisactivatedaminoacidisnexttransferredtothecorrespondingtRNAmolecule.TheAMPandenzymearereleased,andthelat-–4tercanbereutilized.Thechargingreactionshaveanerrorrateoflessthan10andsoareex-tremelyaccurate.
359PROTEINSYNTHESIS&THEGENETICCODE/361mRNApurineischangedtotheotherpurine.TransversionsareCodon5′3′changesfromapurinetoeitherofthetwopyrimidinesU•U•UAnticodonorthechangeofapyrimidineintoeitherofthetwoPu*•A•A•A•Pypurines,asshowninFigure38–3.••NAnticodonPyIfthenucleotidesequenceofthegenecontainingarmthemutationistranscribedintoanRNAmolecule,thentheRNAmoleculewillpossessacomplementaryψCDbasechangeatthiscorrespondinglocus.TarmarmSingle-basechangesinthemRNAmoleculesmayhaveoneofseveraleffectswhentranslatedintoprotein:Phenylalanyl-(1)TheremaybenodetectableeffectbecauseofthetRNAdegeneracyofthecode.Thiswouldbemorelikelyif5′thechangedbaseinthemRNAmoleculeweretobeatC•thethirdnucleotideofacodon;suchmutationsareCAcceptorarmoftenreferredtoassilentmutations.Becauseofwob-•Able,thetranslationofacodonisleastsensitivetoa3′changeatthethirdposition.Phe(2)AmissenseeffectwilloccurwhenadifferentaminoacidisincorporatedatthecorrespondingsiteinFigure38–2.Recognitionofthecodonbytheanti-theproteinmolecule.Thismistakenaminoacid—orcodon.OneofthecodonsforphenylalanineisUUU.missense,dependinguponitslocationinthespecifictRNAchargedwithphenylalanine(Phe)hasthecom-protein—mightbeacceptable,partiallyacceptable,orplementarysequenceAAA;hence,itformsabase-pairunacceptabletothefunctionofthatproteinmolecule.complexwiththecodon.Theanticodonregiontypi-Fromacarefulexaminationofthegeneticcode,onecallyconsistsofasequenceofsevennucleotides:vari-canconcludethatmostsingle-basechangeswouldre-able(N),modifiedpurine((Pu*),X,Y,Z,andtwopyrim-sultinthereplacementofoneaminoacidbyanotheridines(Py)inthe3′to5′direction.withrathersimilarfunctionalgroups.Thisisaneffec-tivemechanismtoavoiddrasticchangeinthephysicalpropertiesofaproteinmolecule.Ifanacceptablemis-bytheWatson-Crickrule.Thisiscalledwobble;thesenseeffectoccurs,theresultingproteinmoleculemaypairingofthecodonandanticodoncan“wobble”atthisnotbedistinguishablefromthenormalone.Apartiallyspecificnucleotide-to-nucleotidepairingsite.Forexam-acceptablemissensewillresultinaproteinmoleculeple,thetwocodonsforarginine,AGAandAGG,canwithpartialbutabnormalfunction.Ifanunacceptablebindtothesameanticodonhavingauracilatits5′endmissenseeffectoccurs,thentheproteinmoleculewill(UCU).Similarly,threecodonsforglycine—GGU,notbecapableoffunctioninginitsassignedrole.GGC,andGGA—canformabasepairfromoneanti-(3)Anonsensecodonmayappearthatwouldthencodon,CCI.Iisaninosinenucleotide,anotheroftheresultintheprematureterminationofaminoacidin-peculiarbasesappearingintRNAmolecules.corporationintoapeptidechainandtheproductionofonlyafragmentoftheintendedproteinmolecule.Theprobabilityishighthataprematurelyterminatedpro-MUTATIONSRESULTWHENCHANGESteinmoleculeorpeptidefragmentwillnotfunctioninOCCURINTHENUCLEOTIDESEQUENCEitsassignedrole.Althoughtheinitialchangemaynotoccurinthetem-platestrandofthedouble-strandedDNAmoleculeforthatgene,afterreplication,daughterDNAmoleculesTCTAATwithmutationsinthetemplatestrandwillsegregateandappearinthepopulationoforganisms.SomeMutationsOccurAGCGGCbyBaseSubstitutionTransitionsTransversionsSingle-basechanges(pointmutations)maybetransi-tionsortransversions.Intheformer,agivenpyrimi-Figure38–3.Diagrammaticrepresentationoftransi-dineischangedtotheotherpyrimidineoragiventionmutationsandtransversionmutations.
360362/CHAPTER38HemoglobinIllustratestheEffectsoftopossessatposition67acodonGUAorGUGinSingle-BaseChangesinStructuralGenesorderthatasinglenucleotidechangecouldprovidefortheappearanceoftheglutamicacidcodonsGAAorSomemutationshavenoapparenteffect.ThegeneGAG.HemoglobinSydney,whichcontainsanalaninesystemthatencodeshemoglobinisoneofthebest-atposition67,couldhavearisenbythechangeofasin-studiedinhumans.Thelackofeffectofasingle-baseglenucleotideinanyofthefourcodonsforvalinechangeisdemonstrableonlybysequencingthenu-(GUU,GUC,GUA,orGUG)tothealaninecodonscleotidesinthemRNAmoleculesorstructuralgenes.(GCU,GCC,GCA,orGCG,respectively).ThesequencingofalargenumberofhemoglobinmRNAsandgenesfrommanyindividualshasshownthatthecodonforvalineatposition67oftheβchainSubstitutionofAminoAcidsCausesofhemoglobinisnotidenticalinallpersonswhopos-MissenseMutationssessanormallyfunctionalβchainofhemoglobin.He-moglobinMilwaukeehasatposition67aglutamicA.ACCEPTABLEMISSENSEMUTATIONSacid;hemoglobinBristolcontainsasparticacidatposi-Anexampleofanacceptablemissensemutation(Figuretion67.Inordertoaccountfortheaminoacidchange38–4,top)inthestructuralgenefortheβchainofhe-bythechangeofasinglenucleotideresidueinthemoglobincouldbedetectedbythepresenceofanelec-codonforaminoacid67,onemustinferthatthetrophoreticallyalteredhemoglobinintheredcellsofanmRNAencodinghemoglobinBristolpossessedaGUUapparentlyhealthyindividual.HemoglobinHikarihasorGUCcodonpriortoalaterchangetoGAUorbeenfoundinatleasttwofamiliesofJapanesepeople.GAC,bothcodonsforasparticacid.However,theThishemoglobinhasasparaginesubstitutedforlysinemRNAencodinghemoglobinMilwaukeewouldhaveatthe61positionintheβchain.ThecorrespondingProteinmoleculeAminoacidCodonsHbA,βchain61LysineAAAorAAGAcceptablemissenseHbHikari,βchainAsparagineAAUorAACHbA,βchain6GlutamateGAAorGAGPartiallyacceptablemissenseHbS,βchainValineGUAorGUGHbA,αchain58HistidineCAUorCACUnacceptablemissenseHbM(Boston),αchainTyrosineUAUorUACFigure38–4.Examplesofthreetypesofmissensemutationsresultinginabnormalhemoglo-binchains.Theaminoacidalterationsandpossiblealterationsintherespectivecodonsareindi-cated.ThehemoglobinHikariβ-chainmutationhasapparentlynormalphysiologicpropertiesbutiselectrophoreticallyaltered.HemoglobinShasaβ-chainmutationandpartialfunction;he-moglobinSbindsoxygenbutprecipitateswhendeoxygenated.HemoglobinMBoston,anα-chainmutation,permitstheoxidationofthehemeferrousirontotheferricstateandsowillnotbindoxygenatall.
361PROTEINSYNTHESIS&THEGENETICCODE/363transversionmightbeeitherAAAorAAGchangedtomalterminationcodon(nonsensecodon),thereadingeitherAAUorAAC.Thereplacementofthespecificly-ofthenormalterminationsignalisdisturbed.Suchasinewithasparagineapparentlydoesnotalterthenor-deletionmightresultinreadingthroughaterminationmalfunctionoftheβchainintheseindividuals.signaluntilanothernonsensecodonisencountered(ex-ample1,Figure38–5).ExamplesofthisphenomenonB.PARTIALLYACCEPTABLEMISSENSEMUTATIONSaredescribedindiscussionsofhemoglobinopathies.Apartiallyacceptablemissensemutation(Figure38–4,Insertionsofoneortwoornonmultiplesofthreenu-center)isbestexemplifiedbyhemoglobinS,whichiscleotidesintoageneresultinanmRNAinwhichthefoundinsicklecellanemia.Hereglutamicacid,thereadingframeisdistortedupontranslation,andthesamenormalaminoacidinposition6oftheβchain,haseffectsthatoccurwithdeletionsarereflectedinthebeenreplacedbyvaline.Thecorrespondingsinglenu-mRNAtranslation.ThismayresultingarbledaminocleotidechangewithinthecodonwouldbeGAAoracidsequencesdistaltotheinsertionandthegenerationGAGofglutamicacidtoGUAorGUGofvaline.ofanonsensecodonatordistaltotheinsertion,orper-Clearly,thismissensemutationhindersnormalfunc-hapsreadingthroughthenormalterminationcodon.tionandresultsinsicklecellanemiawhenthemutantFollowingadeletioninagene,aninsertion(orvicegeneispresentinthehomozygousstate.Thegluta-versa)canreestablishtheproperreadingframe(exam-mate-to-valinechangemaybeconsideredtobepartiallyple4,Figure38–5).ThecorrespondingmRNA,whenacceptablebecausehemoglobinSdoesbindandreleasetranslated,wouldcontainagarbledaminoacidsequenceoxygen,althoughabnormally.betweentheinsertionanddeletion.Beyondthereestab-lishmentofthereadingframe,theaminoacidsequenceC.UNACCEPTABLEMISSENSEMUTATIONSwouldbecorrect.Onecanimaginethatdifferentcom-Anunacceptablemissensemutation(Figure38–4,bot-binationsofdeletions,ofinsertions,orofbothwouldtom)inahemoglobingenegeneratesanonfunctioningresultinformationofaproteinwhereinaportionisab-hemoglobinmolecule.Forexample,thehemoglobinMnormal,butthisportionissurroundedbythenormal2+mutationsgeneratemoleculesthatallowtheFeoftheaminoacidsequences.Suchphenomenahavebeen3+hememoietytobeoxidizedtoFe,producingmethe-demonstratedconvincinglyinanumberofdiseases.moglobin.Methemoglobincannottransportoxygen(seeChapter6).SuppressorMutationsCanCounteractSomeoftheEffectsofMissense,FrameshiftMutationsResultFromNonsense,&FrameshiftMutationsDeletionorInsertionofNucleotidesinDNAThatGeneratesAlteredmRNAsTheabovediscussionofthealteredproteinproductsofgenemutationsisbasedonthepresenceofnormallyThedeletionofasinglenucleotidefromthecodingfunctioningtRNAmolecules.However,inprokaryoticstrandofageneresultsinanalteredreadingframeinandlowereukaryoticorganisms,abnormallyfunction-themRNA.ThemachinerytranslatingthemRNAdoesingtRNAmoleculeshavebeendiscoveredthatarenotrecognizethatabasewasmissing,sincethereisnothemselvestheresultsofmutations.Someoftheseab-punctuationinthereadingofcodons.Thus,amajoral-normaltRNAmoleculesarecapableofbindingtoandterationinthesequenceofpolymerizedaminoacids,asdecodingalteredcodons,therebysuppressingtheeffectsdepictedinexample1,Figure38–5,results.Alteringofmutationsindistantstructuralgenes.Thesesup-thereadingframeresultsinagarbledtranslationofthepressortRNAmolecules,usuallyformedastheresultmRNAdistaltothesinglenucleotidedeletion.Notofalterationsintheiranticodonregions,arecapableofonlyisthesequenceofaminoacidsdistaltothisdele-suppressingmissensemutations,nonsensemutations,tiongarbled,butreadingofthemessagecanalsoresultandframeshiftmutations.However,sincethesuppres-intheappearanceofanonsensecodonandthusthesortRNAmoleculesarenotcapableofdistinguishingproductionofapolypeptidebothgarbledandprema-betweenanormalcodonandoneresultingfromageneturelyterminated(example3,Figure38–5).mutation,theirpresenceinacellusuallyresultsinde-Ifthreenucleotidesoramultipleofthreearedeletedcreasedviability.Forinstance,thenonsensesuppressorfromacodingregion,thecorrespondingmRNAwhentRNAmoleculescansuppressthenormalterminationtranslatedwillprovideaproteinfromwhichismissingsignalstoallowaread-throughwhenitisnotdesirable.thecorrespondingnumberofaminoacids(example2,FrameshiftsuppressortRNAmoleculesmayreadanor-Figure38–5).Becausethereadingframeisatriplet,themalcodonplusacomponentofajuxtaposedcodontoreadingphasewillnotbedisturbedforthosecodonsprovideaframeshift,alsowhenitisnotdesirable.Sup-distaltothedeletion.If,however,deletionofoneorpressortRNAmoleculesmayexistinmammaliancells,twonucleotidesoccursjustpriortoorwithinthenor-sinceread-throughtranscriptionoccurs.
362364/CHAPTER38NormalWildtypemRNA5'...UAGUUUGAUGGCCUCUUGCAAAGGCUAUAGUAGUUAG...3'PolypeptideMetAlaSerCysLysGlyTyrSerSerSTOPExample1Deletion(–1)–1UmRNA5'...UAGUUUGAUGGCCCUUGCAAAGGCUAUAGUAGUUAG...3'PolypeptideMetAlaLeuAlaLysAlaThrValValSerGarbledExample2Deletion(–3)–3UGCmRNA5'...UAGUUUGAUGGCCUCUAAAGGCUAUAGUAGUUAG...3'PolypeptideMetAlaSerLysGlyTrySerSerSTOPExample3Insertion(+1)+1CmRNA5'...UAGUUUGAUGGCCCUCUUGCAAAGGCUAUAGUAGUUAG...3'PolypeptideMetAlaLeuLeuGlnArgLeuSTOPGarbledInsertion(+1)Example4Deletion(–1)+1U–1CmRNA5'...UAGUUUGAUGGCCUCUUUGCAAAGGUAUAGUAGUUAG...3'PolypeptideMetAlaSerLeuGlnArgTyrSerSerSTOPGarbledFigure38–5.ExamplesoftheeffectsofdeletionsandinsertionsinageneonthesequenceofthemRNAtranscriptandofthepolypeptidechaintranslatedtherefrom.Thearrowsindicatethesitesofdeletionsorinser-tions,andthenumbersintheovalsindicatethenumberofnucleotideresiduesdeletedorinserted.Bluetypeindicatesaminoacidsincorrectorder.LIKETRANSCRIPTION,PROTEINThetranslationofthemRNAcommencesnearits5′SYNTHESISCANBEDESCRIBEDterminalwiththeformationofthecorrespondingINTHREEPHASES:INITIATION,aminoterminaloftheproteinmolecule.Themessageisreadfrom5′to3′,concludingwiththeformationofELONGATION,&TERMINATIONthecarboxylterminaloftheprotein.Again,theconceptofpolarityisapparent.AsdescribedinChapter37,theThegeneralstructuralcharacteristicsofribosomesandtranscriptionofageneintothecorrespondingmRNAtheirself-assemblyprocessarediscussedinChapter37.oritsprecursorfirstformsthe5′terminaloftheRNATheseparticulateentitiesserveasthemachineryonmolecule.Inprokaryotes,thisallowsforthebeginningwhichthemRNAnucleotidesequenceistranslatedintoofmRNAtranslationbeforethetranscriptionofthethesequenceofaminoacidsofthespecifiedprotein.geneiscompleted.Ineukaryoticorganisms,theprocess
363PROTEINSYNTHESIS&THEGENETICCODE/365oftranscriptionisanuclearone;mRNAtranslationoc-particularlyinterestinginthisregard.Thiskinaseisac-cursinthecytoplasm.Thisprecludessimultaneoustivatedbyvirusesandprovidesahostdefensemecha-transcriptionandtranslationineukaryoticorganismsnismthatdecreasesproteinsynthesis,therebyinhibit-andmakespossibletheprocessingnecessarytogenerateingviralreplication.PhosphorylatedeIF-2αbindsmaturemRNAfromtheprimarytranscript—hnRNA.tightlytoandinactivatestheGTP-GDPrecyclingpro-teineIF-2B.Thispreventsformationofthe43Spreini-InitiationInvolvesSeveralProtein-RNAtiationcomplexandblocksproteinsynthesis.Complexes(Figure38–6)C.FORMATIONOFTHE43SINITIATIONCOMPLEXInitiationofproteinsynthesisrequiresthatanmRNAThe5′terminalsofmostmRNAmoleculesineukary-moleculebeselectedfortranslationbyaribosome.oticcellsare“capped,”asdescribedinChapter37.ThisOncethemRNAbindstotheribosome,thelatterfindsmethyl-guanosyltriphosphatecapfacilitatesthebind-thecorrectreadingframeonthemRNA,andtransla-ingofmRNAtothe43Spreinitiationcomplex.Acaptionbegins.ThisprocessinvolvestRNA,rRNA,bindingproteincomplex,eIF-4F(4F),whichconsistsmRNA,andatleastteneukaryoticinitiationfactorsofeIF-4EandtheeIF-4G(4G)-eIF4A(4A)complex,(eIFs),someofwhichhavemultiple(threetoeight)bindstothecapthroughthe4Eprotein.TheneIF-4Asubunits.AlsoinvolvedareGTP,ATP,andamino(4A)andeIF-4B(4B)bindandreducethecomplexsec-acids.Initiationcanbedividedintofoursteps:(1)dis-ondarystructureofthe5′endofthemRNAthroughsociationoftheribosomeintoits40Sand60Ssub-ATPaseandATP-dependenthelicaseactivities.Theas-units;(2)bindingofaternarycomplexconsistingofisociationofmRNAwiththe43Spreinitiationcomplexmet-tRNA,GTP,andeIF-2tothe40Sribosometotoformthe48SinitiationcomplexrequiresATPhy-formapreinitiationcomplex;(3)bindingofmRNAtodrolysis.eIF-3isakeyproteinbecauseitbindswiththe40Spreinitiationcomplextoforma43Sinitiationhighaffinitytothe4Gcomponentof4F,anditlinkscomplex;and(4)combinationofthe43Sinitiationthiscomplextothe40Sribosomalsubunit.Followingcomplexwiththe60Sribosomalsubunittoformtheassociationofthe43Spreinitiationcomplexwiththe80Sinitiationcomplex.mRNAcapandreduction(“melting”)ofthesecondaryA.RIBOSOMALDISSOCIATIONstructurenearthe5′endofthemRNA,thecomplexscansthemRNAforasuitableinitiationcodon.Gener-Twoinitiationfactors,eIF-3andeIF-1A,bindtotheallythisisthe5′-mostAUG,butthepreciseinitiationnewlydissociated40Sribosomalsubunit.Thisdelayscodonisdeterminedbyso-calledKozakconsensusse-itsreassociationwiththe60SsubunitandallowsotherquencesthatsurroundtheAUG:translationinitiationfactorstoassociatewiththe40Ssubunit.−31−+4B.FORMATIONOFTHE43SPREINITIATIONCOMPLEXGCCAGCCAUGG/ThefirststepinthisprocessinvolvesthebindingofGTPbyeIF-2.Thisbinarycomplexthenbindstomet-iMostpreferredisthepresenceofapurineatpositionstRNA,atRNAspecificallyinvolvedinbindingtothe−3and+4relativetotheAUG.initiationcodonAUG.(TherearetwotRNAsforme-thionine.Onespecifiesmethioninefortheinitiatorcodon,theotherforinternalmethionines.EachhasaD.ROLEOFTHEPOLY(A)TAILININITIATIONuniquenucleotidesequence.)ThisternarycomplexBiochemicalandgeneticexperimentsinyeasthavere-bindstothe40Sribosomalsubunittoformthe43Svealedthatthe3′poly(A)tailanditsbindingprotein,preinitiationcomplex,whichisstabilizedbyassociationPab1p,arerequiredforefficientinitiationofproteinwitheIF-3andeIF-1A.synthesis.Furtherstudiesshowedthatthepoly(A)taileIF-2isoneoftwocontrolpointsforproteinsyn-stimulatesrecruitmentofthe40Sribosomalsubunittothesisinitiationineukaryoticcells.eIF-2consistsofthemRNAthroughacomplexsetofinteractions.α,β,andγsubunits.eIF-2αisphosphorylated(onPab1p,boundtothepoly(A)tail,interactswitheIF-4G,serine51)byatleastfourdifferentproteinkinaseswhichinturnbindstoeIF-4Ethatisboundtothecap(HCR,PKR,PERK,andGCN2)thatareactivatedstructure.Itispossiblethatacircularstructureiswhenacellisunderstressandwhentheenergyexpen-formedandthatthishelpsdirectthe40Sribosomalditurerequiredforproteinsynthesiswouldbedeleteri-subunittothe5′endofthemRNA.Thishelpsexplainous.Suchconditionsincludeaminoacidandglucosehowthecapandpoly(A)tailstructureshaveasynergis-starvation,virusinfection,misfoldedproteins,serumticeffectonproteinsynthesis.Itappearsthatasimilardeprivation,hyperosmolality,andheatshock.PKRismechanismisatworkinmammaliancells.
364TernarycomplexFormationofthe80SActivationofmRNAformationinitiationcomplex80SRibosomaldissociation31A60SMet40S31AeIF-2C2CapAUG(A)nTernaryATP4F=4E+4G4AcomplexMet243SPreinitiation4FCapAUG(A)complexn32B1A2ATP4A4BADP+PiMet4A4FCapAUG(A)n4B2B24FCapAUG(A)n3GDP1A2MetGTPATPADP+Pi2B2CapAUG(A)n48SInitiation3complex1A22BMeteIF-52+P+1A+3iCap(A)nPsiteAsiteMet80SInitiationcomplexElongationFigure38–6.DiagrammaticrepresentationoftheinitiationofproteinsynthesisonthemRNAtemplatecontain-minga5′cap(GTP-5′)and3′poly(A)terminal[3′(A)n].Thisprocessproceedsinthreesteps:(1)activationofmRNA;i(2)formationoftheternarycomplexconsistingoftRNAmet,initiationfactoreIF-2,andGTP;and(3)formationoftheactive80Sinitiationcomplex.(Seetextfordetails.)GTP,•;GDP,.Thevariousinitiationfactorsappearinabbrevi-atedformascirclesorsquares,eg,eIF-3(3),eIF-4F(4F).4•Fisacomplexconsistingof4Eand4Aboundto4G(seeFigure38–7).Theconstellationofproteinfactorsandthe40Sribosomalsubunitcomprisethe43Spreinitiationcom-plex.WhenboundtomRNA,thisformsthe48Spreinitiationcomplex.
365PROTEINSYNTHESISANDTHEGENETICCODE/367E.FORMATIONOFTHE80SINITIATIONCOMPLEXPO4Thebindingofthe60Sribosomalsubunittothe48SinitiationcomplexinvolveshydrolysisoftheGTP4E-BPPO4boundtoeIF-2byeIF-5.Thisreactionresultsinrelease4E-BPoftheinitiationfactorsboundtothe48Sinitiationcomplex(thesefactorsthenarerecycled)andtherapideIF-4EeIF-4EInsulinassociationofthe40Sand60Ssubunitstoformthe(kinase80Sribosome.Atthispoint,themet-tRNAiisonthePactivation)eIF-4Gsiteoftheribosome,readyfortheelongationcycletocommence.eIF-4ATheRegulationofeIF-4EControlstheRateofInitiationThe4Fcomplexisparticularlyimportantincontrollingtherateofproteintranslation.Asdescribedabove,4FiseIF-4GeIF-4Facomplexconsistingof4E,whichbindstothem7GcomplexeIF-4Ecapstructureatthe5′endofthemRNA,and4G,eIF-4Awhichservesasascaffoldingprotein.InadditiontoPO4binding4E,4GbindstoeIF-3,whichlinksthecom-plextothe40Sribosomalsubunit.Italsobinds4Aand4B,theATPase-helicasecomplexthathelpsunwindthe4FCapAUG(A)nRNA(Figure38–7).4EisresponsibleforrecognitionofthemRNAcapFigure38–7.ActivationofeIF-4Ebyinsulinandfor-structure,whichisarate-limitingstepintranslation.mationofthecapbindingeIF-4Fcomplex.The4F-capThisprocessisregulatedattwolevels.Insulinandmi-mRNAcomplexisdepictedasinFigure38–6.The4FtogenicgrowthfactorsresultinthephosphorylationofcomplexconsistsofeIF-4E(4E),eIF-4A,andeIF-4G.4Eis4Eonser209(orthr210).Phosphorylated4Ebindstoinactivewhenboundbyoneofafamilyofbindingpro-thecapmuchmoreavidlythandoesthenonphospho-teins(4E-BPs).Insulinandmitogenicfactors(eg,IGF-1,rylatedform,thusenhancingtherateofinitiation.APDGF,interleukin-2,andangiotensinII)activateaserinecomponentoftheMAPkinasepathway(seeFigureproteinkinaseinthemTORpathway,andthisresultsin43–8)appearstobeinvolvedinthisphosphorylationthephosphorylationof4E-BP.Phosphorylated4E-BPreaction.dissociatesfrom4E,andthelatteristhenabletoformTheactivityof4Eisregulatedinasecondway,andthe4FcomplexandbindtothemRNAcap.Thesethisalsoinvolvesphosphorylation.Arecentlydiscov-growthpeptidesalsophosphorylate4Eitselfbyactivat-eredsetofproteinsbindtoandinactivate4E.TheseingacomponentoftheMAPkinasepathway.Phos-proteinsinclude4E-BP1(BP1,alsoknownasPHAS-1)phorylated4Ebindsmuchmoreavidlytothecapthanandthecloselyrelatedproteins4E-BP2and4E-BP3.doesnonphosphorylated4E.BP1bindswithhighaffinityto4E.The[4E]•[BP1]as-sociationprevents4Efrombindingto4G(toform4F).Sincethisinteractionisessentialforthebindingof4Ftotheribosomal40Ssubunitandforcorrectlyposi-increaseofproteinsynthesisinliver,adiposetissue,andtioningthisonthecappedmRNA,BP-1effectivelyin-muscle.hibitstranslationinitiation.Insulinandothergrowthfactorsresultinthephos-ElongationAlsoIsaMultistepProcessphorylationofBP-1atfiveuniquesites.Phosphoryla-(Figure38–8)tionofBP-1resultsinitsdissociationfrom4E,anditcannotrebinduntilcriticalsitesaredephosphorylated.ElongationisacyclicprocessontheribosomeinwhichTheproteinkinaseresponsiblehasnotbeenidentified,oneaminoacidatatimeisaddedtothenascentpeptidebutitappearstobedifferentfromtheonethatphos-chain.Thepeptidesequenceisdeterminedbytheorderphorylates4E.AkinaseinthemammaliantargetofofthecodonsinthemRNA.Elongationinvolvesseveralrapamycin(mTOR)pathway,perhapsmTORitself,isstepscatalyzedbyproteinscalledelongationfactors(EFs).involved.Theseeffectsontheactivationof4EexplainThesestepsare(1)bindingofaminoacyl-tRNAtotheAinparthowinsulincausesamarkedposttranscriptionalsite,(2)peptidebondformation,and(3)translocation.
366368/CHAPTER38nn+1A.BINDINGOFAMINOACYL-TRNATOTHEASITEGmTP—5′3′(A)nInthecomplete80Sribosomeformedduringtheprocessofinitiation,theAsite(aminoacyloracceptorsite)isfree.Thebindingoftheproperaminoacyl-PsiteAsitetRNAintheAsiterequirespropercodonrecognition.ElongationfactorEF1AformsaternarycomplexwithnGTPandtheenteringaminoacyl-tRNA(Figure38–8).n-1Peptidyl-n-2Thiscomplexthenallowstheaminoacyl-tRNAtoentertRNAtheAsitewiththereleaseofEF1A•GDPandphos-+metphate.GTPhydrolysisiscatalyzedbyanactivesiteontheribosome.AsshowninFigure38–8,EF1A-GDPthenrecyclestoEF1A-GTPwiththeaidofothersolu-GTP+GTPbleproteinfactorsandGTP.EFIAB.PEPTIDEBONDFORMATIONn+1n+1Theα-aminogroupofthenewaminoacyl-tRNAintheAsitecarriesoutanucleophilicattackontheesterifiedGDPcarboxylgroupofthepeptidyl-tRNAoccupyingthePsite(peptidylorpolypeptidesite).Atinitiation,thissiteiGTPnn+1isoccupiedbyaminoacyl-tRNAmet.Thisreactionis5′3′catalyzedbyapeptidyltransferase,acomponentofthe28SRNAofthe60Sribosomalsubunit.Thisisanotherexampleofribozymeactivityandindicatesanimpor-Pi+GDP+tant—andpreviouslyunsuspected—directroleforEFIARNAinproteinsynthesis(Table38–3).Becausethenn+1aminoacidontheaminoacyl-tRNAisalready“acti-Esiten-1vated,”nofurtherenergysourceisrequiredforthisre-n-2action.Thereactionresultsinattachmentofthegrow-ingpeptidechaintothetRNAintheAsite.metC.TRANSLOCATIONnn+15′3′ThenowdeacylatedtRNAisattachedbyitsanticodontothePsiteatoneendandbytheopenCCAtailtoanexit(E)siteonthelargeribosomalsubunit(Figure38–8).Atthispoint,elongationfactor2(EF2)bindstoanddisplacesthepeptidyltRNAfromtheAsitetothePsite.Inturn,thedeacylatedtRNAisontheEsite,n+1GTP++nfromwhichitleavestheribosome.TheEF2-GTPcom-n-1plexishydrolyzedtoEF2-GDP,effectivelymovingtheEF2n-2mRNAforwardbyonecodonandleavingtheAsiteopenforoccupancybyanotherternarycomplexofmetaminoacidtRNA-EF1A-GTPandanothercycleofelongation.nn+1n+2CodonGmTP—5′3′(A)nPi+GDP++Figure38–8.DiagrammaticrepresentationofthepeptideEF2elongationprocessofproteinsynthesis.Thesmallcirclesla-n+1beledn−1,n,n+1,etc,representtheaminoacidresiduesofnn-1thenewlyformedproteinmolecule.EFIAandEF2representn-2elongationfactors1and2,respectively.Thepeptidyl-tRNAandaminoacyl-tRNAsitesontheribosomearerepresentedbyPsitemetandAsite,respectively.
367PROTEINSYNTHESIS&THEGENETICCODE/369Termination(stop)codonGmTP–5′3′(A)nPsiteAsite+Releasingfactor(RF1)C+GTPReleasingfactor(RF3)N5′3′GTPH2OCNGmTP–5′3′(A)nNC++40S+60S++GDP+PiPeptideRF1RF3tRNAFigure38–9.Diagrammaticrepresentationoftheterminationprocessofproteinsynthesis.Thepeptidyl-tRNAandaminoacyl-tRNAsitesareindicatedasPsiteandAsite,respectively.Thetermination(stop)codonisindicatedbythethreeverticalbars.ReleasingfactorRF1bindstothestopcodon.ReleasingfactorRF3,withboundGTP,bindstoRF1.Hydrolysisofthepeptidyl-tRNAcomplexisshownbytheentryofH2O.NandCindicatetheaminoandcarboxyltermi-nalaminoacids,respectively,andillustratethepolarityofproteinsynthesis.
368370/CHAPTER38Table38–3.EvidencethatrRNAproteinsthathydrolyzethepeptidyl-tRNAbondwhenispeptidyltransferase.astopcodonoccupiestheAsite.ThemRNAisthenre-leasedfromtheribosome,whichdissociatesintoitscomponent40Sand60Ssubunits,andanothercycle•Ribosomescanmakepeptidebondsevenwhenproteinscanberepeated.areremovedorinactivated.•CertainpartsoftherRNAsequencearehighlyconservedinallspecies.PolysomesAreAssembliesofRibosomes•TheseconservedregionsareonthesurfaceoftheRNAManyribosomescantranslatethesamemRNAmole-molecule.•RNAcanbecatalytic.culesimultaneously.Becauseoftheirrelativelylarge•Mutationsthatresultinantibioticresistanceatthelevelofsize,theribosomeparticlescannotattachtoanmRNAproteinsynthesisaremoreoftenfoundinrRNAthanintheanycloserthan35nucleotidesapart.Multipleribo-proteincomponentsoftheribosome.somesonthesamemRNAmoleculeformapolyribo-some,or“polysome.”Inanunrestrictedsystem,thenumberofribosomesattachedtoanmRNA(andthusthesizeofpolyribosomes)correlatespositivelywiththeThechargingofthetRNAmoleculewiththelengthofthemRNAmolecule.ThemassofthemRNAaminoacylmoietyrequiresthehydrolysisofanATPtomoleculeis,ofcourse,quitesmallcomparedwiththeanAMP,equivalenttothehydrolysisoftwoATPstomassofevenasingleribosome.twoADPsandphosphates.Theentryoftheaminoacyl-Asinglemammalianribosomeiscapableofsynthe-tRNAintotheAsiteresultsinthehydrolysisofonesizingabout400peptidebondseachminute.Polyribo-GTPtoGDP.Translocationofthenewlyformedpep-somesactivelysynthesizingproteinscanexistasfreetidyl-tRNAintheAsiteintothePsitebyEF2similarlyparticlesinthecellularcytoplasmormaybeattachedtoresultsinhydrolysisofGTPtoGDPandphosphate.sheetsofmembranouscytoplasmicmaterialreferredtoThus,theenergyrequirementsfortheformationofoneasendoplasmicreticulum.Attachmentoftheparticu-peptidebondincludetheequivalentofthehydrolysisoflatepolyribosomestotheendoplasmicreticulumisre-twoATPmoleculestoADPandoftwoGTPmoleculessponsibleforits“rough”appearanceasseenbyelectrontoGDP,orthehydrolysisoffourhigh-energyphos-microscopy.Theproteinssynthesizedbytheattachedphatebonds.Aeukaryoticribosomecanincorporateaspolyribosomesareextrudedintothecisternalspacebe-manyassixaminoacidspersecond;prokaryoticribo-tweenthesheetsofroughendoplasmicreticulumandsomesincorporateasmanyas18persecond.Thus,theareexportedfromthere.Someoftheproteinproductsprocessofpeptidesynthesisoccurswithgreatspeedandoftheroughendoplasmicreticulumarepackagedbyaccuracyuntilaterminationcodonisreached.theGolgiapparatusintozymogenparticlesforeventualexport(seeChapter46).ThepolyribosomalparticlesTerminationOccursWhenaStopfreeinthecytosolareresponsibleforthesynthesisofCodonIsRecognized(Figure38–9)proteinsrequiredforintracellularfunctions.Incomparisontoinitiationandelongation,termina-TheMachineryofProteinSynthesisCantionisarelativelysimpleprocess.AftermultiplecyclesRespondtoEnvironmentalThreatsofelongationculminatinginpolymerizationofthespe-cificaminoacidsintoaproteinmolecule,thestoporFerritin,aniron-bindingprotein,preventsionizedironterminatingcodonofmRNA(UAA,UAG,UGA)ap-2+(Fe)fromreachingtoxiclevelswithincells.ElementalpearsintheAsite.Normally,thereisnotRNAwithanironstimulatesferritinsynthesisbycausingthereleaseofanticodoncapableofrecognizingsuchaterminationacytoplasmicproteinthatbindstoaspecificregioninsignal.ReleasingfactorRF1recognizesthatastopthe5′nontranslatedregionofferritinmRNA.Disrup-codonresidesintheAsite(Figure38–9).RF1isboundtionofthisprotein-mRNAinteractionactivatesferritinbyacomplexconsistingofreleasingfactorRF3withmRNAandresultsinitstranslation.ThismechanismboundGTP.Thiscomplex,withthepeptidyltrans-providesforrapidcontrolofthesynthesisofaproteinferase,promoteshydrolysisofthebondbetweenthe2+thatsequestersFe,apotentiallytoxicmolecule.peptideandthetRNAoccupyingthePsite.Thus,awatermoleculeratherthananaminoacidisadded.ManyVirusesCo-opttheHostCellThishydrolysisreleasestheproteinandthetRNAfromProteinSynthesisMachinerythePsite.Uponhydrolysisandrelease,the80Sribo-somedissociatesintoits40Sand60Ssubunits,whichTheproteinsynthesismachinerycanalsobemodifiedarethenrecycled.Therefore,thereleasingfactorsareindeleteriousways.Virusesreplicatebyusinghost
369PROTEINSYNTHESIS&THEGENETICCODE/371cellprocesses,includingthoseinvolvedinproteinsyn-4Gthesis.SomeviralmRNAsaretranslatedmuchmoreef-4Eficientlythanthoseofthehostcell(eg,encephalomyo-CapAUGcarditisvirus).Others,suchasreovirusandvesicularstomatitisvirus,replicateabundantly,andtheirmRNAs4G4EhaveacompetitiveadvantageoverhostcellmRNAsfor4Glimitedtranslationfactors.Othervirusesinhibithost4EIRESAUGcellproteinsynthesisbypreventingtheassociationofPoliovirusmRNAwiththe40Sribosome.proteasePoliovirusandotherpicornavirusesgainaselectiveadvantagebydisruptingthefunctionofthe4FcomplexNiltotheiradvantage.ThemRNAsofthesevirusesdonot4GCapAUGhaveacapstructuretodirectthebindingofthe40Sri-4E4Gbosomalsubunit(seeabove).Instead,the40Sribosomalsubunitcontactsaninternalribosomalentrysite(IRES)inareactionthatrequires4Gbutnot4E.TheIRESAUGvirusgainsaselectiveadvantagebyhavingaproteasethatFigure38–10.Picornavirusesdisruptthe4Fcom-attacks4Gandremovestheaminoterminal4Ebindingplex.The4E-4Gcomplex(4F)directsthe40Sribosomalsite.Nowthe4E-4Gcomplex(4F)cannotform,sothesubunittothetypicalcappedmRNA(seetext).4G40SribosomalsubunitcannotbedirectedtocappedmRNAs.Hostcelltranslationisthusabolished.The4Galoneissufficientfortargetingthe40Ssubunittothefragmentcandirectbindingofthe40Sribosomalsub-internalribosomalentrysite(IRES)ofviralmRNAs.TounittoIRES-containingmRNAs,soviralmRNAtrans-gainselectiveadvantage,certainviruses(eg,poliovirus)lationisveryefficient(Figure38–10).Thesevirusesalsohaveaproteasethatcleavesthe4EbindingsitefrompromotethedephosphorylationofBP1(PHAS-1),theaminoterminalendof4G.Thistruncated4Gcandi-therebydecreasingcap(4E)-dependenttranslation.rectthe40SribosomalsubunittomRNAsthathaveanIRESbutnottothosethathaveacap.ThewidthsofthearrowsindicatetherateoftranslationinitiationfromPOSTTRANSLATIONALPROCESSINGtheAUGcodonineachexample.AFFECTSTHEACTIVITYOFMANYPROTEINSSomeanimalviruses,notablypoliovirusandhepatitisAlagenpolypeptidemolecules,frequentlynotidenticalinvirus,synthesizelongpolycistronicproteinsfromonesequence,alignthemselvesinaparticularwaythatislongmRNAmolecule.Theseproteinmoleculesaredependentupontheexistenceofspecificaminotermi-subsequentlycleavedatspecificsitestoprovidethesev-nalpeptides.Specificenzymesthencarryouthydrox-eralspecificproteinsrequiredforviralfunction.Inani-ylationsandoxidationsofspecificaminoacidresiduesmalcells,manyproteinsaresynthesizedfromthewithintheprocollagenmoleculestoprovidecross-linksmRNAtemplateasaprecursormolecule,whichthenforgreaterstability.Aminoterminalpeptidesaremustbemodifiedtoachievetheactiveprotein.Thecleavedoffthemoleculetoformthefinalproduct—aprototypeisinsulin,whichisalow-molecular-weightstrong,insolublecollagenmolecule.Manyotherpost-proteinhavingtwopolypeptidechainswithinterchaintranslationalmodificationsofproteinsoccur.Covalentandintrachaindisulfidebridges.Themoleculeissyn-modificationbyacetylation,phosphorylation,methyla-thesizedasasinglechainprecursor,orprohormone,tion,ubiquitinylation,andglycosylationiscommon,whichfoldstoallowthedisulfidebridgestoform.Aforexample.specificproteasethenclipsoutthesegmentthatcon-nectsthetwochainswhichformthefunctionalinsulinMANYANTIBIOTICSWORKBECAUSEmolecule(seeFigure42–12).THEYSELECTIVELYINHIBITPROTEINManyotherpeptidesaresynthesizedasproproteinsSYNTHESISINBACTERIAthatrequiremodificationsbeforeattainingbiologicac-tivity.Manyoftheposttranslationalmodificationsin-Ribosomesinbacteriaandinthemitochondriaofvolvetheremovalofaminoterminalaminoacidhighereukaryoticcellsdifferfromthemammalianribo-residuesbyspecificaminopeptidases.Collagen,ansomedescribedinChapter35.Thebacterialribosomeabundantproteinintheextracellularspacesofhigherissmaller(70Sratherthan80S)andhasadifferent,eukaryotes,issynthesizedasprocollagen.Threeprocol-somewhatsimplercomplementofRNAandprotein
370372/CHAPTER38molecules.Thisdifferenceisexploitedforclinicalpur-Otherantibioticsinhibitproteinsynthesisonallri-posesbecausemanyeffectiveantibioticsinteractspecifi-bosomes(puromycin)oronlyonthoseofeukaryoticcallywiththeproteinsandRNAsofprokaryoticribo-cells(cycloheximide).Puromycin(Figure38–11)isasomesandthusinhibitproteinsynthesis.Thisresultsinstructuralanalogoftyrosinyl-tRNA.Puromycinisin-growtharrestordeathofthebacterium.Themostuse-corporatedviatheAsiteontheribosomeintothecar-fulmembersofthisclassofantibiotics(eg,tetracy-boxylterminalpositionofapeptidebutcausesthepre-clines,lincomycin,erythromycin,andchlorampheni-maturereleaseofthepolypeptide.Puromycin,asacol)donotinteractwithcomponentsofeukaryotictyrosinyl-tRNAanalog,effectivelyinhibitsproteinsyn-ribosomalparticlesandthusarenottoxictoeukaryotes.thesisinbothprokaryotesandeukaryotes.Cyclohex-Tetracyclinepreventsthebindingofaminoacyl-tRNAsimideinhibitspeptidyltransferaseinthe60SribosomaltotheAsite.Chloramphenicolandthemacrolideclasssubunitineukaryotes,presumablybybindingtoanofantibioticsworkbybindingto23SrRNA,whichisrRNAcomponent.interestinginviewofthenewlyappreciatedroleofDiphtheriatoxin,anexotoxinofCorynebacteriumrRNAinpeptidebondformationthroughitspeptidyl-diphtheriaeinfectedwithaspecificlysogenicphage,cat-transferaseactivity.ItshouldbementionedthatthealyzestheADP-ribosylationofEF-2ontheuniqueclosesimilaritybetweenprokaryoticandmitochondrialaminoaciddiphthamideinmammaliancells.ThisribosomescanleadtocomplicationsintheuseofsomemodificationinactivatesEF-2andtherebyspecificallyantibiotics.inhibitsmammalianproteinsynthesis.Manyanimals(eg,mice)areresistanttodiphtheriatoxin.Thisresis-tanceisduetoinabilityofdiphtheriatoxintocrossthecellmembraneratherthantoinsensitivityofmouseN(CH3)2EF-2todiphtheriatoxin-catalyzedADP-ribosylationNbyNAD.NRicin,anextremelytoxicmoleculeisolatedfromthecastorbean,inactivateseukaryotic28SribosomalRNANNbyprovidingtheN-glycolyticcleavageorremovalofaHOCH2Osingleadenine.Manyofthesecompounds—puromycinandcyclo-HHheximideinparticular—arenotclinicallyusefulbutHHhavebeenimportantinelucidatingtheroleofproteinNHOHsynthesisintheregulationofmetabolicprocesses,par-ticularlyenzymeinductionbyhormones.OCCHCH2OCH3NH2SUMMARY•TheflowofgeneticinformationfollowsthesequenceDNA→RNA→protein.NH•Thegeneticinformationinthestructuralregionofa2geneistranscribedintoanRNAmoleculesuchthatNNthesequenceofthelatteriscomplementarytothatin–theDNA.O•SeveraldifferenttypesofRNA,includingribosomalNtRNAOPOCH2ONRNA(rRNA),transferRNA(tRNA),andmessengerRNA(mRNA),areinvolvedinproteinsynthesis.OHH•TheinformationinmRNAisinatandemarrayofHHcodons,eachofwhichisthreenucleotideslong.OOH•ThemRNAisreadcontinuouslyfromastartcodon(AUG)toaterminationcodon(UAA,UAG,UGA).OCCHCH2OH•TheopenreadingframeofthemRNAistheseriesofNHcodons,eachspecifyingacertainaminoacid,thatde-2terminesthepreciseaminoacidsequenceofthepro-Figure38–11.Thecomparativestructuresofthean-tein.tibioticpuromycin(top)andthe3′terminalportionof•Proteinsynthesis,likeDNAandRNAsynthesis,fol-tyrosinyl-tRNA(bottom).lowsa5′to3′polarityandcanbedividedintothree
371PROTEINSYNTHESIS&THEGENETICCODE/373processes:initiation,elongation,andtermination.KozakM:StructuralfeaturesineukaryoticmRNAsthatmodulateMutantproteinsarisewhensingle-basesubstitutionstheinitiationoftranslation.JBiolChem1991;266:1986.resultincodonsthatspecifyadifferentaminoacidatLawrenceJC,AbrahamRT:PHAS/4E-BPsasregulatorsofmRNAagivenposition,whenastopcodonresultsinatrun-translationandcellproliferation.TrendsBiochemSci1997;22:345.catedprotein,orwhenbaseadditionsordeletionsSachsAB,BuratowskiS:Commonthemesintranslationalandalterthereadingframe,sodifferentcodonsareread.transcriptionalregulation.TrendsBiochemSci1997;22:189.•Avarietyofcompounds,includingseveralantibi-SachsAB,SarnowP,HentzeMW:Startingatthebeginning,mid-otics,inhibitproteinsynthesisbyaffectingoneordleandend:translationinitiationineukaryotes.Cell1997;moreofthestepsinvolvedinproteinsynthesis.98:831.WeatherallDJetal:Thehemoglobinopathies.In:TheMetabolicREFERENCESandMolecularBasesofInheritedDisease,8thed.ScriverCRetal(editors).McGraw-Hill,2001.CrickFetal:Thegeneticcode.Nature1961;192:1227.GreenR,NollerHF:Ribosomesandtranslation.AnnuRevBiochem1997;66:679.
372RegulationofGeneExpression39DarylK.Granner,MD,&P.AnthonyWeil,PhDBIOMEDICALIMPORTANCEsuringthattheorganismcanrespondtocomplexenvi-ronmentalchallenges.OrganismsadapttoenvironmentalchangesbyalteringInsimpleterms,thereareonlytwotypesofgenegeneexpression.Theprocessofalterationofgeneex-regulation:positiveregulationandnegativeregula-pressionhasbeenstudiedindetailandofteninvolvestion(Table39–1).Whentheexpressionofgeneticin-modulationofgenetranscription.Controloftranscrip-formationisquantitativelyincreasedbythepresenceoftionultimatelyresultsfromchangesintheinteractionaspecificregulatoryelement,regulationissaidtobeofspecificbindingregulatoryproteinswithvariousre-positive;whentheexpressionofgeneticinformationisgionsofDNAinthecontrolledgene.Thiscanhaveadiminishedbythepresenceofaspecificregulatoryele-positiveornegativeeffectontranscription.Transcrip-ment,regulationissaidtobenegative.Theelementortioncontrolcanresultintissue-specificgeneexpres-moleculemediatingnegativeregulationissaidtobeasion,andgeneregulationisinfluencedbyhormones,negativeregulatororrepressor;thatmediatingpositiveheavymetals,andchemicals.Inadditiontotranscrip-regulationisapositiveregulatororactivator.However,tionlevelcontrols,geneexpressioncanalsobemodu-adoublenegativehastheeffectofactingasapositive.latedbygeneamplification,generearrangement,post-Thus,aneffectorthatinhibitsthefunctionofanega-transcriptionalmodifications,andRNAstabilization.tiveregulatorwillappeartobringaboutapositiveregu-Manyofthemechanismsthatcontrolgeneexpressionlation.Manyregulatedsystemsthatappeartobein-areusedtorespondtohormonesandtherapeuticducedareinfactderepressedatthemolecularlevel.agents.Thus,amolecularunderstandingofthese(SeeChapter9forexplanationoftheseterms.)processeswillleadtodevelopmentofagentsthatalterpathophysiologicmechanismsorinhibitthefunctionorarrestthegrowthofpathogenicorganisms.BIOLOGICSYSTEMSEXHIBITTHREETYPESOFTEMPORALRESPONSESREGULATEDEXPRESSIONOFGENESTOAREGULATORYSIGNALISREQUIREDFORDEVELOPMENT,DIFFERENTIATION,&ADAPTATIONFigure39–1depictstheextentoramountofgeneex-pressioninthreetypesoftemporalresponsetoanin-Thegeneticinformationpresentineachsomaticcellofducingsignal.AtypeAresponseischaracterizedbyanametazoanorganismispracticallyidentical.Theexcep-increasedextentofgeneexpressionthatisdependenttionsarefoundinthosefewcellsthathaveamplifiedoruponthecontinuedpresenceoftheinducingsignal.rearrangedgenesinordertoperformspecializedcellularWhentheinducingsignalisremoved,theamountoffunctions.Expressionofthegeneticinformationmustgeneexpressiondiminishestoitsbasallevel,buttheberegulatedduringontogenyanddifferentiationoftheamountrepeatedlyincreasesinresponsetothereap-organismanditscellularcomponents.Furthermore,inpearanceofthespecificsignal.Thistypeofresponseisorderfortheorganismtoadapttoitsenvironmentandcommonlyobservedinprokaryotesinresponsetosud-toconserveenergyandnutrients,theexpressionofdenchangesoftheintracellularconcentrationofanu-geneticinformationmustbecuedtoextrinsicsignalstrient.Itisalsoobservedinmanyhigherorganismsandrespondonlywhennecessary.Asorganismshaveafterexposuretoinducerssuchashormones,nutrients,evolved,moresophisticatedregulatorymechanismsorgrowthfactors(Chapter43).haveappearedwhichprovidetheorganismanditscellsAtypeBresponseexhibitsanincreasedamountofwiththeresponsivenessnecessaryforsurvivalinacom-geneexpressionthatistransienteveninthecontinuedplexenvironment.Mammaliancellspossessabout1000presenceoftheregulatorysignal.Aftertheregulatorytimesmoregeneticinformationthandoesthebac-signalhasterminatedandthecellhasbeenallowedtoteriumEscherichiacoli.Muchofthisadditionalgeneticrecover,asecondtransientresponsetoasubsequentinformationisprobablyinvolvedinregulationofgeneregulatorysignalmaybeobserved.Thisphenomenonexpressionduringthedifferentiationoftissuesandbio-ofresponse-desensitization-recoverycharacterizesthelogicprocessesinthemulticellularorganismandinen-actionofmanypharmacologicagents,butitisalsoa374
373REGULATIONOFGENEEXPRESSION/375Table39–1.Effectsofpositiveandnegativefeatureofmanynaturallyoccurringprocesses.Thistyperegulationongeneexpression.ofresponsecommonlyoccursduringdevelopmentofanorganism,whenonlythetransientappearanceofaspecificgeneproductisrequiredalthoughthesignalRateofGeneExpressionpersists.NegativePositiveThetypeCresponsepatternexhibits,inresponseRegulationRegulationtotheregulatorysignal,anincreasedextentofgeneex-RegulatorpresentDecreasedIncreasedpressionthatpersistsindefinitelyevenafterterminationofthesignal.Thesignalactsasatriggerinthispattern.RegulatorabsentIncreasedDecreasedOnceexpressionofthegeneisinitiatedinthecell,itcannotbeterminatedeveninthedaughtercells;itisthereforeanirreversibleandinheritedalteration.Thistypeofresponsetypicallyoccursduringthedevelop-mentofdifferentiatedfunctioninatissueororgan.TypeAProkaryotesProvideModelsfortheStudyofGeneExpressioninMammalianCellsAnalysisoftheregulationofgeneexpressioninprokaryoticcellshelpedestablishtheprinciplethatin-formationflowsfromthegenetoamessengerRNAtoaGeneexpressionspecificproteinmolecule.Thesestudieswereaidedbytheadvancedgeneticanalysesthatcouldbeperformedinprokaryoticandlowereukaryoticorganisms.Inre-Timecentyears,theprinciplesestablishedintheseearlystud-Signalies,coupledwithavarietyofmolecularbiologytech-TypeBniques,haveledtoremarkableprogressintheanalysisofgeneregulationinhighereukaryoticorganisms,in-cludingmammals.Inthischapter,theinitialdiscussionwillcenteronprokaryoticsystems.Theimpressivege-neticstudieswillnotbedescribed,butthephysiologyofgeneexpressionwillbediscussed.However,nearlyalloftheconclusionsaboutthisphysiologyhavebeenGeneexpressionderivedfromgeneticstudiesandconfirmedbymolecu-largeneticandbiochemicalstudies.RecoveryTimeSignalSomeFeaturesofProkaryoticGeneExpressionAreUniqueTypeCBeforethephysiologyofgeneexpressioncanbeex-plained,afewspecializedgeneticandregulatorytermsmustbedefinedforprokaryoticsystems.Inprokary-otes,thegenesinvolvedinametabolicpathwayareoftenpresentinalineararraycalledanoperon,eg,thelacoperon.Anoperoncanberegulatedbyasinglepro-moterorregulatoryregion.ThecistronisthesmallestGeneexpressionunitofgeneticexpression.AsdescribedinChapter9,someenzymesandotherproteinmoleculesarecom-posedoftwoormorenonidenticalsubunits.Thus,theTimeSignal“onegene,oneenzyme”conceptisnotnecessarilyvalid.ThecistronisthegeneticunitcodingfortheFigure39–1.Diagrammaticrepresentationsofthestructureofthesubunitofaproteinmolecule,actingasresponsesoftheextentofexpressionofagenetospe-itdoesasthesmallestunitofgeneticexpression.Thus,cificregulatorysignalssuchasahormone.theonegene,oneenzymeideamightmoreaccurately
374376/CHAPTER39beregardedasaonecistron,onesubunitconcept.APromoterOperatorsinglemRNAthatencodesmorethanoneseparatelysitetranslatedproteinisreferredtoasapolycistroniclacIlacZlacYlacAmRNA.Forexample,thepolycistroniclacoperonmRNAistranslatedintothreeseparateproteins(seelacoperonbelow).OperonsandpolycistronicmRNAsarecom-moninbacteriabutnotineukaryotes.Figure39–2.ThepositionalrelationshipsoftheAninduciblegeneisonewhoseexpressionincreasesstructuralandregulatorygenesofthelacoperon.lacZinresponsetoaninduceroractivator,aspecificposi-encodesβ-galactosidase,lacYencodesapermease,andtiveregulatorysignal.Ingeneral,induciblegeneshavelacAencodesathiogalactosidetransacetylase.lacIen-relativelylowbasalratesoftranscription.Bycontrast,codesthelacoperonrepressorprotein.geneswithhighbasalratesoftranscriptionareoftensubjecttodown-regulationbyrepressors.Theexpressionofsomegenesisconstitutive,mean-encodestheprimarytranscript.Althoughthereareingthattheyareexpressedatareasonablyconstantratemanyhistoricalexceptions,ageneisgenerallyitalicizedandnotknowntobesubjecttoregulation.Theseareinlowercaseandtheencodedprotein,whenabbrevi-oftenreferredtoashousekeepinggenes.Asaresultofated,isexpressedinromantypewiththefirstlettercap-mutation,someinduciblegeneproductsbecomecon-italized.Forexample,thegenelacIencodestherepres-stitutivelyexpressed.Amutationresultinginconstitu-sorproteinLacI.WhenEcoliispresentedwithlactosetiveexpressionofwhatwasformerlyaregulatedgeneisorsomespecificlactoseanalogsunderappropriatenon-calledaconstitutivemutation.repressingconditions(eg,highconcentrationsoflac-tose,noorverylowglucoseinmedia;seebelow),theAnalysisofLactoseMetabolisminEcoliexpressionoftheactivitiesofβ-galactosidase,galacto-sidepermease,andthiogalactosidetransacetylaseisin-LedtotheOperonHypothesiscreased100-foldto1000-fold.ThisisatypeAre-JacobandMonodin1961describedtheiroperonsponse,asdepictedinFigure39–1.Thekineticsofmodelinaclassicpaper.Theirhypothesiswastoainductioncanbequiterapid;lac-specificmRNAsarelargeextentbasedonobservationsontheregulationoffullyinducedwithin5–6minutesafteradditionoflac-lactosemetabolismbytheintestinalbacteriumEcoli.tosetoaculture;β-galactosidaseproteinismaximalThemolecularmechanismsresponsiblefortheregula-within10minutes.Underfullyinducedconditions,tionofthegenesinvolvedinthemetabolismoflactosetherecanbeupto5000β-galactosidasemoleculesperarenowamongthebest-understoodinanyorganism.cell,anamountabout1000timesgreaterthantheβ-Galactosidasehydrolyzestheβ-galactosidelactosetobasal,uninducedlevel.Uponremovalofthesignal,ie,galactoseandglucose.Thestructuralgeneforβ-galac-theinducer,thesynthesisofthesethreeenzymesde-tosidase(lacZ)isclusteredwiththegenesresponsibleclines.forthepermeationofgalactoseintothecell(lacY)andWhenEcoliisexposedtobothlactoseandglucoseforthiogalactosidetransacetylase(lacA).Thestructuralassourcesofcarbon,theorganismsfirstmetabolizegenesforthesethreeenzymes,alongwiththelacpro-theglucoseandthentemporarilystopgrowinguntilthemoterandlacoperator(aregulatoryregion),arephysi-genesofthelacoperonbecomeinducedtoprovidethecallyassociatedtoconstitutethelacoperonasdepictedabilitytometabolizelactoseasausableenergysource.inFigure39–2.Thisgeneticarrangementofthestruc-Althoughlactoseispresentfromthebeginningoftheturalgenesandtheirregulatorygenesallowsforcoordi-bacterialgrowthphase,thecelldoesnotinducethosenateexpressionofthethreeenzymesconcernedwithenzymesnecessaryforcatabolismoflactoseuntilthelactosemetabolism.Eachoftheselinkedgenesistran-glucosehasbeenexhausted.ThisphenomenonwasfirstscribedintoonelargemRNAmoleculethatcontainsthoughttobeattributabletorepressionofthelacmultipleindependenttranslationstart(AUG)andstopoperonbysomecataboliteofglucose;hence,itwas(UAA)codonsforeachcistron.Thus,eachproteinistermedcataboliterepression.Itisnowknownthattranslatedseparately,andtheyarenotprocessedfromacataboliterepressionisinfactmediatedbyacatabolitesinglelargeprecursorprotein.ThistypeofmRNAmol-geneactivatorprotein(CAP)inconjunctionwitheculeiscalledapolycistronicmRNA.PolycistroniccAMP(Figure18–5).ThisproteinisalsoreferredtoasmRNAsarepredominantlyfoundinprokaryoticorgan-thecAMPregulatoryprotein(CRP).Theexpressionofisms.manyinducibleenzymesystemsoroperonsinEcoliItisnowconventionaltoconsiderthatagenein-andotherprokaryotesissensitivetocataboliterepres-cludesregulatorysequencesaswellastheregionthatsion,asdiscussedbelow.
375REGULATIONOFGENEEXPRESSION/377AOperatorPromoterlacIgenelacZgenelacYgenelacAgeneRNApolymeraseNoinducercannottranscribe↑RNAoperatorordistalorpolymerasegenes(Z,Y,A)InducerandglucoseRepressorsubunitsRepressor(tetramer)BCAP-cAMPlacIlacZlacYlacAWithinducerandRNApolymerasestranscribinggenesnoglucoseInactiverepressormRNAβ-Galacto-PermeaseTransacetylaseInducerssidaseproteinproteinproteinFigure39–3.Themechanismofrepressionandderepressionofthelacoperon.Wheneithernoinducerispresentorinducerispresentwithglucose(A),thelacIgeneproductsthataresynthesizedconstitutivelyformarepressortetramermoleculewhichbindsattheoperatorlocustopreventtheefficientinitiationoftranscrip-tionbyRNApolymeraseatthepromoterlocusandthustopreventthesubsequenttranscriptionofthelacZ,lacY,andlacAstructuralgenes.Wheninducerispresent(B),theconstitutivelyexpressedlacIgeneformsre-pressormoleculesthatareconformationallyalteredbytheinducerandcannotefficientlybindtotheoperatorlocus(affinityofbindingreduced>1000-fold).InthepresenceofcAMPanditsbindingprotein(CAP),theRNApolymerasecantranscribethestructuralgeneslacZ,lacY,andlacA,andthepolycistronicmRNAmoleculeformedcanbetranslatedintothecorrespondingproteinmoleculesβ-galactosidase,permease,andtransacetylase,allowingforthecatabolismoflactose.Thephysiologyofinductionofthelacoperonisaninvertedpalindrome(indicatedbysolidlinesaboutwellunderstoodatthemolecularlevel(Figure39–3).thedottedaxis)inaregionthatis21basepairslong,asExpressionofthenormallacIgeneofthelacoperonisshownbelow:constitutive;itisexpressedataconstantrate,resultinginformationofthesubunitsofthelacrepressor.Four:identicalsubunitswithmolecularweightsof38,000as-5′−AATTGTGAGCGGATAACAATTsembleintoalacrepressormolecule.TheLacIrepressor3′−TTAACACTCGCCTATTGTTAAproteinmolecule,theproductoflacI,hasahighaffinity−13:(Kdabout10mol/L)fortheoperatorlocus.Theop-eratorlocusisaregionofdouble-strandedDNA27TheminimumeffectivesizeofanoperatorforLacIbasepairslongwithatwofoldrotationalsymmetryandrepressorbindingis17basepairs(boldfacelettersinthe
376378/CHAPTER39abovesequence).Atanyonetime,onlytwosubunitsofpriatebioengineeredconstructsiscommonlyusedtoex-therepressorsappeartobindtotheoperator,andwithinpressmammalianrecombinantproteinsinEcoli.the17-base-pairregionatleastonebaseofeachbaseInorderfortheRNApolymerasetoefficientlyformpairisinvolvedinLacIrecognitionandbinding.TheaPICatthepromotersite,theremustalsobepresentbindingoccursmostlyinthemajorgroovewithoutin-thecatabolitegeneactivatorprotein(CAP)towhichterruptingthebase-paired,double-helicalnatureofthecAMPisbound.Byanindependentmechanism,theoperatorDNA.Theoperatorlocusisbetweenthepro-bacteriumaccumulatescAMPonlywhenitisstarvedmotersite,atwhichtheDNA-dependentRNApolym-forasourceofcarbon.Inthepresenceofglucose—oreraseattachestocommencetranscription,andthetran-ofglycerolinconcentrationssufficientforgrowth—thescriptioninitiationsiteofthelacZgene,thestructuralbacteriawilllacksufficientcAMPtobindtoCAPbe-geneforβ-galactosidase(Figure39–2).Whenattachedcausetheglucoseinhibitsadenylylcyclase,theenzymetotheoperatorlocus,theLacIrepressormoleculepre-thatconvertsATPtocAMP(seeChapter42).Thus,inventstranscriptionoftheoperatorlocusaswellasofthethepresenceofglucoseorglycerol,cAMP-saturateddistalstructuralgenes,lacZ,lacY,andlacA.Thus,theCAPislacking,sothattheDNA-dependentRNALacIrepressormoleculeisanegativeregulator;initspolymerasecannotinitiatetranscriptionofthelacpresence(andintheabsenceofinducer;seebelow),ex-operon.InthepresenceoftheCAP-cAMPcomplex,pressionfromthelacZ,lacY,andlacAgenesispre-whichbindstoDNAjustupstreamofthepromotervented.Therearenormally20–40repressortetramersite,transcriptionthenoccurs(Figure39–3).Studiesmoleculesinthecell,aconcentrationoftetramersuffi-indicatethataregionofCAPcontactstheRNApolym-cienttoeffect,atanygiventime,>95%occupancyoferaseαsubunitandfacilitatesbindingofthisenzymetotheonelacoperatorelementinabacterium,thusensur-thepromoter.Thus,theCAP-cAMPregulatorisactinginglow(butnotzero)basallacoperongenetranscrip-asapositiveregulatorbecauseitspresenceisrequiredtionintheabsenceofinducingsignals.forgeneexpression.Thelacoperonisthereforecon-Alactoseanalogthatiscapableofinducingthelactrolledbytwodistinct,ligand-modulatedDNAbind-operonwhilenotitselfservingasasubstrateforβ-galac-ingtransfactors;onethatactspositively(cAMP-CRPtosidaseisanexampleofagratuitousinducer.Anex-complex)andonethatactsnegatively(LacIrepressor).ampleisisopropylthiogalactoside(IPTG).TheadditionMaximalactivityofthelacoperonoccurswhenglucoseoflactoseorofagratuitousinducersuchasIPTGtolevelsarelow(highcAMPwithCAPactivation)andbacteriagrowingonapoorlyutilizedcarbonsourcelactoseispresent(LacIispreventedfrombindingtothe(suchassuccinate)resultsinpromptinductionoftheoperator).lacoperonenzymes.Smallamountsofthegratuitousin-WhenthelacIgenehasbeenmutatedsothatitsduceroroflactoseareabletoenterthecellevenintheproduct,LacI,isnotcapableofbindingtooperatorabsenceofpermease.TheLacIrepressormolecules—DNA,theorganismwillexhibitconstitutiveexpres-boththoseattachedtotheoperatorlociandthosefreeinsionofthelacoperon.Inacontrarymanner,anorgan-thecytosol—haveahighaffinityfortheinducer.Bind-ismwithalacIgenemutationthatproducesaLacIpro-ingoftheinducertoarepressormoleculeattachedtoteinwhichpreventsthebindingofaninducertothetheoperatorlocusinducesaconformationalchangeinrepressorwillremainrepressedeveninthepresenceofthestructureoftherepressorandcausesittodissociatetheinducermolecule,becausetheinducercannotbindfromtheDNAbecauseitsaffinityfortheoperatoristotherepressorontheoperatorlocusinordertodere-3−9now10timeslower(Kdabout10mol/L)thanthatofpresstheoperon.Similarly,bacteriaharboringmuta-LacIintheabsenceofIPTG.IfDNA-dependentRNAtionsintheirlacoperatorlocussuchthattheoperatorpolymerasehasalreadyattachedtothecodingstrandatsequencewillnotbindanormalrepressormoleculethepromotersite,transcriptionwillbegin.Thepolym-constitutivelyexpressthelacoperongenes.MechanismserasegeneratesapolycistronicmRNAwhose5′terminalofpositiveandnegativeregulationcomparabletothoseiscomplementarytothetemplatestrandoftheoperator.describedhereforthelacsystemhavebeenobservedinInsuchamanner,aninducerderepressesthelaceukaryoticcells(seebelow).operonandallowstranscriptionofthestructuralgenesforβ-galactosidase,galactosidepermease,andthiogalac-TheGeneticSwitchofBacteriophagetosidetransacetylase.TranslationofthepolycistronicLambda()ProvidesaParadigmmRNAcanoccurevenbeforetranscriptioniscom-forProtein-DNAInteractionspleted.DerepressionofthelacoperonallowsthecelltoinEukaryoticCellssynthesizetheenzymesnecessarytocatabolizelactoseasanenergysource.Basedonthephysiologyjustde-Likesomeeukaryoticviruses(eg,herpessimplex,HIV),scribed,IPTG-inducedexpressionoftransfectedplas-somebacterialvirusescaneitherresideinadormantmidsbearingthelacoperator-promoterligatedtoappro-statewithinthehostchromosomesorcanreplicate
377REGULATIONOFGENEEXPRESSION/379withinthebacteriumandeventuallyleadtolysisand1killingofthebacterialhost.SomeEcoliharborsucha“temperate”virus,bacteriophagelambda(λ).Whenlambdainfectsanorganismofthatspeciesitinjectsits45,000-bp,double-stranded,linearDNAgenomeintothecell(Figure39–4).Dependinguponthenutritional2stateofthecell,thelambdaDNAwilleitherintegrateintothehostgenome(lysogenicpathway)andremaindormantuntilactivated(seebelow),oritwillcom-mencereplicatinguntilithasmadeabout100copiesofcomplete,protein-packagedvirus,atwhichpointitcauseslysisofitshost(lyticpathway).Thenewlygen-eratedvirusparticlescantheninfectothersusceptible3hosts.Whenintegratedintothehostgenomeinitsdor-mantstate,lambdawillremaininthatstateuntilacti-LysogenicLyticvatedbyexposureofitslysogenicbacterialhosttopathwaypathwayDNA-damagingagents.Inresponsetosuchanoxious46stimulus,thedormantbacteriophagebecomes“in-duced”andbeginstotranscribeandsubsequentlytrans-latethosegenesofitsowngenomewhicharenecessaryforitsexcisionfromthehostchromosome,itsDNAreplication,andthesynthesisofitsproteincoatandlysisenzymes.ThiseventactslikeatriggerortypeC5107(Figure39–1)response;ie,oncelambdahascommitteditselftoinduction,thereisnoturningbackuntilthecellislysedandthereplicatedbacteriophagereleased.ThisswitchfromadormantorprophagestatetoaUltravioletradiationInductionlyticinfectioniswellunderstoodatthegeneticand98molecularlevelsandwillbedescribedindetailhere.Theswitchingeventinlambdaiscenteredaroundan80-bpregioninitsdouble-strandedDNAgenomereferredtoasthe“rightoperator”(OR)(Figure39–5A).TherightoperatorisflankedonitsleftsidebytheFigure39–4.InfectionofthebacteriumEcolibystructuralgeneforthelambdarepressorprotein,thecIprotein,andonitsrightsidebythestructuralgeneen-phagelambdabeginswhenavirusparticleattachesit-codinganotherregulatoryproteincalledCro.Whenselftothebacterialcell(1)andinjectsitsDNA(shadedlambdaisinitsprophagestate—ie,integratedintotheline)intothecell(2,3).Infectioncantakeeitheroftwohostgenome—thecIrepressorgeneistheonlylambdacoursesdependingonwhichoftwosetsofviralgenesgenecIproteinthatisexpressed.Whenthebacterio-isturnedon.Inthelysogenicpathway,theviralDNAphageisundergoinglyticgrowth,thecIrepressorgenebecomesintegratedintothebacterialchromosome(4,isnotexpressed,butthecrogene—aswellasmany5),whereitreplicatespassivelyasthebacterialcelldi-othergenesinlambda—isexpressed.Thatis,whenthevides.Thedormantvirusiscalledaprophage,andtherepressorgeneison,thecrogeneisoff,andwhencellthatharborsitiscalledalysogen.Inthealternativethecrogeneison,therepressorgeneisoff.Aswelyticmodeofinfection,theviralDNAreplicatesitself(6)shallsee,thesetwogenesregulateeachother’sexpres-anddirectsthesynthesisofviralproteins(7).About100sionandthus,ultimately,thedecisionbetweenlyticnewvirusparticlesareformed.Theproliferatingvirusesandlysogenicgrowthoflambda.Thisdecisionbe-lyse,orburst,thecell(8).Aprophagecanbe“induced”tweenrepressorgenetranscriptionandcrogenebyaDNAdamagingagentsuchasultravioletradiationtranscriptionisaparadigmaticexampleofamolecu-(9).Theinducingagentthrowsaswitch,sothatadiffer-larswitch.entsetofgenesisturnedon.ViralDNAloopsoutoftheThe80-bpλrightoperator,OR,canbesubdividedchromosome(10)andreplicates;thevirusproceedsintothreediscrete,evenlyspaced,17-bpcis-activealongthelyticpathway.(Reproduced,withpermission,DNAelementsthatrepresentthebindingsitesforei-fromPtashneM,JohnsonAD,PaboCO:Ageneticswitchtheroftwobacteriophageλregulatoryproteins.Impor-inabacterialvirus.SciAm[Nov]1982;247:128.)
378380/CHAPTER39Geneforrepressor(cl)GeneforCroAORRepressorRNAOR3OR2OR1BRepressorpromotercroPromotercroRNATACCTCTGGCGGTGATACATGGGAACCGCCACTATFigure39–5.Rightoperator(OR)isshowninincreasingdetailinthisseriesofdrawings.TheoperatorisaregionoftheviralDNAsome80basepairslong(A).Toitsleftliesthegeneencodinglambdarepressor(cI),toitsrightthegene(cro)encodingtheregulatorpro-teinCro.Whentheoperatorregionisenlarged(B),itisseentoincludethreesubregions,OR1,OR2,andOR3,each17basepairslong.Theyarerecognitionsitestowhichbothrepres-sorandCrocanbind.Therecognitionsitesoverlaptwopromoters—sequencesofbasestowhichRNApolymerasebindsinordertotranscribethesegenesintomRNA(wavylines),thataretranslatedintoprotein.SiteOR1isenlarged(C)toshowitsbasesequence.Notethatinthisregionoftheλchromosome,bothstrandsofDNAactasatemplatefortran-scription(Chapter39).(Reproduced,withpermission,fromPtashneM,JohnsonAD,PaboCO:Ageneticswitchinabacterialvirus.SciAm[Nov]1982;247:128.)tantly,thenucleotidesequencesofthesethreetandemly39–6D).TheCroprotein’ssingledomainmediatesarrangedsitesaresimilarbutnotidentical(Figurebothoperatorbindinganddimerization.39–5B).Thethreerelatedciselements,termedopera-Inalysogenicbacterium—ie,abacteriumcontainingtorsOR1,OR2,andOR3,canbeboundbyeithercIoralambdaprophage—thelambdarepressordimerbindsCroproteins.However,therelativeaffinitiesofcIandpreferentiallytoOR1butinsodoing,byacooperativeCroforeachofthesitesvaries,andthisdifferentialinteraction,enhancesthebinding(byafactorof10)ofbindingaffinityiscentraltotheappropriateoperationanotherrepressordimertoOR2(Figure39–7).Theoftheλphagelyticorlysogenic“molecularswitch.”affinityofrepressorforOR3istheleastofthethreeoper-TheDNAregionbetweenthecroandrepressorgenesatorsubregions.ThebindingofrepressortoOR1hastwoalsocontainstwopromotersequencesthatdirectthemajoreffects.TheoccupationofOR1byrepressorbindingofRNApolymeraseinaspecifiedorientation,blocksthebindingofRNApolymerasetotheright-whereitcommencestranscribingadjacentgenes.OnewardpromoterandinthatwaypreventsexpressionofpromoterdirectsRNApolymerasetotranscribeinthecro.Second,asmentionedabove,repressordimerboundrightwarddirectionand,thus,totranscribecroandtoOR1enhancesthebindingofrepressordimertoOR2.otherdistalgenes,whiletheotherpromoterdirectstheThebindingofrepressortoOR2hastheimportanttranscriptionoftherepressorgeneintheleftwarddi-addedeffectofenhancingthebindingofRNApolym-rection(Figure39–5B).erasetotheleftwardpromoterthatoverlapsOR2andTheproductoftherepressorgene,the236-amino-therebyenhancestranscriptionandsubsequentexpres-acid,27kDarepressorprotein,existsasatwo-sionoftherepressorgene.Thisenhancementoftran-domainmoleculeinwhichtheaminoterminaldomainscriptionisapparentlymediatedthroughdirectprotein-bindstooperatorDNAandthecarboxylterminalproteininteractionsbetweenpromoter-boundRNAdomainpromotestheassociationofonerepressorpolymeraseandOR2-boundrepressor.Thus,thelambdaproteinwithanothertoformadimer.Adimerofre-repressorisbothanegativeregulator,bypreventingpressormoleculesbindstooperatorDNAmuchmoretranscriptionofcro,andapositiveregulator,byenhanc-tightlythandoesthemonomericform(Figure39–6Aingtranscriptionofitsowngene,therepressorgene.to39–6C).ThisdualeffectofrepressorisresponsibleforthestableTheproductofthecrogene,the66-amino-acid,stateofthedormantlambdabacteriophage;notonly9kDaCroprotein,hasasingledomainbutalsobindsdoestherepressorpreventexpressionofthegenesneces-theoperatorDNAmoretightlyasadimer(Figuresaryforlysis,butitalsopromotesexpressionofitselfto
379REGULATIONOFGENEEXPRESSION/381ABCDAminoacidsCOOHCOOHCOOHCOOHCOOH132–236CroNH2AminoacidsNH2NH2NH2NH21–92OR1OR3Figure39–6.SchematicmolecularstructuresofcI(lambdarepressor,showninA,B,andC)andCro(D).Lambdarepressorproteinisapolypeptidechain236aminoacidslong.Thechainfoldsitselfintoadumbbellshapewithtwosubstructures:anaminoterminal(NH2)domainandacarboxylterminal(COOH)domain.Thetwodomainsarelinkedbyaregionofthechainthatissusceptibletocleavagebyproteases(indicatedbythetwoarrowsinA).Singlerepressormole-cules(monomers)tendtoassociatetoformdimers(B);adimercandissociatetoformmonomersagain.Adimerisheldtogethermainlybycontactbetweenthecarboxylterminaldomains(hatching).Repressordimersbindto(andcandissociatefrom)therecognitionsitesintheoperatorregion;theydisplaythegreatestaffinityforsiteOR1(C).ItistheaminoterminaldomainoftherepressormoleculethatmakescontactwiththeDNA(hatching).Cro(D)hasasingledomainwithsitesthatpromotedimerizationandothersitesthatpromotebindingofdimerstooperator,preferentiallytoOR3.(Reproduced,withpermission,fromPtashneM,JohnsonAD,PaboCO:Ageneticswitchinabacterialvirus.SciAm[Nov]1982;247:128.)stabilizethisstateofdifferentiation.Intheeventthatin-TheresultingnewlysynthesizedCroproteinalsotracellularrepressorproteinconcentrationbecomesverybindstotheoperatorregionasadimer,butitsorderofhigh,thisexcessrepressorwillbindtoOR3andbysopreferenceisoppositetothatofrepressor(Figuredoingdiminishtranscriptionoftherepressorgenefrom39–7).Thatis,CrobindsmosttightlytoOR3,buttheleftwardpromoteruntiltherepressorconcentrationthereisnocooperativeeffectofCroatOR3onthedropsandrepressordissociatesitselffromOR3.bindingofCrotoOR2.Atincreasinglyhigherconcen-Withsuchastable,repressive,cI-mediated,lyso-trationsofCro,theproteinwillbindtoOR2andeven-genicstate,onemightwonderhowthelyticcyclecouldtuallytoOR1.everbeentered.However,thisprocessdoesoccurquiteOccupancyofOR3byCroimmediatelyturnsoffefficiently.WhenaDNA-damagingsignal,suchasul-transcriptionfromtheleftwardpromoterandinthattravioletlight,strikesthelysogenichostbacterium,waypreventsanyfurtherexpressionoftherepressorfragmentsofsingle-strandedDNAaregeneratedthatgene.Themolecularswitchisthuscompletelyactivateaspecificproteasecodedbyabacterialgene“thrown”inthelyticdirection.Thecrogeneisnowex-andreferredtoasrecA(Figure39–7).Theactivatedpressed,andtherepressorgeneisfullyturnedoff.ThisrecAproteasehydrolyzestheportionoftherepressoreventisirreversible,andtheexpressionofotherlambdaproteinthatconnectstheaminoterminalandcarboxylgenesbeginsaspartofthelyticcycle.WhenCrorepres-terminaldomainsofthatmolecule(seeFigure39–6A).sorconcentrationbecomesquitehigh,itwilleventuallySuchcleavageoftherepressordomainscausesthere-occupyOR1andinsodoingreducetheexpressionofitspressordimerstodissociate,whichinturncausesdis-owngene,aprocessthatisnecessaryinordertoeffectsociationoftherepressormoleculesfromOR2andthefinalstagesofthelyticcycle.eventuallyfromOR1.Theeffectsofremovalofrepres-Thethree-dimensionalstructuresofCroandofthesorfromOR1andOR2arepredictable.RNApolym-lambdarepressorproteinhavebeendeterminedbyeraseimmediatelyhasaccesstotherightwardpromoterx-raycrystallography,andmodelsfortheirbindingandef-andcommencestranscribingthecrogene,andtheen-fectingtheabove-describedmolecularandgeneticeventshancementeffectoftherepressoratOR2onleftwardhavebeenproposedandtested.BothbindtoDNAusingtranscriptionislost(Figure39–7).helix-turn-helixDNAbindingdomainmotifs(seebelow).
380ProphageOR3OR2OR1RNApolymeraseRepressorpromotercropromoterOR3OR2OR1Induction(1)RNApolymeraserecARepressorpromotercropromoterUltravioletradiationOR3OR2OR1RNApolymeraseInduction(2)RepressorpromotercropromoterEarlylyticgrowthOR3OR2OR1RNApolymeraseRepressorpromotercropromoterFigure39–7.Configurationoftheswitchisshownatfourstagesoflambda’slifecycle.Thelysogenicpathway(inwhichthevirusremainsdormantasaprophage)isselectedwhenarepressordimerbindstoOR1,therebymakingitlikelythatOR2willbefilledimmediatelybyanotherdimer.Intheprophage(top),therepressordimersboundatOR1andOR2preventRNApolymerasefrombindingtotherightwardpromoterandsoblockthesynthesisofCro(nega-tivecontrol).Therepressorsalsoenhancethebindingofpolymerasetotheleftwardpromoter(positivecontrol),withtheresultthattherepressorgeneistranscribedintoRNA(wavyline)andmorerepressorissynthesized,main-tainingthelysogenicstate.TheprophageisinducedwhenultravioletradiationactivatestheproteaserecA,whichcleavesrepressormonomers.Theequilibriumoffreemonomers,freedimers,andbounddimersistherebyshifted,anddimersleavetheoperatorsites.RNApolymeraseisnolongerencouragedtobindtotheleftwardpromoter,sothatrepressorisnolongersynthesized.Asinductionproceeds,alltheoperatorsitesbecomevacant,andsopolym-erasecanbindtotherightwardpromoterandCroissynthesized.Duringearlylyticgrowth,asingleCrodimerbindstoOR3shadedcircles,thesiteforwhichithasthehighestaffinity.Consequently,RNApolymerasecannotbindtotheleftwardpromoter,buttherightwardpromoterremainsaccessible.Polymerasecontinuestobindthere,transcribingcroandotherearlylyticgenes.Lyticgrowthensues.(Reproduced,withpermission,fromPtashneM,JohnsonAD,PaboCO:Ageneticswitchinabacterialvirus.SciAm[Nov]1982;247:128.)382
381REGULATIONOFGENEEXPRESSION/383Todate,thissystemprovidesthebestunderstandingoftorswithspecificDNAregions.Thedynamicsofthefor-themoleculareventsinvolvedingeneregulation.mationanddisruptionofnucleosomestructurearethere-Detailedanalysisofthelambdarepressorledtotheforeanimportantpartofeukaryoticgeneregulation.importantconceptthattranscriptionregulatoryproteinsHistoneacetylationanddeacetylationisanim-haveseveralfunctionaldomains.Forexample,lambdaportantdeterminantofgeneactivity.ThesurprisingrepressorbindstoDNAwithhighaffinity.Repressordiscoverythathistoneacetylaseactivityisassociatedmonomersformdimers,dimersinteractwitheachwithTAFsandthecoactivatorsinvolvedinhormonalother,andrepressorinteractswithRNApolymerase.regulationofgenetranscription(seeChapter43)hasTheprotein-DNAinterfaceandthethreeprotein-providedanewconceptofgeneregulation.Acetylationproteininterfacesallinvolveseparateanddistinctdo-isknowntooccuronlysineresiduesintheaminoter-mainsoftherepressormolecule.Aswillbenotedbelowminaltailsofhistonemolecules.Thismodificationre-(seeFigure39–17),thisisacharacteristicsharedbyducesthepositivechargeofthesetailsanddecreasesthemost(perhapsall)moleculesthatregulatetranscription.bindingaffinityofhistoneforthenegativelychargedDNA.Accordingly,theacetylationofhistonecouldre-sultindisruptionofnucleosomalstructureandallowSPECIALFEATURESAREINVOLVEDreadieraccessoftranscriptionfactorstocognateregula-INREGULATIONOFEUKARYOTICtoryDNAelements.Asdiscussedpreviously,thisGENETRANSCRIPTIONwouldenhancebindingofthebasaltranscriptionma-chinerytothepromoter.HistonedeacetylationwouldMostoftheDNAinprokaryoticcellsisorganizedintohavetheoppositeeffect.Differentproteinswithspecificgenes,andthetemplatescanalwaysbetranscribed.Aacetylaseanddeacetylaseactivitiesareassociatedwithverydifferentsituationexistsinmammaliancells,invariouscomponentsofthetranscriptionapparatus.ThewhichrelativelylittleofthetotalDNAisorganizedspecificityoftheseprocessesisunderinvestigation,asintogenesandtheirassociatedregulatoryregions.Theareavarietyofmechanismsofaction.Somespecificex-functionoftheextraDNAisunknown.Inaddition,asamplesareillustratedinChapter43.describedinChapter36,theDNAineukaryoticcellsisThereisevidencethatthemethylationofdeoxycy-extensivelyfoldedandpackedintotheprotein-DNAtidineresidues(inthesequence5′-mCpG-3′)inDNAcomplexcalledchromatin.Histonesareanimportantmayeffectgrosschangesinchromatinsoastoprecludepartofthiscomplexsincetheybothformthestructuresitsactivetranscription,asdescribedinChapter36.Forknownasnucleosomes(seeChapter36)andalsofactorexample,inmouseliver,onlytheunmethylatedriboso-significantlyintogeneregulatorymechanismsasout-malgenescanbeexpressed,andthereisevidencethatlinedbelow.manyanimalvirusesarenottranscribedwhentheirDNAismethylated.Acutedemethylationofdeoxycyti-ChromatinRemodelingIsanImportantdineresiduesinaspecificregionofthetyrosineamino-transferasegene—inresponsetoglucocorticoidhor-AspectofEukaryoticGeneExpressionmones—hasbeenassociatedwithanincreasedrateofChromatinstructureprovidesanadditionalleveloftranscriptionofthegene.However,itisnotpossibletocontrolofgenetranscription.AsdiscussedinChaptergeneralizethatmethylatedDNAistranscriptionallyin-36,largeregionsofchromatinaretranscriptionallyinac-active,thatallinactivechromatinismethylated,orthattivewhileothersareeitheractiveorpotentiallyactive.activeDNAisnotmethylated.Withfewexceptions,eachcellcontainsthesamecom-Finally,thebindingofspecifictranscriptionfactorsplementofgenes(antibody-producingcellsareanotabletocognateDNAelementsmayresultindisruptionofexception).Thedevelopmentofspecializedorgans,tis-nucleosomalstructure.Manyeukaryoticgeneshavesues,andcellsandtheirfunctionintheintactorganismmultipleprotein-bindingDNAelements.Theserialdependuponthedifferentialexpressionofgenes.bindingoftranscriptionfactorstotheseelements—inaSomeofthisdifferentialexpressionisachievedbycombinatorialfashion—mayeitherdirectlydisruptthehavingdifferentregionsofchromatinavailablefortran-structureofthenucleosomeorpreventitsre-formationscriptionincellsfromvarioustissues.Forexample,theorrecruit,viaprotein-proteininteractions,multipro-DNAcontainingtheβ-globingeneclusterisin“active”teincoactivatorcomplexesthathavetheabilitytocova-chromatininthereticulocytebutin“inactive”chro-lentlymodifyorremodelnucleosomes.Thesereactionsmatininmusclecells.Allthefactorsinvolvedinthede-resultinchromatin-levelstructuralchangesthatintheterminationofactivechromatinhavenotbeeneluci-endincreaseDNAaccessibilitytootherfactorsandthedated.Thepresenceofnucleosomesandofcomplexesoftranscriptionmachinery.histonesandDNA(seeChapter36)certainlyprovidesaEukaryoticDNAthatisinan“active”regionofbarrieragainstthereadyassociationoftranscriptionfac-chromatincanbetranscribed.Asinprokaryoticcells,a
382384/CHAPTER39promoterdictateswheretheRNApolymerasewillini-(EnhancerPromoterStructuralgenetiatetranscription,butthispromotercannotbeneatlyresponseelement)definedascontaininga−35and−10box,particularlyinmammaliancells(Chapter37).Inaddition,theASV40βglobinβglobintrans-actingfactorsgenerallycomefromotherchromo-somes(andsoactintrans),whereasthisconsiderationismootinthecaseofthesinglechromosome-contain-ingprokaryoticcells.AdditionalcomplexityisaddedbyBSV40βglobinβglobinelementsorfactorsthatenhanceorrepresstranscrip-tion,definetissue-specificexpression,andmodulatetheactionsofmanyeffectormolecules.CmttkhGHCertainDNAElementsEnhanceorRepressTranscriptionofEukaryoticGenesInadditiontogrosschangesinchromatinaffectingDGREPEPCKCATtranscriptionalactivity,certainDNAelementsfacilitateorenhanceinitiationatthepromoter.Forexample,inFigure39–8.Aschematicexplanationoftheactionsimianvirus40(SV40)thereexistsabout200bpup-ofenhancersandothercis-actingregulatoryelements.streamfromthepromoteroftheearlygenesaregionofThesemodelchimericgenesconsistofareportertwoidentical,tandem72-bplengthsthatcangreatlyin-(structural)genethatencodesaproteinwhichcanbecreasetheexpressionofgenesinvivo.Eachofthesereadilyassayed,apromoterthatensuresaccurateinitia-72-bpelementscanbesubdividedintoaseriesoftionoftranscription,andtheputativeregulatoryele-smallerelements;therefore,someenhancershaveaveryments.Inallcases,high-leveltranscriptionfromthein-complexstructure.Enhancerelementsdifferfromthedicatedchimerasdependsuponthepresenceofpromoterintworemarkableways.Theycanexerttheirenhancers,whichstimulatetranscription≥100-foldpositiveinfluenceontranscriptionevenwhenseparatedoverbasaltranscriptionallevels(ie,transcriptionofthebythousandsofbasepairsfromapromoter;theyworksamechimericgenescontainingjustpromotersfusedwhenorientedineitherdirection;andtheycanworktothestructuralgenes).ExamplesAandBillustratetheupstream(5′)ordownstream(3′)fromthepromoter.factthatenhancers(eg,SV40)workineitherorientationEnhancersarepromiscuous;theycanstimulateanyanduponaheterologouspromoter.ExampleCillus-promoterinthevicinityandmayactonmorethanonetratesthatthemetallothionein(mt)regulatoryelementpromoter.TheSV40enhancerelementcanexertanin-(whichundertheinfluenceofcadmiumorzincinducesfluenceon,forexample,thetranscriptionofβ-globintranscriptionoftheendogenousmtgeneandhencebyincreasingitstranscription200-foldincellscontain-themetal-bindingmtprotein)willworkthroughtheingboththeenhancerandtheβ-globingeneonthethymidinekinase(tk)promotertoenhancetranscrip-sameplasmid(seebelowandFigure39–8).Theen-tionofthehumangrowthhormone(hGH)gene.Thehancerelementdoesnotproduceaproductthatinturnactsonthepromoter,sinceitisactiveonlywhenitex-engineeredgeneticconstructionswereintroducedintoistswithinthesameDNAmoleculeas(ie,cisto)thethemalepronucleiofsingle-cellmouseembryosandpromoter.Enhancerbindingproteinsareresponsibletheembryosplacedintotheuterusofasurrogateforthiseffect.Theexactmechanismsbywhichthesemothertodevelopastransgenicanimals.Offspringtranscriptionactivatorsworkaresubjecttomuchde-havebeengeneratedundertheseconditions,andinbate.Certainly,enhancerbindingtransfactorshavesometheadditionofzincionstotheirdrinkingwaterbeenshowntointeractwithaplethoraofothertran-effectsanincreaseinlivergrowthhormone.Inthisscriptionproteins.Theseinteractionsincludechro-case,thesetransgenicanimalshaverespondedtothematin-modifyingcoactivatorsaswellastheindividualhighlevelsofgrowthhormonebybecomingtwiceascomponentsofthebasalRNApolymeraseIItranscrip-largeastheirnormallittermates.ExampleDillustratestionmachinery.Ultimately,trans-factor-enhancerDNAthataglucocorticoidresponseelement(GRE)willworkbindingeventsresultinanincreaseinthebindingofthroughhomologous(PEPCKgene)orheterologousthebasaltranscriptionmachinerytothepromoter.En-promoters(notshown;ie,tkpromoter,SV40promoter,hancerelementsandassociatedbindingproteinsoftenβ-globinpromoter,etc).conveynucleasehypersensitivitytothoseregionswheretheyreside(Chapter36).AsummaryofthepropertiesofenhancersispresentedinTable39–2.Oneofthe
383REGULATIONOFGENEEXPRESSION/385Table39–2.Summaryofthepropertiesducesβ-interferongenetranscription—rather,itistheofenhancers.formationoftheenhanceosomeproperthatprovidesappropriatesurfacesfortherecruitmentofcoactivatorsthatresultsintheenhancedformationofthePICon•Workwhenlocatedlongdistancesfromthepromoterthecis-linkedpromoterandthustranscriptionactiva-•Workwhenupstreamordownstreamfromthepromotertion.•Workwhenorientedineitherdirection•WorkthroughheterologouspromotersThecis-actingelementsthatdecreaseorrepressthe•Workbybindingoneormoreproteinsexpressionofspecificgeneshavealsobeenidentified.•Workbyfacilitatingbindingofthebasaltranscriptioncom-Becausefeweroftheseelementshavebeenstudied,itisplextothepromoternotpossibletoformulategeneralizationsabouttheirmechanismofaction—thoughagain,asforgeneactiva-tion,chromatinlevelcovalentmodificationsofhistonesandotherproteinsby(repressor)-recruitedmultisub-best-understoodmammalianenhancersystemsisthatunitcorepressorshavebeenimplicated.oftheβ-interferongene.Thisgeneisinduceduponviralinfectionofmammaliancells.Onegoalofthecell,Tissue-SpecificExpressionMayoncevirallyinfected,istoattempttomountanantivi-ResultFromtheActionralresponse—ifnottosavetheinfectedcell,thentohelptosavetheentireorganismfromviralinfection.ofEnhancersorRepressorsInterferonproductionisonemechanismbywhichthisManygenesarenowrecognizedtoharborenhancerorisaccomplished.Thisfamilyofproteinsissecretedbyactivatorelementsinvariouslocationsrelativetotheirvirallyinfectedcells.Theyinteractwithneighboringcodingregions.Inadditiontobeingabletoenhancecellstocauseaninhibitionofviralreplicationbyavari-genetranscription,someoftheseenhancerelementsetyofmechanisms,therebylimitingtheextentofviralclearlypossesstheabilitytodosoinatissue-specificinfection.Theenhancerelementcontrollinginductionmanner.Thus,theenhancerelementassociatedwithofthisgene,locatedbetweennucleotides−110and−45theimmunoglobulingenesbetweentheJandCregionsoftheβ-interferongene,iswellcharacterized.Thisen-enhancestheexpressionofthosegenespreferentiallyinhanceriscomposedoffourdistinctclusteredcisele-lymphoidcells.SimilarlytotheSV40enhancer,whichments,eachofwhichisboundbydistincttransfactors.iscapableofpromiscuouslyactivatingavarietyofcis-Oneciselementisboundbythetrans-actingfactorlinkedgenes,enhancerelementsassociatedwiththeNF-κB,onebyamemberoftheIRF(interferonregula-genesforpancreaticenzymesarecapableofenhancingtoryfactor)familyoftransfactors,andathirdbytheevenunrelatedbutphysicallylinkedgenespreferentiallyheterodimericleucinezipperfactorATF-2/c-Jun.Theinthepancreaticcellsofmiceintowhichthespecifi-fourthfactoristheubiquitous,architecturaltranscrip-callyengineeredgeneconstructionswereintroducedtionfactorknownasHMGI(Y).Uponbindingtoitsmicrosurgicallyatthesingle-cellembryostage.Thisdegenerate,A+T-richbindingsites,HMGI(Y)inducestransgenicanimalapproachhasprovedusefulinasignificantbendintheDNA.Therearefoursuchstudyingtissue-specificgeneexpression.Forexample,HMGI(Y)bindingsitesinterspersedthroughouttheDNAcontainingapancreaticBcelltissue-specificen-enhancer.Thesesitesplayacriticalroleinformingthehancer(fromtheinsulingene),whenligatedinavectorenhanceosome,alongwiththeaforementionedthreetopolyomalarge-Tantigen,anoncogene,producedtransfactors,byinducingaseriesofcriticallyspacedBcelltumorsintransgenicmice.Tumorsdidnotde-DNAbends.Consequently,HMGI(Y)inducestheco-velopinanyothertissue.Tissue-specificgeneexpres-operativeformationofaunique,stereospecific,threesionmaythereforebemediatedbyenhancersoren-dimensionalstructurewithinwhichallfourfactorsarehancer-likeelements.activewhenviralinfectionsignalsaresensedbythecell.ThestructureformedbythecooperativeassemblyofReporterGenesAreUsedtoDefinethesefourfactorsistermedtheβ-interferonenhanceo-Enhancers&OtherRegulatoryElementssome(seeFigure39–9),sonamedbecauseofitsobvi-ousstructuralsimilaritytothenucleosome,alsoaByligatingregionsofDNAsuspectedofharboringreg-uniquethree-dimensionalproteinDNAstructurethatulatorysequencestovariousreportergenes(there-wrapsDNAaboutanassemblyofproteins(seeFiguresporterorchimericgeneapproach)(Figures39–1036–1and36–2).Theenhanceosome,onceformed,in-and39–11),onecandeterminewhichregionsintheducesalargeincreaseinβ-interferongenetranscriptionvicinityofstructuralgeneshaveaninfluenceontheiruponvirusinfection.Itisnotsimplytheproteinoccu-expression.PiecesofDNAthoughttoharborregula-pancyofthelinearlyapposedciselementsitesthatin-toryelementsareligatedtoasuitablereportergeneand
384386/CHAPTER39HMGPRDIVHMGPRDI-IIIPRDIIHMGNRDIHMGHMGI-YATF-2cJunNF-κBIRF(IRF3/7)HMGIcJunATF-2HMGINF-κBHMGIIRF3IRF7HMGIFigure39–9.Formationandputativestructureoftheenhanceosomeformedonthehumanβ-interferongeneenhancer.Diagramaticallyrepresentedatthetopisthedistributionofthemultiplecis-elements(HMG,PRDIV,PRDI-III,PRDII,NRDI)composingtheβ-interferongeneenhancer.Theintactenhancermediatestranscriptionalin-ductionoftheβ-interferongene(over100-fold)uponvirusinfectionofhumancells.Thecis-elementsofthismodu-larenhancerrepresentthebindingsitesforthetrans-factorsHMGI(Y),cJun-ATF-2,IRF3,IRF7,andNF-κB,respec-tively.ThefactorsinteractwiththeseDNAelementsinanobligatory,ordered,andhighlycooperativefashionasindicatedbythearrow.InitialbindingoffourHMGI(Y)proteinsinducessharpDNAbendsintheenhancer,causingtheentire70–80bpregiontoassumeahighlevelofcurvature.Thiscurvatureisintegraltothesubsequenthighlycooperativebindingoftheothertrans-factorssincethisenablestheDNA-boundfactorstomakeimportant,directprotein-proteininteractionsthatbothcontributetotheformationandstabilityoftheenhanceosomeandgenerateauniquethree-dimensionalsurfacethatservestorecruitchromatin-modifyingactivities(eg,Swi/SnfandP/CAF)aswellasthegeneraltranscriptionmachinery(RNApolymeraseIIandGTFs).Althoughfourofthefivecis-elements(PRDIV,PRDI-III,PRDII,NRDI)independentlycanmodestlystimulate(~tenfold)transcriptionofareportergeneintransfectedcells(seeFigures39–10and39–12),allfivecis-elements,inappropriateorder,arerequiredtoformanenhancerthatcanappropriatelystimulatemRNAgenetranscription(ie,≥100-fold)inresponsetoviralinfectionofahumancell.Thisdistinctionindicatesthestrictrequirementforappropriateenhanceosomearchitectureforefficienttrans-activation.Similarenhanceosomes,involvingdistinctcis-andtrans-factors,areproposedtoformonmanyothermammaliangenes.introducedintoahostcell(Figure39–10).Basalex-Thisstrategy,usingtransfectedcellsinculturepressionofthereportergenewillbeincreasediftheandtransgenicanimals,hasledtotheidentificationDNAcontainsanenhancer.Additionofahormoneorofdozensofenhancers,repressors,tissue-specificele-heavymetaltotheculturemediumwillincreaseexpres-ments,andhormone,heavymetal,anddrug-responsesionofthereportergeneiftheDNAcontainsahor-elements.Theactivityofageneatanymomentre-moneormetalresponseelement(Figure39–11).Theflectstheinteractionofthesenumerouscis-actinglocationoftheelementcanbepinpointedbyusingpro-DNAelementswiththeirrespectivetrans-actingfac-gressivelyshorterpiecesofDNA,deletions,orpointtors.Thechallengenowistofigureouthowthisoc-mutations(Figure39–11).curs.
385REGULATIONOFGENEEXPRESSION/387TestpromoterReportergenener,buttheprocessinmostgenes,especiallyinmam-5′3′5′3′mals,ismuchmorecomplicated.SignalsrepresentingaGENECATnumberofcomplexenvironmentalstimulimaycon-ENHANCER-PROMOTERREPORTERGENE:vergeonasinglegene.TheresponseofthegenetoTESTENHANCER-PROMOTERDRIVINGTRANSCRIPTIONthesesignalscanhaveseveralphysiologiccharacteristics.CATGENEFirst,theresponsemayextendoveraconsiderablerange.Thisisaccomplishedbyhavingadditiveandsyn-CATergisticpositiveresponsescounterbalancedbynegativeorrepressingeffects.Insomecases,eitherthepositiveorthenegativeresponsecanbedominant.Alsore-quiredisamechanismwherebyaneffectorsuchasaTRANSFECTCELLSUSINGCaPO4PRECIPITATEDDNAhormonecanactivatesomegenesinacellwhilerepress-ingothersandleavingstillothersunaffected.Whenalloftheseprocessesarecoupledwithtissue-specificele-Divideandre-platementfactors,considerableflexibilityisafforded.Thesephysiologicvariablesobviouslyrequireanarrangementmuchmorecomplicatedthananon-offswitch.TheCellsarrayofDNAelementsinapromoterspecifies—withassociatedfactors—howagivengenewillrespond.ControlHormonesSomesimpleexamplesareillustratedinFigure39–12.HARVEST24HOURSLATERASSAYFORCATACTIVITYTranscriptionDomainsCanBeDefinedbyIdentificationofcontrolelementsLocusControlRegions&InsulatorsFigure39–10.TheuseofreportergenestodefineThelargenumberofgenesineukaryoticcellsandtheDNAregulatoryelements.ADNAfragmentfromthecomplexarraysoftranscriptionregulatoryfactorspre-geneinquestion—inthisexample,approximately2kbsentsanorganizationalproblem.Whyaresomegenesof5′-flankingDNAandcognatepromoter—isligatedavailablefortranscriptioninagivencellwhereasothersintoaplasmidvectorthatcontainsasuitablereporterarenot?Ifenhancerscanregulateseveralgenesandaregene—inthiscase,thebacterialenzymechlorampheni-notposition-andorientation-dependent,howaretheypreventedfromtriggeringtranscriptionrandomly?Partcoltransferase(CAT).Theenzymeluciferase(abbrevi-ofthesolutiontotheseproblemsisarrivedatbyhavingatedLUC)isanotherpopularreportergene.NeitherthechromatinarrangedinfunctionalunitsthatrestrictLUCnorCATispresentinmammaliancells;hence,de-patternsofgeneexpression.Thismaybeachievedbytectionoftheseactivitiesinacellextractmeansthathavingthechromatinformastructurewiththenuclearthecellwassuccessfullytransfectedbytheplasmid.Anmatrixorotherphysicalentity,orcompartmentsincreaseofCATactivityoverthebasallevel,eg,afterwithinthenucleus.Alternatively,someregionsarecon-additionofoneormorehormones,meansthatthere-trolledbycomplexDNAelementscalledlocuscontrolgionofDNAinsertedintothereportergeneplasmidregions(LCRs).AnLCR—withassociatedboundpro-containsfunctionalhormoneresponseelements(HRE).teins—controlstheexpressionofaclusterofgenes.TheProgressivelyshorterpiecesofDNA,regionswithinter-best-definedLCRregulatesexpressionoftheglobinnaldeletions,orregionswithpointmutationscanbegenefamilyoveralargeregionofDNA.Anothermech-constructedandinsertedtopinpointtheresponseele-anismisprovidedbyinsulators.TheseDNAelements,ment(seeFigure39–11fordeletionmappingoftherel-alsoinassociationwithoneormoreproteins,preventevantHREs).anenhancerfromactingonapromoterontheothersideofaninsulatorinanothertranscriptiondomain.CombinationsofDNAElementsSEVERALMOTIFSMEDIATETHEBINDING&AssociatedProteinsProvideOFREGULATORYPROTEINSTODNADiversityinResponsesThespecificityinvolvedinthecontroloftranscriptionProkaryoticgenesareoftenregulatedinanon-offman-requiresthatregulatoryproteinsbindwithhighaffinitynerinresponsetosimpleenvironmentalcues.Someeu-tothecorrectregionofDNA.Threeuniquemotifs—karyoticgenesareregulatedinthesimpleon-offman-thehelix-turn-helix,thezincfinger,andtheleucine
386388/CHAPTER39REPORTERGENECONSTRUCTSHORMONE-DEPENDENTWITHVARIABLEAMOUNTSTRANSCRIPTIONOF5'-FLANKINGDNAINDUCTIONABC+++++++++Figure39–11.Locationofhormoneresponseele-CATments(HREs)A,B,andCusingthereporter5′gene–transfectionapproach.Afamilyofreporter20001000+1genes,constructedasdescribedinFigure39–10,canNucleotidepositionbetransfectedindividuallyintoarecipientcell.Byan-alyzingwhencertainhormoneresponsesarelostinHREHREHREcomparisontothe5′deletion,specifichormone-ABCresponsiveelementscanbelocated.zipper—accountformanyofthesespecificprotein-DNAinteractions.Examplesofproteinscontaining312thesemotifsaregiveninTable39–3.ComparisonofthebindingactivitiesoftheproteinsGeneAthatcontainthesemotifsleadstoseveralimportant4generalizations.31(1)BindingmustbeofhighaffinitytothespecificGeneBsiteandoflowaffinitytootherDNA.(2)Smallregionsoftheproteinmakedirectcontact2withDNA;therestoftheprotein,inadditiontopro-315GeneCTable39–3.ExamplesoftranscriptionregulatoryFigure39–12.CombinationsofDNAelementsandproteinsthatcontainthevariousbindingmotifs.proteinsprovidediversityintheresponseofagene.GeneAisactivated(thewidthofthearrowindicatesBindingMotifOrganismRegulatoryProteintheextent)bythecombinationofactivators1,2,and3Helix-turn-helixEcolilacrepressor(probablywithcoactivators,asshowninFigure37–10).CAPGeneBisactivated,inthiscasemoreeffectively,bythePhageλcI,cro,andtryptophanandcombinationof1,3,and4;notethat4doesnotcontact434repressorsDNAdirectlyinthisexample.TheactivatorscouldformMammalshomeoboxproteinsalinearbridgethatlinksthebasalmachinerytothePit-1,Oct1,Oct2promoter,orthiscouldbeaccomplishedbyloopingZincfingerEcoliGene32proteinoutoftheDNA.Ineithercase,thepurposeistodirectYeastGaI4thebasaltranscriptionmachinerytothepromoter.DrosophilaSerendipity,HunchbackGeneCisinactivatedbythecombinationof1,5,and3;XenopusTFIIIAinthiscase,factor5isshowntoprecludetheessentialMammalssteroidreceptorfamily,Sp1bindingoffactor2toDNA,asoccursinexampleA.IfLeucinezipperYeastGCN4activator1helpsrepressor5bindandifactivator1MammalsC/EBP,fos,Jun,Fra-1,bindingrequiresaligand(soliddot),itcanbeseenhowCREbindingprotein,theligandcouldactivateonegeneinacell(geneA)c-myc,n-myc,I-mycandrepressanother(geneC).
387REGULATIONOFGENEEXPRESSION/389vidingthetrans-activationdomains,maybeinvolvedinTheHelix-Turn-HelixMotifthedimerizationofmonomersofthebindingprotein,mayprovideacontactsurfacefortheformationofhet-Thefirstmotifdescribed—andtheonestudiedmosterodimers,mayprovideoneormoreligand-bindingextensively—isthehelix-turn-helix.Analysisofthesites,ormayprovidesurfacesforinteractionwithcoac-three-dimensionalstructureoftheλCrotranscriptiontivatorsorcorepressors.regulatorhasrevealedthateachmonomerconsistsof(3)Theprotein-DNAinteractionsaremaintainedthreeantiparallelβsheetsandthreeαhelices(FigurebyhydrogenbondsandvanderWaalsforces.39–13).Thedimerformsbyassociationoftheantipar-(4)Themotifsfoundintheseproteinsareunique;allelβ3sheets.Theα3helicesformtheDNArecogni-theirpresenceinaproteinofunknownfunctionsug-tionsurface,andtherestofthemoleculeappearstobegeststhattheproteinmaybindtoDNA.involvedinstabilizingthesestructures.Theaveragedi-(5)Proteinswiththehelix-turn-helixorleucinezip-ameterofanαhelixis1.2nm,whichistheapproxi-permotifsformsymmetricdimers,andtheirrespectivematewidthofthemajorgrooveintheBformofDNA.DNAbindingsitesaresymmetricpalindromes.Inpro-TheDNArecognitiondomainofeachCromonomerteinswiththezincfingermotif,thebindingsiteisre-interactswith5bpandthedimerbindingsitesspanpeatedtwotoninetimes.Thesefeaturesallowforco-3.4nm,allowingfitintosuccessivehalfturnsoftheoperativeinteractionsbetweenbindingsitesandmajorgrooveonthesamesurface(Figure39–13).X-rayenhancethedegreeandaffinityofbinding.analysesoftheλcIrepressor,CAP(thecAMPreceptorα2α3α1NCαβ2134Åβ2β3α3Twofoldaxisofβ3symmetryβ2βα13α2CNαα3134Åα2Figure39–13.Aschematicrepresentationofthethree-dimensionalstructureofCroproteinanditsbindingtoDNAbyitshelix-turn-helixmotif.TheCromonomerconsistsofthreeantiparallelβsheets(β1–β3)andthreeα-helices(α1–α3).Thehelix-turn-helixmotifisformedbecausetheα3andα2helicesareheldatabout90degreestoeachotherbyaturnoffouraminoacids.Theα3helixofCroistheDNArecognitionsurface(shaded).Twomonomersassociatethroughtheantiparallelβ3sheetstoformadimerthathasatwofoldaxisofsymmetry(right).ACrodimerbindstoDNAthroughitsα3helices,eachofwhichcontactsabout5bponthesamesurfaceofthemajorgroove.ThedistancebetweencomparablepointsonthetwoDNAα-helicesis34Å,whichisthedis-tancerequiredforonecompleteturnofthedoublehelix.(CourtesyofBMathews.)
388390/CHAPTER39proteinofEcoli),tryptophanrepressor,andphage434TheLeucineZipperMotifrepressorallalsodisplaythisdimerichelix-turn-helixCarefulanalysisofa30-amino-acidsequenceinthecar-structurethatispresentineukaryoticDNAproteinsasboxylterminalregionoftheenhancerbindingproteinwell(seeTable39–3).C/EBPrevealedanovelstructure.AsillustratedinFig-ure39–15,thisregionoftheproteinformsanαhelixTheZincFingerMotifinwhichthereisaperiodicrepeatofleucineresiduesateveryseventhposition.ThisoccursforeighthelicalThezincfingerwasthesecondDNAbindingmotifturnsandfourleucinerepeats.Similarstructureshavewhoseatomicstructurewaselucidated.ItwasknownbeenfoundinanumberofotherproteinsassociatedthattheproteinTFIIIA,apositiveregulatorof5SRNAwiththeregulationoftranscriptioninmammalianandtranscription,requiredzincforactivity.Structuralandyeastcells.ItisthoughtthatthisstructureallowstwobiophysicalanalysesrevealedthateachTFIIIAmoleculeidenticalmonomersorheterodimers(eg,Fos-JunorcontainsninezincionsinarepeatingcoordinationJun-Jun)to“ziptogether”inacoiledcoilandformacomplexformedbycloselyspacedcysteine-cysteinetightdimericcomplex(Figure39–15).Thisprotein-residuesfollowed12–13aminoacidslaterbyahisti-proteininteractionmayservetoenhancetheassocia-dine-histidinepair(Figure39–14).Insomeinstances—tionoftheseparateDNAbindingdomainswiththeirnotablythesteroid-thyroidreceptorfamily—theHis-target(Figure39–15).HisdoubletisreplacedbyasecondCys-Cyspair.TheproteincontainingzincfingersappearstolieononeTHEDNABINDING&TRANS-ACTIVATIONfaceoftheDNAhelix,withsuccessivefingersalterna-tivelypositionedinoneturninthemajorgroove.AsisDOMAINSOFMOSTREGULATORYthecasewiththerecognitiondomaininthehelix-turn-PROTEINSARESEPARATEhelixprotein,eachTFIIIAzincfingercontactsabout&NONINTERACTIVE5bpofDNA.Theimportanceofthismotifintheac-tionofsteroidhormonesisunderscoredbyan“experi-DNAbindingcouldresultinageneralconformationalmentofnature.”Asingleaminoacidmutationineitherchangethatallowstheboundproteintoactivatetran-ofthetwozincfingersofthe1,25(OH)2-D3receptorscription,orthesetwofunctionscouldbeservedbyproteinresultsinresistancetotheactionofthishor-separateandindependentdomains.Domainswapex-moneandtheclinicalsyndromeofrickets.perimentssuggestthatthelatteristhecase.TheGAL1geneproductisinvolvedingalactoseme-tabolisminyeast.TranscriptionofthisgeneispositivelyregulatedbytheGAL4protein,whichbindstoanup-streamactivatorsequence(UAS),orenhancer,throughanaminoterminaldomain.Theaminoterminal73-amino-acidDNA-bindingdomain(DBD)ofGAL4wasremovedandreplacedwiththeDBDofLexA,anEcoliDNA-bindingprotein.ThisdomainswapresultedinamoleculethatdidnotbindtotheGAL1UASand,ofcourse,didnotactivatetheGAL1gene(Figure39–16).CCCHIf,however,thelexAoperator—theDNAsequencenor-ZnZnmallyboundbythelexADBD—wasinsertedintotheCCCHpromoterregionoftheGALgene,thehybridproteinboundtothispromoter(atthelexAoperator)anditac-Cys-CyszincfingerCys-HiszincfingertivatedtranscriptionofGAL1.Thisexperiment,whichhasbeenrepeatedanumberoftimes,affordssolidevi-Figure39–14.ZincfingersareaseriesofrepeateddencethatthecarboxylterminalregionofGAL4causesdomains(twotonine)inwhicheachiscenteredonatranscriptionalactivation.Thesedataalsodemonstratetetrahedralcoordinationwithzinc.InthecaseofTFIIIA,thattheDNA-bindingDBDandtrans-activationdo-thecoordinationisprovidedbyapairofcysteinemains(ADs)areindependentandnoninteractive.Theresidues(C)separatedby12–13aminoacidsfromahierarchyinvolvedinassemblinggenetranscriptionacti-pairofhistidine(H)residues.Inotherzincfingerpro-vatingcomplexesincludesproteinsthatbindDNAandteins,thesecondpairalsoconsistsofCresidues.Zinctrans-activate;othersthatformprotein-proteincom-fingersbindinthemajorgroove,withadjacentfingersplexeswhichbridgeDNA-bindingproteinstotrans-makingcontactwith5bpalongthesamefaceoftheactivatingproteins;andothersthatformprotein-proteinhelix.complexeswithcomponentsofthebasaltranscription
389REGULATIONOFGENEEXPRESSION/391ABLL22L15NH2L81FLIEVRDN45COOHCOOH2TR7RQSTRQNH2S36DKERDGRFigure39–15.Theleucinezippermotif.AshowsahelicalwheelanalysisofacarboxylterminalportionoftheDNAbindingproteinC/EBP.Theaminoacidsequenceisdisplayedend-to-enddowntheaxisofaschematicα-helix.Thehelicalwheelconsistsofsevenspokesthatcorrespondtothesevenaminoacidsthatcompriseeverytwoturnsoftheα-helix.Notethatleucineresidues(L)occurateveryseventhposition.Otherproteinswith“leucinezippers”haveasimilarhelicalwheelpattern.BisaschematicmodeloftheDNAbindingdomainofC/EBP.TwoidenticalC/EBPpolypeptidechainsareheldindimerformationbytheleucinezipperdomainofeachpolypeptide(denotedbytherectanglesandattachedovals).ThisassociationisapparentlyrequiredtoholdtheDNAbindingdomainsofeachpolypeptide(theshadedrectangles)intheproperconformationforDNAbinding.(CourtesyofSMcKnight.)apparatus.Agivenproteinmaythushaveseveralsur-prokaryotes.TheseRNAprocessingstepsineukaryotes,facesordomainsthatservedifferentfunctions(seeFig-describedindetailinChapter37,includecappingofure39–17).AsdescribedinChapter37,theprimarythe5′endsoftheprimarytranscripts,additionofapurposeofthesecomplexassembliesistofacilitatethepolyadenylatetailtothe3′endsoftranscripts,andexci-assemblyofthebasaltranscriptionapparatusonthecis-sionofintronregionstogeneratesplicedexonsinthelinkedpromoter.maturemRNAmolecule.Todate,analysesofeukary-oticgeneexpressionprovideevidencethatregulationoccursattheleveloftranscription,nuclearRNApro-GENEREGULATIONINPROKARYOTEScessing,andmRNAstability.Inaddition,geneampli-&EUKARYOTESDIFFERSINficationandrearrangementinfluencegeneexpression.IMPORTANTRESPECTSOwingtotheadventofrecombinantDNAtechnol-ogy,muchprogresshasbeenmadeinrecentyearsinInadditiontotranscription,eukaryoticcellsemployatheunderstandingofeukaryoticgeneexpression.How-varietyofmechanismstoregulategeneexpressionever,becausemosteukaryoticorganismscontainso(Table39–4).Thenuclearmembraneofeukaryoticmuchmoregeneticinformationthandoprokaryotescellsphysicallysegregatesgenetranscriptionfromtrans-andbecausemanipulationoftheirgenesissomuchlation,sinceribosomesexistonlyinthecytoplasm.morelimited,molecularaspectsofeukaryoticgenereg-Manymoresteps,especiallyinRNAprocessing,arein-ulationarelesswellunderstoodthantheexamplesvolvedintheexpressionofeukaryoticgenesthanofdiscussedearlierinthischapter.Thissectionbrieflyde-prokaryoticgenes,andthesestepsprovideadditionalscribesafewdifferenttypesofeukaryoticgeneregula-sitesforregulatoryinfluencesthatcannotexistintion.
390392/CHAPTER39GAL4+1ActiveAUASGAL1geneLexA–GAL4+1InactiveBUASGAL1geneLexA–GAL4+1ActivelexAGAL1geneCoperatorFigure39–16.Domain-swapexperimentsdemonstratetheindependentnatureofDNAbindingandtranscrip-tionactivationdomains.TheGAL1genepromotercontainsanupstreamactivatingsequence(UAS)orenhancerthatbindstheregulatoryproteinGAL4(A).ThisinteractionresultsinastimulationofGAL1genetranscription.Achimericprotein,inwhichtheaminoterminalDNAbindingdomainofGAL4isremovedandreplacedwiththeDNAbindingregionoftheEcoliproteinLexA,failstostimulateGAL1transcriptionbecausetheLexAdomaincannotbindtotheUAS(B).TheLexA–GAL4fusionproteindoesincreaseGAL1transcriptionwhenthelexAoperator(itsnaturaltarget)isinsertedintotheGAL1promoterregion(C).EukaryoticGenesCanBeAmplifiedorRearrangedDuringDevelopmentorinResponsetoDrugsDuringearlydevelopmentofmetazoans,thereisanabruptincreaseintheneedforspecificmoleculessuch2asribosomalRNAandmessengerRNAmoleculesforActivationproteinsthatmakeupsuchorgansastheeggshell.Onedomains1–4waytoincreasetherateatwhichsuchmoleculescanbeLigand-bindingdomain3formedistoincreasethenumberofgenesavailablefor1transcriptionofthesespecificmolecules.AmongtherepetitiveDNAsequencesarehundredsofcopiesofri-4bosomalRNAgenesandtRNAgenes.Thesegenespre-DNA-bindingdomainexistrepetitivelyinthegenomicmaterialofthegametesTable39–4.GeneexpressionisregulatedbytranscriptionandinnumerousotherwaysinFigure39–17.Proteinsthatregulatetranscriptionhaveseveraldomains.Thishypotheticaltranscriptioneukaryoticcells.factorhasaDNA-bindingdomain(DBD)thatisdistinctfromaligand-bindingdomain(LBD)andseveralactiva-•Geneamplificationtiondomains(ADs)(1–4).Otherproteinsmaylackthe•GenerearrangementDBDorLBDandallmayhavevariablenumbersof•RNAprocessing•AlternatemRNAsplicingdomainsthatcontactotherproteins,including•TransportofmRNAfromnucleustocytoplasmco-regulatorsandthoseofthebasaltranscription•RegulationofmRNAstabilitycomplex(seealsoChapters42and43).
391REGULATIONOFGENEEXPRESSION/393andthusaretransmittedinhighcopynumbersfrombothimmunologicflexibilityandspecificity.However,generationtogeneration.InsomespecificorganismsagivenfunctionalIgGlightchaintranscriptionunit—suchasthefruitfly(drosophila),thereoccursduringlikeallother“normal”mammaliantranscriptionoogenesisanamplificationofafewpreexistinggenesunits—containsonlythecodingsequencesforasinglesuchasthoseforthechorion(eggshell)proteins.Subse-protein.Thus,beforeaparticularIgGlightchaincanquently,theseamplifiedgenes,presumablygeneratedbeexpressed,singleVL,JL,andCLcodingsequencesbyaprocessofrepeatedinitiationsduringDNAsyn-mustberecombinedtogenerateasingle,contiguousthesis,providemultiplesitesforgenetranscriptiontranscriptionunitexcludingthemultiplenonutilized(Figures36–16and39–18).segments(ie,theotherapproximately300unusedVLAsnotedinChapter37,thecodingsequencesre-segments,theotherfourunusedJLsegments,andthesponsibleforthegenerationofspecificproteinmole-othernineunusedCLsegments).Thisdeletionofun-culesarefrequentlynotcontiguousinthemammalianusedgeneticinformationisaccomplishedbyselectivegenome.Inthecaseofantibodyencodinggenes,thisisDNArecombinationthatremovestheunwantedcod-particularlytrue.AsdescribedindetailinChapter50,ingDNAwhileretainingtherequiredcodingse-immunoglobulinsarecomposedoftwopolypeptides,quences:oneVL,oneJL,andoneCLsequence.(VLse-theso-calledheavy(about50kDa)andlight(about25quencesaresubjectedtoadditionalpointmutagenesiskDa)chains.ThemRNAsencodingthesetwoproteintogenerateevenmorevariability—hencethename.)subunitsareencodedbygenesequencesthataresub-ThenewlyrecombinedsequencesthusformasinglejectedtoextensiveDNAsequence-codingchanges.transcriptionunitthatiscompetentforRNApolym-TheseDNAcodingchangesareintegraltogeneratingeraseII-mediatedtranscription.AlthoughtheIgGtherequisiterecognitiondiversitycentraltoappropriategenesrepresentoneofthebest-studiedinstancesofdi-immunefunction.rectedDNArearrangementmodulatinggeneexpres-IgGheavyandlightchainmRNAsareencodedbysion,othercasesofgeneregulatoryDNArearrange-severaldifferentsegmentsthataretandemlyrepeatedinmenthavebeendescribedintheliterature.Indeed,asthegermline.Thus,forexample,theIgGlightchainisdetailedbelow,drug-inducedgeneamplificationisancomposedofvariable(VL),joining(JL),andconstantimportantcomplicationofcancerchemotherapy.(CL)domainsorsegments.ForparticularsubsetsofInrecentyears,ithasbeenpossibletopromotetheIgGlightchains,thereareroughly300tandemlyre-amplificationofspecificgeneticregionsinculturedpeatedVLgenecodingsegments,fivetandemlymammaliancells.Insomecases,aseveralthousand-foldarrangedJLcodingsequences,androughlytenCLgeneincreaseinthecopynumberofspecificgenescanbecodingsegments.Allofthesemultiple,distinctcodingachievedoveraperiodoftimeinvolvingincreasingdosesregionsarelocatedinthesameregionofthesamechro-ofselectivedrugs.Infact,ithasbeendemonstratedinmosome,andeachtypeofcodingsegment(VL,JL,andpatientsreceivingmethotrexateforcancerthatmalig-CL)istandemlyrepeatedinhead-to-tailfashionwithinnantcellscandevelopdrugresistancebyincreasingthethesegmentrepeatregion.ByhavingmultipleVL,JL,numberofgenesfordihydrofolatereductase,thetargetandCLsegmentstochoosefrom,animmunecellhasaofmethotrexate.Geneamplificationeventssuchasthesegreaterrepertoireofsequencestoworkwithtodevelopoccurspontaneouslyinvivo—ie,intheabsenceofex-ogenouslysuppliedselectiveagents—andtheseunsched-uledextraroundsofreplicationcanbecome“frozen”inthegenomeunderappropriateselectivepressures.Unamplifieds36s38AlternativeRNAProcessings36s38IsAnotherControlMechanismInadditiontoaffectingtheefficiencyofpromoteruti-lization,eukaryoticcellsemployalternativeRNApro-Amplifiedcessingtocontrolgeneexpression.Thiscanresultwhenalternativepromoters,intron-exonsplicesites,orpolyadenylationsitesareused.Occasionally,hetero-geneitywithinacellresults,butmorecommonlythesameprimarytranscriptisprocesseddifferentlyindif-Figure39–18.Schematicrepresentationoftheam-ferenttissues.Afewexamplesofeachofthesetypesofplificationofchorionproteingeness36ands38.(Repro-regulationarepresentedbelow.duced,withpermission,fromChisholmR:Geneamplifica-Theuseofalternativetranscriptionstartsitesre-tionduringdevelopment.TrendsBiochemSci1982;7:161.)sultsinadifferent5′exononmRNAscorrespondingto
392394/CHAPTER39mouseamylaseandmyosinlightchain,ratglucokinase,capstructureineukaryoticmRNApreventsattackby5′anddrosophilaalcoholdehydrogenaseandactin.Alter-exonucleases,andthepoly(A)tailprohibitstheactionnativepolyadenylationsitesintheμimmunoglobulinof3′exonucleases.InmRNAmoleculeswiththoseheavychainprimarytranscriptresultinmRNAsthatstructures,itispresumedthatasingleendonucleolyticareeither2700baseslong(μm)or2400baseslong(μs).cutallowsexonucleasestoattackanddigesttheentireThisresultsinadifferentcarboxylterminalregionofmolecule.Otherstructures(sequences)inthe5′non-theencodedproteinssuchthattheμmproteinremainscodingsequence(5′NCS),thecodingregion,andtheattachedtothemembraneoftheBlymphocyteandthe3′NCSarethoughttopromoteorpreventthisinitialμsimmunoglobulinissecreted.Alternativesplicingendonucleolyticaction(Figure39–19).Afewillustra-andprocessingresultsintheformationofseventiveexampleswillbecited.uniqueα-tropomyosinmRNAsinsevendifferenttis-Deletionofthe5′NCSresultsinathreefoldtofive-sues.Itisnotclearhowtheseprocessing-splicingdeci-foldprolongationofthehalf-lifeofc-mycmRNA.Short-sionsaremadeorwhetherthesestepscanberegulated.eningthecodingregionofhistonemRNAresultsinaprolongedhalf-life.AformofautoregulationofmRNARegulationofMessengerRNAStabilitystabilityindirectlyinvolvesthecodingregion.Freetubu-linbindstothefirstfouraminoacidsofanascentchainProvidesAnotherControlMechanismoftubulinasitemergesfromtheribosome.ThisappearsAlthoughmostmRNAsinmammaliancellsareverytoactivateanRNaseassociatedwiththeribosome(RNP)stable(half-livesmeasuredinhours),someturnoverwhichthendigeststhetubulinmRNA.veryrapidly(half-livesof10–30minutes).Incertainin-Structuresatthe3′end,includingthepoly(A)tail,stances,mRNAstabilityissubjecttoregulation.ThisenhanceordiminishthestabilityofspecificmRNAs.hasimportantimplicationssincethereisusuallyadi-Theabsenceofapoly(A)tailisassociatedwithrapidrectrelationshipbetweenmRNAamountandthedegradationofmRNA,andtheremovalofpoly(A)translationofthatmRNAintoitscognateprotein.fromsomeRNAsresultsintheirdestabilization.His-ChangesinthestabilityofaspecificmRNAcanthere-tonemRNAslackapoly(A)tailbuthaveasequenceforehavemajoreffectsonbiologicprocesses.nearthe3′terminalthatcanformastem-loopstruc-MessengerRNAsexistinthecytoplasmasribonu-ture,andthisappearstoprovideresistancetoexonucle-cleoproteinparticles(RNPs).Someoftheseproteinsolyticattack.HistoneH4mRNA,forexample,isde-protectthemRNAfromdigestionbynucleases,whilegradedinthe3′to5′directionbutonlyafterasingleothersmayundercertainconditionspromotenucleaseendonucleolyticcutoccursaboutninenucleotidesfromattack.ItisthoughtthatmRNAsarestabilizedordesta-the3′endintheregionoftheputativestem-loopstruc-bilizedbytheinteractionofproteinswiththesevariousture.Stem-loopstructuresinthe3′noncodingse-structuresorsequences.Certaineffectors,suchashor-quencearealsocriticalfortheregulation,byiron,ofmones,mayregulatemRNAstabilitybyincreasingorthemRNAencodingthetransferrinreceptor.Stem-decreasingtheamountoftheseproteins.loopstructuresarealsoassociatedwithmRNAstabilityItappearsthattheendsofmRNAmoleculesareinbacteria,suggestingthatthismechanismmaybeinvolvedinmRNAstability(Figure39–19).The5′commonlyemployed.Cap5′NCSCoding3′NCSA–A–A–A–AnAUUUAFigure39–19.StructureofatypicaleukaryoticmRNAshowingelementsthatareinvolvedinregulatingmRNAstability.ThetypicaleukaryoticmRNAhasa5′noncodingsequence(5′NCS),acodingregion,anda3′NCS.Allarecappedatthe5′end,andmosthaveapolyadenylatesequenceatthe3′end.The5′capand3′poly(A)tailprotectthemRNAagainstexonucleaseattack.Stem-loopstructuresinthe5′and3′NCS,featuresinthecodingsequence,andtheAU-richregioninthe3′NCSarethoughttoplayrolesinmRNAstability.
393REGULATIONOFGENEEXPRESSION/395Othersequencesinthe3′endsofcertaineukaryoticREFERENCESmRNAsappeartobeinvolvedinthedestabilizationofthesemolecules.OfparticularinterestareAU-richre-AlbrightSR,TjianR:TAFsrevisited:moredatarevealnewtwistsandconfirmoldideas.Gene2000;242:1.gions,manyofwhichcontainthesequenceAUUUA.BirdAP,WolffeAP:Methylation-inducedrepression—belts,bracesThissequenceappearsinmRNAsthathaveaveryshortandchromatin.Cell1999;99:451.half-life,includingsomeencodingoncogeneproteinsBergerSL,FelsenfeldG:Chromatingoesglobal.MolCell2001;andcytokines.Theimportanceofthisregionisunder-8:263.scoredbyanexperimentinwhichasequencecorre-BusbyS,EbrightRH:Promoterstructure,promoterrecognition,spondingtothe3′noncodingregionoftheshort-half-andtranscriptionactivationinprokaryotes.Cell1994;79:lifecolony-stimulatingfactor(CSF)mRNA,which743.containstheAUUUAmotif,wasaddedtothe3′endofBusbyS,EbrightRH:Transcriptionactivationbycataboliteactiva-theβ-globinmRNA.Insteadofbecomingverystable,torprotein(CAP).JMolBiol1999;293:199.thishybridβ-globinmRNAnowhadtheshort-half-lifeCowellIG:Repressionversusactivationinthecontrolofgenetran-characteristicofCSFmRNA.scription.TrendsBiochemSci1994;1:38.Fromthefewexamplescited,itisclearthatanum-EbrightRH:RNApolymerase:structuralsimilaritiesbetweenbac-berofmechanismsareusedtoregulatemRNAstabil-terialRNApolymeraseandeukaryoticRNApolymeraseII.ity—justasseveralmechanismsareusedtoregulatetheJMolBiol2000;304:687.synthesisofmRNA.CoordinateregulationofthesetwoFugmanSD:RAG1andRAG2inV(D)Jrecombinationandtrans-position.ImmunolRes2001;23:23.processesconfersonthecellremarkableadaptability.JacobF,MonodJ:Geneticregulatorymechanismsinproteinsyn-thesis.JMolBiol1961;3:318.SUMMARYLemonB,TjianR:Orchestratedresponse:asymphonyoftran-scriptionfactorsforgenecontrol.GenesDev2000;14:2551.•Thegeneticconstitutionsofnearlyallmetazoanso-maticcellsareidentical.LetchmanDS:Transcriptionfactormutationsanddisease.NEnglJMed1996;334:28.•Phenotype(tissueorcellspecificity)isdictatedbyMerikaM,ThanosD:Enhanceosomes.CurrOpinGenetDevdifferencesingeneexpressionofthiscomplementof2001;11:205.genes.NaarAM,LemonBD,TjianR:Transcriptionalcoactivatorcom-•Alterationsingeneexpressionallowacelltoadapttoplexes.AnnuRevBiochem2001;70:475.environmentalchanges.NarlikarGJ,FanHY,KingstonRE:Cooperationbetweencom-•Geneexpressioncanbecontrolledatmultiplelevelsplexesthatregulatechromatinstructureandtranscription.Cell2002;108:475.bychangesintranscription,RNAprocessing,local-OltzEM:Regulationofantigenreceptorgeneassemblyinlympho-ization,andstabilityorutilization.Geneamplifica-cytes.ImmunolRes2001;23:121.tionandrearrangementsalsoinfluencegeneexpres-PtashneM:Controlofgenetranscription:anoutline.NatMedsion.1997;3:1069.•Transcriptioncontrolsoperateatthelevelofprotein-PtashneM:AGeneticSwitch,2nded.CellPressandBlackwellSci-DNAandprotein-proteininteractions.Theseinter-entificPublications,1992.actionsdisplayproteindomainmodularityandhighSternerDE,BergerSL:Acetylationofhistonesandtranscription-specificity.relatedfactors.MicrobiolMolBiolRev2000;64:435.•SeveraldifferentclassesofDNA-bindingdomainsWuR,BahlCP,NarangSA:Lactoseoperator-repressorinterac-havebeenidentifiedintranscriptionfactors.tion.CurrTopCellRegul1978;13:137.•Chromatinmodificationsareimportantineukary-otictranscriptioncontrol.
394MolecularGenetics,RecombinantDNA,&GenomicTechnology40DarylK.Granner,MD,&P.AnthonyWeil,PhDBIOMEDICALIMPORTANCE*ELUCIDATIONOFTHEBASICFEATURESThedevelopmentofrecombinantDNA,high-density,OFDNALEDTORECOMBINANThigh-throughputscreening,andothermolecularge-DNATECHNOLOGYneticmethodologieshasrevolutionizedbiologyandisDNAIsaComplexBiopolymerhavinganincreasingimpactonclinicalmedicine.OrganizedasaDoubleHelixMuchhasbeenlearnedabouthumangeneticdiseasefrompedigreeanalysisandstudyofaffectedproteins,Thefundamentalorganizationalelementisthese-butinmanycaseswherethespecificgeneticdefectisquenceofpurine(adenine[A]orguanine[G])andunknown,theseapproachescannotbeused.Thenewpyrimidine(cytosine[C]orthymine[T])bases.Thesetechnologiescircumventtheselimitationsbygoingdi-basesareattachedtotheC-1′positionofthesugarde-rectlytotheDNAmoleculeforinformation.Manipu-oxyribose,andthebasesarelinkedtogetherthroughlationofaDNAsequenceandtheconstructionofjoiningofthesugarmoietiesattheir3′and5′positionschimericmolecules—so-calledgeneticengineering—viaaphosphodiesterbond(Figure35–1).Thealternat-providesameansofstudyinghowaspecificsegmentofingdeoxyriboseandphosphategroupsformtheback-DNAworks.Novelmoleculargenetictoolsallowinves-boneofthedoublehelix(Figure35–2).These3′–5′tigatorstoqueryandmanipulategenomicsequencesaslinkagesalsodefinetheorientationofagivenstrandofwellastoexaminebothcellularmRNAandproteintheDNAmolecule,and,sincethetwostrandsruninprofilesatthemolecularlevel.oppositedirections,theyaresaidtobeantiparallel.Understandingthistechnologyisimportantforsev-eralreasons:(1)Itoffersarationalapproachtounder-standingthemolecularbasisofanumberofdiseasesBasePairingIsaFundamentalConcept(eg,familialhypercholesterolemia,sicklecelldisease,ofDNAStructure&Functionthethalassemias,cysticfibrosis,musculardystrophy).(2)HumanproteinscanbeproducedinabundanceforAdenineandthyminealwayspair,byhydrogenbonding,therapy(eg,insulin,growthhormone,tissueplasmino-asdoguanineandcytosine(Figure35–3).Thesebasegenactivator).(3)Proteinsforvaccines(eg,hepatitisB)pairsaresaidtobecomplementary,andtheguanineandfordiagnostictesting(eg,AIDStests)canbeob-contentofafragmentofdouble-strandedDNAwillal-tained.(4)Thistechnologyisusedtodiagnoseexistingwaysequalitscytosinecontent;likewise,thethyminediseasesandpredicttheriskofdevelopingagivendis-andadeninecontentsareequal.Basepairingandhy-ease.(5)Specialtechniqueshaveledtoremarkablead-drophobicbase-stackinginteractionsholdthetwoDNAvancesinforensicmedicine.(6)Genetherapyforsicklestrandstogether.Theseinteractionscanbereducedbycelldisease,thethalassemias,adenosinedeaminasedefi-heatingtheDNAtodenatureit.Thelawsofbasepairingciency,andotherdiseasesmaybedevised.predictthattwocomplementaryDNAstrandswillrean-nealexactlyinregisteruponrenaturation,ashappenswhenthetemperatureofthesolutionisslowlyreducedtonormal.Indeed,thedegreeofbase-pairmatching(or*Seeglossaryoftermsattheendofthischapter.mismatching)canbeestimatedfromthetemperaturere-396
395MOLECULARGENETICS,RECOMBINANTDNA,&GENOMICTECHNOLOGY/397quiredfordenaturation-renaturation.SegmentsofDNAexons.Regulatoryregionsforspecificeukaryoticgeneswithhighdegreesofbase-pairmatchingrequiremoreen-areusuallylocatedintheDNAthatflanksthetran-ergyinput(heat)toaccomplishdenaturation—or,toputscriptioninitiationsiteatits5′end(5′flanking-itanotherway,acloselymatchedsegmentwillwithstandsequenceDNA).Occasionally,suchsequencesaremoreheatbeforethestrandsseparate.Thisreactionisfoundwithinthegeneitselforintheregionthatflanksusedtodeterminewhethertherearesignificantdiffer-the3′endofthegene.Inmammaliancells,eachgeneencesbetweentwoDNAsequences,anditunderliesthehasitsownregulatoryregion.Manyeukaryoticgenesconceptofhybridization,whichisfundamentaltothe(andsomevirusesthatreplicateinmammaliancells)processesdescribedbelow.havespecialregions,calledenhancers,thatincreasethe9Thereareabout310basepairs(bp)ineachrateoftranscription.SomegenesalsohaveDNAse-humanhaploidgenome.Ifanaveragegenelengthisquences,knownassilencers,thatrepresstranscription.33×10bp(3kilobases[kb]),thegenomecouldconsistMammaliangenesareobviouslycomplicated,multi-6of10genes,assumingthatthereisnooverlapandthatcomponentstructures.transcriptionproceedsinonlyonedirection.Itis5GenesAreTranscribedIntoRNAthoughtthatthereare<10genesinthehumanandthatonly1–2%oftheDNAcodesforproteins.TheInformationgenerallyflowsfromDNAtomRNAtoexactfunctionoftheremaining~98%ofthehumanprotein,asillustratedinFigure40–1anddiscussedingenomehasnotyetbeendefined.moredetailinChapter39.ThisisarigidlycontrolledThedouble-helicalDNAispackagedintoamoreprocessinvolvinganumberofcomplexsteps,eachofcompactstructurebyanumberofproteins,mostwhichnodoubtisregulatedbyoneormoreenzymesornotablythebasicproteinscalledhistones.Thiscon-factors;faultyfunctionatanyofthesestepscancausedensationmayservearegulatoryroleandcertainlyhasdisease.apracticalpurpose.TheDNApresentwithinthenu-cleusofacell,ifsimplyextended,wouldbeaboutRECOMBINANTDNATECHNOLOGY1meterlong.ThechromosomalproteinscompactthislongstrandofDNAsothatitcanbepackagedintoaINVOLVESISOLATION&MANIPULATIONnucleuswithavolumeofafewcubicmicrometers.OFDNATOMAKECHIMERICMOLECULESIsolationandmanipulationofDNA,includingend-to-DNAIsOrganizedIntoGenesendjoiningofsequencesfromverydifferentsourcestomakechimericmolecules(eg,moleculescontainingIngeneral,prokaryoticgenesconsistofasmallregula-bothhumanandbacterialDNAsequencesinase-toryregion(100–500bp)andalargeprotein-codingquence-independentfashion),istheessenceofrecom-segment(500–10,000bp).Severalgenesareoftencon-binantDNAresearch.Thisinvolvesseveraluniquetrolledbyasingleregulatoryunit.Mostmammaliantechniquesandreagents.genesaremorecomplicatedinthatthecodingregionsareinterruptedbynoncodingregionsthatareelimi-RestrictionEnzymesCutDNAnatedwhentheprimaryRNAtranscriptisprocessedChainsatSpecificLocationsintomaturemessengerRNA(mRNA).Thecodingre-gions(thoseregionsthatappearinthematureRNACertainendonucleases—enzymesthatcutDNAatspe-species)arecalledexons,andthenoncodingregions,cificDNAsequenceswithinthemolecule(asopposedwhichinterposeorintervenebetweentheexons,aretoexonucleases,whichdigestfromtheendsofDNAcalledintrons(Figure40–1).Intronsarealwaysre-molecules)—areakeytoolinrecombinantDNAre-movedfromprecursorRNAbeforetransportintothesearch.Theseenzymeswerecalledrestrictionenzymescytoplasmoccurs.Theprocessbywhichintronsarere-becausetheirpresenceinagivenbacteriumrestrictedmovedfromprecursorRNAandbywhichexonsarethegrowthofcertainbacterialvirusescalledbacterio-ligatedtogetheriscalledRNAsplicing.Incorrectpro-phages.RestrictionenzymescutDNAofanysourcecessingoftheprimarytranscriptintothematureintoshortpiecesinasequence-specificmanner—inmRNAcanresultindiseaseinhumans(seebelow);thiscontrasttomostotherenzymatic,chemical,orphysicalunderscorestheimportanceoftheseposttranscriptionalmethods,whichbreakDNArandomly.Thesedefensiveprocessingsteps.Thevariationinsizeandcomplexityenzymes(hundredshavebeendiscovered)protecttheofsomehumangenesisillustratedinTable40–1.Al-hostbacterialDNAfromDNAfromforeignorganismsthoughthereisa300-folddifferenceinthesizesofthe(primarilyinfectivephages).However,theyarepresentgenesillustrated,themRNAsizesvaryonlyabout20-onlyincellsthatalsohaveacompanionenzymewhichfold.ThisisbecausemostoftheDNAingenesispres-methylatesthehostDNA,renderingitanunsuitableentasintrons,andintronstendtobemuchlargerthansubstratefordigestionbytherestrictionenzyme.Thus,
396398/CHAPTER40RegulatoryBasalTranscriptionPoly(A)regionpromoterstartsiteadditionregionsiteExonExonDNA5′CAATTATAAATAAA3′5′Intron3′NoncodingNoncodingregionregionTranscriptionNUCLEUSPrimaryRNAtranscriptPPPModificationof5′and3′endsModifiedtranscriptAAA---APoly(A)tailCapRemovalofintronsandsplicingofexonsProcessednuclearmRNAAAA---ATransmembraneCYTOPLASMtransportmRNAAAA---ATranslationProteinNH2COOHFigure40–1.Organizationofaeukaryotictranscriptionunitandthepathwayofeukaryoticgeneexpres-sion.Eukaryoticgeneshavestructuralandregulatoryregions.ThestructuralregionconsistsofthecodingDNAand5′and3′noncodingDNAsequences.Thecodingregionsaredividedintotwoparts:(1)exons,whicheventuallyareligatedtogethertobecomematureRNA,and(2)introns,whichareprocessedoutofthepri-marytranscript.Thestructuralregionisboundedatits5′endbythetranscriptioninitiationsiteandatits3′endbythepolyadenylateadditionorterminationsite.Thepromoterregion,whichcontainsspecificDNAsequencesthatinteractwithvariousproteinfactorstoregulatetranscription,isdiscussedindetailinChap-ters37and39.Theprimarytranscripthasaspecialstructure,acap,atthe5′endandastretchofAsatthe3′end.Thistranscriptisprocessedtoremovetheintrons;andthematuremRNAisthentransportedtothecyto-plasm,whereitistranslatedintoprotein.site-specificDNAmethylasesandrestrictionenzymesHpaI)oroverlapping(sticky)ends(eg,BamHI)(Figurealwaysexistinpairsinabacterium.40–2),dependingonthemechanismusedbytheen-Restrictionenzymesarenamedafterthebac-zyme.Stickyendsareparticularlyusefulinconstructingteriumfromwhichtheyareisolated.Forexample,hybridorchimericDNAmolecules(seebelow).IftheEcoRIisfromEscherichiacoli,andBamHIisfromBacil-fournucleotidesaredistributedrandomlyinagivenlusamyloliquefaciens(Table40–2).ThefirstthreelettersDNAmolecule,onecancalculatehowfrequentlyaintherestrictionenzymenameconsistofthefirstlettergivenenzymewillcutalengthofDNA.Foreachposi-ofthegenus(E)andthefirsttwolettersofthespeciestionintheDNAmolecule,therearefourpossibilities(co).Thesemaybefollowedbyastraindesignation(R)(A,C,G,andT);therefore,arestrictionenzymethatandaromannumeral(I)toindicatetheorderofdiscov-recognizesa4-bpsequencecuts,onaverage,onceevery4ery(eg,EcoRI,EcoRII).Eachenzymerecognizesand256bp(4),whereasanotherenzymethatrecognizesa6cleavesaspecificdouble-strandedDNAsequencethatis6-bpsequencecutsonceevery4096bp(4).Agiven4–7bplong.TheseDNAcutsresultinbluntends(eg,pieceofDNAhasacharacteristiclineararrayofsitesfor
397MOLECULARGENETICS,RECOMBINANTDNA,&GENOMICTECHNOLOGY/399Table40–1.VariationsinthesizeandcomplexityTable40–2.Selectedrestrictionendonucleases11ofsomehumangenesandmRNAs.andtheirsequencespecificities.mRNASequenceRecognizedBacterialGeneSizeNumberSizeEndonucleaseCleavageSitesShownSourceGene(kb)ofIntrons(kb)↓β-Globin1.520.6BamHIGGATCCBacillusamylo-Insulin1.720.4CCTAGGliquefaciensHβ-Adrenergicreceptor302.2↑Albumin25142.1↓LDLreceptor45175.5BgIIIAGATCTBacillusglolbigiiFactorVIII186259.0TCTAGAThyroglobulin300368.7↑1Thesizesaregiveninkilobases(kb).Thesizesofthegenesin-↓cludesomeproximalpromoterandregulatoryregionsequences;EcoRIGAATTCEscherichiacolithesearegenerallyaboutthesamesizeforallgenes.GenesvaryCTTAAGRY136insizefromabout1500basepairs(bp)toover2×10bp.Thereis↑alsogreatvariationinthenumberofintronsandexons.The↓β-adrenergicreceptorgeneisintronless,andthethyroglobulinEcoRIICCTGGEscherichiacoligenehas36introns.AsnotedbythesmallerdifferenceinmRNAGGACCR245sizes,intronscomprisemostofthegenesequence.↑↓HindIIIAAGCTTHaemophilusthevariousenzymesdictatedbythelinearsequenceofTTCGAAinfluenzaeRditsbases;hence,arestrictionmapcanbeconstructed.↑WhenDNAisdigestedwithagivenenzyme,theendsofallthefragmentshavethesameDNAsequence.The↓HhalGCGCHaemophilusfragmentsproducedcanbeisolatedbyelectrophoresisCGCGhaemolyticusonagaroseorpolyacrylamidegels(seethediscussionof↑blottransfer,below);thisisanessentialstepincloningandamajoruseoftheseenzymes.↓AnumberofotherenzymesthatactonDNAandHpalGTTAACHaemophilusCAATTGparainfluenzaeRNAareanimportantpartofrecombinantDNAtech-↑nology.Manyofthesearereferredtointhisandsubse-quentchapters(Table40–3).↓MstIICCTNAGGMicrocoleusGGANTCCstrainRestrictionEnzymes&DNALigaseAre↑UsedtoPrepareChimericDNAMolecules↓Sticky-endligationistechnicallyeasy,butsomespecialPstICTGCAGProvidenciatechniquesareoftenrequiredtoovercomeproblemsin-GACGTCstuartii164herentinthisapproach.Stickyendsofavectormayre-↑connectwiththemselves,withnonetgainofDNA.↓Stickyendsoffragmentscanalsoanneal,sothattandemTaqlTCGAThermusheterogeneousinsertsform.Also,sticky-endsitesmayAGCTaquaticusYTInotbeavailableorinaconvenientposition.Tocircum-↑venttheseproblems,anenzymethatgeneratesblunt1A,adenine;C,cytosine;G,guanine,T,thymine.Arrowsshowthesiteendsisused,andnewendsareaddedusingtheenzymeofcleavage;dependingonthesite,stickyends(BamHI)orbluntendsterminaltransferase.Ifpolyd(G)isaddedtothe3′ends(Hpal)mayresult.Thelengthoftherecognitionsequencecanbe4bpofthevectorandpolyd(C)isaddedtothe3′endsof(Taql),5bp(EcoRII),6bp(EcoRI),or7bp(MstII)orlonger.Byconven-theforeignDNA,thetwomoleculescanonlyannealtotion,thesearewritteninthe5′or3′directionfortheupperstrandofeachrecognitionsequence,andthelowerstrandisshownwiththeeachother,thuscircumventingtheproblemslistedopposite(ie,3′or5′)polarity.Notethatmostrecognitionsequencesabove.Thisprocedureiscalledhomopolymertailing.arepalindromes(ie,thesequencereadsthesameinoppositedirec-Sometimes,syntheticblunt-endedduplexoligonu-tionsonthetwostrands).AresiduedesignatedNmeansthatanynu-cleotidelinkerswithaconvenientrestrictionenzymese-cleotideispermitted.
398400/CHAPTER40A.Stickyorstaggeredends5’GGATCC3’GGATCCBamHIFigure40–2.Resultsofrestrictionen-+donucleasedigestion.Digestionwithare-3’CCTAGG5’CCTAGGstrictionendonucleasecanresultinthefor-B.BluntendsmationofDNAfragmentswithsticky,or5’GTTAAC3’GTTAACHpaIcohesive,ends(A)orbluntends(B).Thisis+animportantconsiderationindevising3’CAATTG5’CAATTGcloningstrategies.quenceareligatedtotheblunt-endedDNA.Directterizedorusedforotherpurposes.Thistechniqueisblunt-endligationisaccomplishedusingtheenzymebasedonthefactthatchimericorhybridDNAmoleculesbacteriophageT4DNAligase.Thistechnique,thoughcanbeconstructedincloningvectors—typicallybacter-lessefficientthansticky-endligation,hastheadvantageialplasmids,phages,orcosmids—whichthencontinueofjoiningtogetheranypairsofends.Thedisadvantagestoreplicateinahostcellundertheirowncontrolsystems.arethatthereisnocontrolovertheorientationofinser-Inthisway,thechimericDNAisamplified.Thegeneraltionorthenumberofmoleculesannealedtogether,andprocedureisillustratedinFigure40–3.thereisnoeasywaytoretrievetheinsert.Bacterialplasmidsaresmall,circular,duplexDNAmoleculeswhosenaturalfunctionistoconferantibioticresistancetothehostcell.Plasmidshaveseveralproper-CloningAmplifiesDNAtiesthatmakethemextremelyusefulascloningvectors.Acloneisalargepopulationofidenticalmolecules,bac-Theyexistassingleormultiplecopieswithinthebac-teria,orcellsthatarisefromacommonancestor.Molec-teriumandreplicateindependentlyfromthebacterialularcloningallowsfortheproductionofalargenumberDNA.ThecompleteDNAsequenceofmanyplasmidsisofidenticalDNAmolecules,whichcanthenbecharac-known;hence,thepreciselocationofrestrictionenzyme1Table40–3.SomeoftheenzymesusedinrecombinantDNAresearch.EnzymeReactionPrimaryUseAlkalinephosphataseDephosphorylates5′endsofRNAandDNA.Removalof5′-PO4groupspriortokinaselabelingtopreventself-ligation.BAL31nucleaseDegradesboththe3′and5′endsofDNA.ProgressiveshorteningofDNAmolecules.DNAligaseCatalyzesbondsbetweenDNAmolecules.JoiningofDNAmolecules.DNApolymeraseISynthesizesdouble-strandedDNAfromSynthesisofdouble-strandedcDNA;nicktranslation;gener-single-strandedDNA.ationofbluntendsfromstickyends.DNaseIUnderappropriateconditions,producesNicktranslation;mappingofhypersensitivesites;mappingsingle-strandednicksinDNA.protein-DNAinteractions.ExonucleaseIIIRemovesnucleotidesfrom3′endsofDNA.DNAsequencing;mappingofDNA-proteininteractions.λexonucleaseRemovesnucleotidesfrom5′endsofDNA.DNAsequencing.32PolynucleotidekinaseTransfersterminalphosphate(γposition)PlabelingofDNAorRNA.fromATPto5′-OHgroupsofDNAorRNA.ReversetranscriptaseSynthesizesDNAfromRNAtemplate.SynthesisofcDNAfrommRNA;RNA(5′end)mappingstudies.S1nucleaseDegradessingle-strandedDNA.Removalof“hairpin”insynthesisofcDNA;RNAmappingstudies(both5′and3′ends).TerminaltransferaseAddsnucleotidestothe3′endsofDNA.Homopolymertailing.1Adaptedandreproduced,withpermission,fromEmeryAEH:Page41in:AnIntroductiontoRecombinantDNA.Wiley,1984.
399MOLECULARGENETICS,RECOMBINANTDNA,&GENOMICTECHNOLOGY/401EcoRITrestrictionTAendonucleaseAAATHumanDNATCircularplasmidDNALinearplasmidDNAwithstickyendsEcoRIrestrictionendonucleaseAATTAAAATTTTAATTTTTATAAnnealPieceofhumanDNAcutwithAADNAsamerestrictionnucleaseandligasecontainingsamestickyendsTTTTAAAAAAAATTTTPlasmidDNAmoleculewithhumanDNAinsert(recombinantDNAmolecule)Figure40–3.UseofrestrictionnucleasestomakenewrecombinantorchimericDNAmolecules.Whenin-sertedbackintoabacterialcell(bytheprocesscalledtransformation),typicallyonlyasingleplasmidistakenupbyasinglecell,andtheplasmidDNAreplicatesnotonlyitselfbutalsothephysicallylinkednewDNAinsert.Sincerecombiningthestickyends,asindicated,regeneratesthesameDNAsequencerecognizedbytheoriginalrestric-tionenzyme,theclonedDNAinsertcanbecleanlycutbackoutoftherecombinantplasmidcirclewiththisen-donuclease.IfamixtureofalloftheDNApiecescreatedbytreatmentoftotalhumanDNAwithasinglerestrictionnucleaseisusedasthesourceofhumanDNA,amillionorsodifferenttypesofrecombinantDNAmoleculescanbeobtained,eachpureinitsownbacterialclone.(Modifiedandreproduced,withpermission,fromCohenSN:Themanipulationofgenes.SciAm[July]1975;233:34.)cleavagesitesforinsertingtheforeignDNAisavailable.LargerfragmentsofDNAcanbeclonedincosmids,Plasmidsaresmallerthanthehostchromosomeandarewhichcombinethebestfeaturesofplasmidsandthereforeeasilyseparatedfromthelatter,andthedesiredphages.CosmidsareplasmidsthatcontaintheDNAse-plasmid-insertedDNAisreadilyremovedbycuttingthequences,so-calledcossites,requiredforpackagingplasmidwiththeenzymespecificfortherestrictionsitelambdaDNAintothephageparticle.ThesevectorsintowhichtheoriginalpieceofDNAwasinserted.growintheplasmidforminbacteria,butsincemuchofPhagesusuallyhavelinearDNAmoleculesintotheunnecessarylambdaDNAhasbeenremoved,morewhichforeignDNAcanbeinsertedatseveralrestric-chimericDNAcanbepackagedintotheparticlehead.tionenzymesites.ThechimericDNAiscollectedafterItisnotunusualforcosmidstocarryinsertsofchimericthephageproceedsthroughitslyticcycleandproducesDNAthatare35–50kblong.Evenlargerpiecesofmature,infectivephageparticles.AmajoradvantageofDNAcanbeincorporatedintobacterialartificialchro-phagevectorsisthatwhileplasmidsacceptDNApiecesmosome(BAC),yeastartificialchromosome(YAC),orabout6–10kblong,phagescanacceptDNAfragmentsE.colibacteriophageP1-based(PAC)vectors.These10–20kblong,alimitationimposedbytheamountofvectorswillacceptandpropagateDNAinsertsofsev-DNAthatcanbepackedintothephagehead.eralhundredkilobasesormoreandhavelargelyre-
400402/CHAPTER40Table40–4.Cloningcapacitiesofcommonferentrecombinantclonesiscalledalibrary.Agenomiccloningvectors.libraryispreparedfromthetotalDNAofacelllineortissue.AcDNAlibrarycomprisescomplementaryDNAcopiesofthepopulationofmRNAsinatissue.VectorDNAInsertSizeGenomicDNAlibrariesareoftenpreparedbyperform-PlasmidpBR3220.01–10kbingpartialdigestionoftotalDNAwitharestrictionen-Lambdacharon4A10–20kbzymethatcutsDNAfrequently(eg,afourbasecutterCosmids35–50kbsuchasTaqI).Theideaistogenerateratherlargefrag-BAC,P150–250kbmentssothatmostgeneswillbeleftintact.TheBAC,YAC500–3000kbYAC,andP1vectorsarepreferredsincetheycanacceptverylargefragmentsofDNAandthusofferabetterplacedtheplasmid,phage,andcosmidvectorsforsomechanceofisolatinganintactgeneonasingleDNAcloningandgenemappingapplications.Acomparisonfragment.ofthesevectorsisshowninTable40–4.Avectorinwhichtheproteincodedbythegenein-BecauseinsertionofDNAintoafunctionalregiontroducedbyrecombinantDNAtechnologyisactuallyofthevectorwillinterferewiththeactionofthisre-synthesizedisknownasanexpressionvector.Suchgion,caremustbetakennottointerruptanessentialvectorsarenowcommonlyusedtodetectspecificfunctionofthevector.Thisconceptcanbeexploited,cDNAmoleculesinlibrariesandtoproduceproteinshowever,toprovideaselectiontechnique.Forexample,bygeneticengineeringtechniques.ThesevectorsarethecommonplasmidvectorpBR322hasbothtetracy-speciallyconstructedtocontainveryactiveinduciblecline(tet)andampicillin(amp)resistancegenes.Apromoters,properin-phasetranslationinitiationsinglePstIrestrictionenzymesitewithintheampresis-codons,bothtranscriptionandtranslationterminationtancegeneiscommonlyusedastheinsertionsiteforasignals,andappropriateproteinprocessingsignals,ifpieceofforeignDNA.Inadditiontohavingstickyendsneeded.Someexpressionvectorsevencontaingenes(Table40–2andFigure40–2),theDNAinsertedatthatcodeforproteaseinhibitors,sothatthefinalyieldthissitedisruptstheampresistancegeneandmakestheofproductisenhanced.bacteriumcarryingthisplasmidamp-sensitive(Figure40–4).Thus,theparentalplasmid,whichprovidesre-ProbesSearchLibrariesforSpecificsistancetobothantibiotics,canbereadilyseparatedGenesorcDNAMoleculesfromthechimericplasmid,whichisresistantonlytotetracycline.YACscontainreplicationandsegregationAvarietyofmoleculescanbeusedto“probe”librariesinfunctionsthatworkinbothbacteriaandyeastcellsandsearchofaspecificgeneorcDNAmoleculeortodefinethereforecanbepropagatedineitherorganism.andquantitateDNAorRNAseparatedbyelectrophore-InadditiontothevectorsdescribedinTable40–4sisthroughvariousgels.ProbesaregenerallypiecesofthataredesignedprimarilyforpropagationinbacterialDNAorRNAlabeledwitha32P-containingnu-cells,vectorsformammaliancellpropagationandinsertcleotide—orfluorescentlylabelednucleotides(more32gene(cDNA)/proteinexpressionhavealsobeendevel-commonlynow).Importantly,neithermodification(Poped.Thesevectorsareallbaseduponvariouseukary-orfluorescent-label)affectsthehybridizationpropertiesoticvirusesthatarecomposedofRNAorDNAoftheresultinglabelednucleicacidprobes.Theprobegenomes.Notableexamplesofsuchviralvectorsaremustrecognizeacomplementarysequencetobeeffec-thoseutilizingadenoviral(DNA-based)andretroviraltive.AcDNAsynthesizedfromaspecificmRNAcanbe(RNA-based)genomes.ThoughsomewhatlimitedinusedtoscreeneitheracDNAlibraryforalongercDNAthesizeofDNAsequencesthatcanbeinserted,suchoragenomiclibraryforacomplementarysequenceinmammalianviralcloningvectorsmakeupforthisthecodingregionofagene.Apopulartechniqueforshortcomingbecausetheywillefficientlyinfectawidefindingspecificgenesentailstakingashortaminoacidrangeofdifferentcelltypes.Forthisreason,varioussequenceand,employingthecodonusageforthatmammalianviralvectorsarebeinginvestigatedforusespecies(seeChapter38),makinganoligonucleotideingenetherapyexperiments.probethatwilldetectthecorrespondingDNAfragmentinagenomiclibrary.Ifthesequencesmatchexactly,probes15–20nucleotideslongwillhybridize.cDNAALibraryIsaCollectionprobesareusedtodetectDNAfragmentsonSouthernofRecombinantClonesblottransfersandtodetectandquantitateRNAonThecombinationofrestrictionenzymesandvariousNorthernblottransfers.Specificantibodiescanalsobecloningvectorsallowstheentiregenomeofanorgan-usedasprobesprovidedthatthevectorusedsynthesizesismtobepackedintoavector.Acollectionofthesedif-proteinmoleculesthatarerecognizedbythem.
401MOLECULARGENETICS,RECOMBINANTDNA,&GENOMICTECHNOLOGY/403AmpicillinTetracyclineresistancegeneresistancegeneEcoRIEcoRITetracyclineHindIIIresistancegeneHindIIIPstIBamHIBamHICutopenwithPstISalIPstIPstISalITheninsertrPstI-cutDNAsAmpAmpTetrTetrHostpBR322ChimericpBR322Figure40–4.AmethodofscreeningrecombinantsforinsertedDNAfragments.UsingtheplasmidpBR322,apieceofDNAisinsertedintotheuniquePstIsite.Thisinsertiondisruptsthegenecodingforaproteinthatpro-videsampicillinresistancetothehostbacterium.Hence,thechimericplasmidwillnolongersurvivewhenplatedonasubstratemediumthatcontainsthisantibiotic.Thedifferentialsensitivitytotetracyclineandampicillincanthereforebeusedtodistinguishclonesofplasmidthatcontainaninsert.Asimilarschemerelyinguponproduc-tionofanin-framefusionofanewlyinsertedDNAproducingapeptidefragmentcapableofcomplementinganinactive,deletedformoftheenzymeβ-galactosidaseallowsforblue-whitecolonyformationonagarplatescon-tainingadyehydrolyzablebyβ-galactoside.β-Galactosidase-positivecoloniesareblue.Blotting&HybridizationTechniquesAllowrenatured,andanalyzedforaninteractionbyhybridiza-VisualizationofSpecificFragmentstionwithaspecificlabeledDNAprobe.ColonyorplaquehybridizationisthemethodbyVisualizationofaspecificDNAorRNAfragmentwhichspecificclonesareidentifiedandpurified.Bacte-amongthemanythousandsof“contaminating”mole-riaaregrownoncoloniesonanagarplateandoverlaidculesrequirestheconvergenceofanumberoftech-withnitrocellulosefilterpaper.Cellsfromeachcolonyniques,collectivelytermedblottransfer.Figure40–5sticktothefilterandarepermanentlyfixedtheretobyillustratestheSouthern(DNA),Northern(RNA),andheat,whichwithNaOHtreatmentalsolysesthecellsWestern(protein)blottransferprocedures.(ThefirstisanddenaturestheDNAsothatitwillhybridizewithnamedforthepersonwhodevisedthetechnique,andtheprobe.Aradioactiveprobeisaddedtothefilter,theothernamesbeganaslaboratoryjargonbutarenowand(afterwashing)thehybridcomplexislocalizedbyacceptedterms.)Theseproceduresareusefulindeter-exposingthefiltertox-rayfilm.Bymatchingthespotmininghowmanycopiesofageneareinagiventissueontheautoradiographtoacolony,thelattercanbeorwhetherthereareanygrossalterationsinagenepickedfromtheplate.Asimilarstrategyisusedtoiden-(deletions,insertions,orrearrangements).Occasionally,tifyfragmentsinphagelibraries.Successiveroundsofifaspecificbaseischangedandarestrictionsiteisal-thisprocedureresultinaclonalisolate(bacterialtered,theseprocedurescandetectapointmutation.colony)orindividualphageplaque.TheNorthernandWesternblottransfertechniquesareAllofthehybridizationproceduresdiscussedinthisusedtosizeandquantitatespecificRNAandproteinsectiondependonthespecificbase-pairingpropertiesmolecules,respectively.Afourthhybridizationtech-ofcomplementarynucleicacidstrandsdescribedabove.nique,theSouthwesternblot,examinesprotein•DNAPerfectmatcheshybridizereadilyandwithstandhighinteractions.Proteinsareseparatedbyelectrophoresis,temperaturesinthehybridizationandwashingreac-
402404/CHAPTER40SouthernNorthernWesterntions.Specificcomplexesalsoforminthepresenceoflowsaltconcentrations.LessthanperfectmatchesdoDNARNAProteinnottoleratethesestringentconditions(ie,elevatedtemperaturesandlowsaltconcentrations);thus,hy-bridizationeitherneveroccursorisdisruptedduringGelthewashingstep.Genefamilies,inwhichthereissomeelectrophoresisdegreeofhomology,canbedetectedbyvaryingthestringencyofthehybridizationandwashingsteps.Cross-speciescomparisonsofagivengenecanalsobemadeusingthisapproach.Hybridizationconditionsca-pableofdetectingjustasinglebasepairmismatchbe-tweenprobeandtargethavebeendevised.TransfertopaperManual&AutomaticTechniquesAreAvailabletoDeterminecDNA*cDNA*Antibody*AddprobetheSequenceofDNAThesegmentsofspecificDNAmoleculesobtainedbyAutoradiographrecombinantDNAtechnologycanbeanalyzedtode-terminetheirnucleotidesequence.Thismethodde-pendsuponhavingalargenumberofidenticalDNAmolecules.ThisrequirementcanbesatisfiedbycloningFigure40–5.Theblottransferprocedure.Inathefragmentofinterest,usingthetechniquesdescribedSouthern,orDNA,blottransfer,DNAisolatedfromaabove.Themanualenzymaticmethod(Sanger)em-celllineortissueisdigestedwithoneormorerestric-ploysspecificdideoxynucleotidesthatterminateDNAtionenzymes.Thismixtureispipettedintoawellinanstrandsynthesisatspecificnucleotidesasthestrandisagaroseorpolyacrylamidegelandexposedtoadirectsynthesizedonpurifiedtemplatenucleicacid.Thereac-electricalcurrent.DNA,beingnegativelycharged,mi-tionsareadjustedsothatapopulationofDNAfrag-gratestowardtheanode;thesmallerfragmentsmovementsrepresentingterminationateverynucleotideisthemostrapidly.Afterasuitabletime,theDNAisdena-obtained.Byhavingaradioactivelabelincorporatedatturedbyexposuretomildalkaliandtransferredtoni-theendoppositetheterminationsite,onecanseparatetrocelluloseornylonpaper,inanexactreplicaofthethefragmentsaccordingtosizeusingpolyacrylamidepatternonthegel,bytheblottingtechniquedevisedgelelectrophoresis.Anautoradiographismade,andbySouthern.TheDNAisboundtothepaperbyexpo-eachofthefragmentsproducesanimage(band)onansuretoheat,andthepaperisthenexposedtothex-rayfilm.ThesearereadinordertogivetheDNAse-labeledcDNAprobe,whichhybridizestocomplemen-quence(Figure40–6).Anothermanualmethod,thatoftaryfragmentsonthefilter.Afterthoroughwashing,MaxamandGilbert,employschemicalmethodstothepaperisexposedtox-rayfilm,whichisdevelopedcleavetheDNAmoleculeswheretheycontainthespe-torevealseveralspecificbandscorrespondingtothecificnucleotides.TechniquesthatdonotrequiretheDNAfragmentthatrecognizedthesequencesintheuseofradioisotopesarecommonlyemployedinauto-cDNAprobe.TheRNA,orNorthern,blotisconceptuallymatedDNAsequencing.Mostcommonlyemployedissimilar.RNAissubjectedtoelectrophoresisbeforeblotanautomatedprocedureinwhichfourdifferentfluo-rescentlabels—onerepresentingeachnucleotide—aretransfer.Thisrequiressomedifferentstepsfromthoseused.EachemitsaspecificsignaluponexcitationbyaofDNAtransfer,primarilytoensurethattheRNAre-laserbeam,andthiscanberecordedbyacomputer.mainsintact,andisgenerallysomewhatmoredifficult.Intheprotein,orWestern,blot,proteinsareelec-trophoresedandtransferredtonitrocelluloseandthenOligonucleotideSynthesisIsNowRoutineprobedwithaspecificantibodyorotherprobemole-Theautomatedchemicalsynthesisofmoderatelylongcule.(Asteriskssignifylabeling,eitherradioactiveoroligonucleotides(about100nucleotides)ofprecisese-fluorescent.)quenceisnowaroutinelaboratoryprocedure.Eachsyntheticcycletakesbutafewminutes,soanentiremoleculecanbemadebysynthesizingrelativelyshortsegmentsthatcanthenbeligatedtooneanother.OligonucleotidesarenowindispensableforDNAse-
403MOLECULARGENETICS,RECOMBINANTDNA,&GENOMICTECHNOLOGY/405Reactioncontainingradiolabel:Sequenceoforiginalstrand:ddGTPddATPddTTPddCTP––––––––––––AGTCTTGGAGCT–3′ElectrophoresisSlabgelGATCAGTCTTGGAGCTBasesterminatedFigure40–6.SequencingofDNAbythemethoddevisedbySanger.Theladder-likearraysrepresentfrombot-tomtotopallofthesuccessivelylongerfragmentsoftheoriginalDNAstrand.Knowingwhichspecificdideoxynu-cleotidereactionwasconductedtoproduceeachmixtureoffragments,onecandeterminethesequenceofnu-cleotidesfromthelabeledend(asterisk)towardtheunlabeledendbyreadingupthegel.Automatedsequencinginvolvesthereadingofchemicallymodifieddeoxynucleotides.Thebase-pairingrulesofWatsonandCrick(A–T,G–C)dictatethesequenceoftheother(complementary)strand.(Asteriskssignifyradiolabeling.)quencing,libraryscreening,protein-DNAbinding,quences,andextensionoftheannealedprimerswithDNAmobilityshiftassays,thepolymerasechainreac-DNApolymeraseresultintheexponentialamplifica-tion(seebelow),site-directedmutagenesis,andnumer-tionofDNAsegmentsofdefinedlength.EarlyPCRre-ousotherapplications.actionsusedanEcoliDNApolymerasethatwasde-stroyedbyeachheatdenaturationcycle.SubstitutionofThePolymeraseChainReactionaheat-stableDNApolymerasefromThermusaquaticus(orthecorrespondingDNApolymerasefromother(PCR)AmplifiesDNASequencesthermophilicbacteria),anorganismthatlivesandrepli-Thepolymerasechainreaction(PCR)isamethodofcatesat70–80°C,obviatesthisproblemandhasmadeamplifyingatargetsequenceofDNA.PCRprovidesapossibleautomationofthereaction,sincethepolym-sensitive,selective,andextremelyrapidmeansofampli-erasereactionscanberunat70°C.Thishasalsoim-fyingadesiredsequenceofDNA.SpecificityisbasedprovedthespecificityandtheyieldofDNA.ontheuseoftwooligonucleotideprimersthathy-DNAsequencesasshortas50–100bpandaslongbridizetocomplementarysequencesonoppositeas10kbcanbeamplified.Twentycyclesprovidean69strandsofDNAandflankthetargetsequence(Figureamplificationof10and30cyclesof10.ThePCRal-40–7).TheDNAsampleisfirstheatedtoseparatethelowstheDNAinasinglecell,hairfollicle,orspermato-twostrands;theprimersareallowedtobindtothezoontobeamplifiedandanalyzed.Thus,theapplica-DNA;andeachstrandiscopiedbyaDNApolymerase,tionsofPCRtoforensicmedicineareobvious.Thestartingattheprimersite.ThetwoDNAstrandseachPCRisalsoused(1)todetectinfectiousagents,espe-serveasatemplateforthesynthesisofnewDNAfromciallylatentviruses;(2)tomakeprenatalgeneticdiag-thetwoprimers.Repeatedcyclesofheatdenaturation,noses;(3)todetectallelicpolymorphisms;(4)toestab-annealingoftheprimerstotheircomplementaryse-lishprecisetissuetypesfortransplants;and(5)tostudy
404406/CHAPTER40evolution,usingDNAfromarcheologicalsamplesafterTargetedsequenceRNAcopyingandmRNAquantitationbytheso-calledRT-PCRmethod(cDNAcopiesofmRNAgeneratedSTARTbyaretroviralreversetranscriptase).ThereareanequalnumberofapplicationsofPCRtoproblemsinbasicscience,andnewusesaredevelopedeveryyear.CYCLE1PRACTICALAPPLICATIONSOFRECOMBINANTDNATECHNOLOGYARENUMEROUSTheisolationofaspecificgenefromanentiregenomeCYCLE2requiresatechniquethatwilldiscriminateonepartinamillion.Theidentificationofaregulatoryregionthatmaybeonly10bpinlengthrequiresasensitivityofonepartin3×108;adiseasesuchassicklecellanemia9iscausedbyasinglebasechange,oronepartin3×10.RecombinantDNAtechnologyispowerfulenoughtoaccomplishallthesethings.GeneMappingLocalizesSpecificGenestoDistinctChromosomesGenelocalizingthuscandefineamapofthehumangenome.ThisisalreadyyieldingusefulinformationinCYCLE3thedefinitionofhumandisease.Somaticcellhybridiza-tionandinsituhybridizationaretwotechniquesusedtoaccomplishthis.Ininsituhybridization,thesim-plerandmoredirectprocedure,aradioactiveprobeisaddedtoametaphasespreadofchromosomesonaglassslide.Theexactareaofhybridizationislocalizedbylay-eringphotographicemulsionovertheslideand,afterexposure,liningupthegrainswithsomehistologicidentificationofthechromosome.Fluorescenceinsituhybridization(FISH)isaverysensitivetechniquethatisalsousedforthispurpose.Thisoftenplacesthegeneatalocationonagivenbandorregiononthechromo-some.SomeofthehumangeneslocalizedusingthesetechniquesarelistedinTable40–5.Thistablerepre-sentsonlyasampling,sincethousandsofgeneshavebeenmappedasaresultoftherecentsequencingoftheFigure40–7.Thepolymerasechainreactionisusedtoamplifyspecificgenesequences.Double-strandedDNAisheatedtoseparateitintoindividualstrands.Thesebindtwodistinctprimersthataredirectedatspecificsequencesonoppositestrandsandthatdefinethesegmenttobeampli-fied.DNApolymeraseextendstheprimersineachdirectionandsynthesizestwostrandscomplementarytotheoriginaltwo.Thiscycleisrepeatedseveraltimes,givinganamplifiedproductofdefinedlengthandsequence.NotethatthetwoCYCLES4–nprimersarepresentinexcess.
405MOLECULARGENETICS,RECOMBINANTDNA,&GENOMICTECHNOLOGY/4071Table40–5.Localizationofhumangenes.GeneChromosomeDiseaseInsulin11p15Prolactin6p23-q12Growthhormone17q21-qterGrowthhormonedeficiencyα-Globin16p12-pterα-Thalassemiaβ-Globin11p12β-Thalassemia,sicklecellAdenosinedeaminase20q13-qterAdenosinedeaminasedeficiencyPhenylalaninehydroxylase12q24PhenylketonuriaHypoxanthine-guanineXq26-q27Lesch-NyhansyndromephosphoribosyltransferaseDNAsegmentG84pHuntington’schorea1Thistableindicatesthechromosomallocationofseveralgenesandthediseasesasso-ciatedwithdeficientorabnormalproductionofthegeneproducts.Thechromosomeinvolvedisindicatedbythefirstnumberorletter.Theothernumbersandlettersrefertopreciselocalizations,asdefinedinMcKusickVA:MendelianInheritanceinMan,6thed.JohnHopkinsUnivPress,1983.humangenome.OncethedefectislocalizedtoaregionRecombinantDNATechnologyIsUsedofDNAthathasthecharacteristicstructureofageneintheMolecularAnalysisofDisease(Figure40–1),asyntheticgenecanbeconstructedandexpressedinanappropriatevectoranditsfunctioncanA.NORMALGENEVARIATIONSbeassessed—ortheputativepeptide,deducedfromtheThereisanormalvariationofDNAsequencejustasisopenreadingframeinthecodingregion,canbesynthe-trueofmoreobviousaspectsofhumanstructure.Varia-sized.AntibodiesdirectedagainstthispeptidecanbetionsofDNAsequence,polymorphisms,occurap-usedtoassesswhetherthispeptideisexpressedinnor-proximatelyonceinevery500nucleotides,oraboutmalpersonsandwhetheritisabsentinthosewiththe710timespergenome.Therearewithoutdoubtdele-geneticsyndrome.tionsandinsertionsofDNAaswellassingle-basesub-stitutions.Inhealthypeople,thesealterationsobviouslyoccurinnoncodingregionsofDNAoratsitesthatProteinsCanBeProducedcausenochangeinfunctionoftheencodedprotein.forResearch&DiagnosisThisheritablepolymorphismofDNAstructurecanbeassociatedwithcertaindiseaseswithinalargekindredApracticalgoalofrecombinantDNAresearchistheandcanbeusedtosearchforthespecificgeneinvolved,productionofmaterialsforbiomedicalapplications.asisillustratedbelow.ItcanalsobeusedinavarietyofThistechnologyhastwodistinctmerits:(1)Itcansup-applicationsinforensicmedicine.plylargeamountsofmaterialthatcouldnotbeob-tainedbyconventionalpurificationmethods(eg,inter-feron,tissueplasminogenactivatingfactor).(2)ItcanB.GENEVARIATIONSCAUSINGDISEASEprovidehumanmaterial(eg,insulin,growthhormone).ClassicgeneticstaughtthatmostgeneticdiseaseswereTheadvantagesinbothcasesareobvious.Althoughtheduetopointmutationswhichresultedinanimpairedprimaryaimistosupplyproducts—generallypro-protein.Thismaystillbetrue,butifonreadingtheteins—fortreatment(insulin)anddiagnosis(AIDSinitialsectionsofthischapteronepredictedthatge-testing)ofhumanandotheranimaldiseasesandforneticdiseasecouldresultfromderangementofanyofdiseaseprevention(hepatitisBvaccine),thereareotherthestepsillustratedinFigure40–1,onewouldhavepotentialcommercialapplications,especiallyinagricul-madeaproperassessment.Thispointisnicelyillus-ture.Anexampleofthelatteristheattempttoengineertratedbyexaminationoftheβ-globingene.Thisgeneplantsthataremoreresistanttodroughtortemperatureislocatedinaclusteronchromosome11(Figureextremes,moreefficientatfixingnitrogen,orthatpro-40–8),andanexpandedversionofthegeneisillus-duceseedscontainingthecompletecomplementofes-tratedinFigure40–9.Defectiveproductionofβ-glo-sentialaminoacids(rice,wheat,corn,etc).binresultsinavarietyofdiseasesandisduetomany
406408/CHAPTER40∋GγAγΨβδβ5′LCR3′10kbHemoglobinopathyβ0-Thalassemiaβ0-ThalassemiaHemoglobinLeporeInverted(Aγδβ)0-ThalassemiaFigure40–8.Schematicrepresentationoftheβ-globingeneclusterandofthelesionsinsomege-neticdisorders.Theβ-globingeneislocatedonchromosome11incloseassociationwiththetwoγ-glo-bingenesandtheδ-globingene.Theβ-genefamilyisarrangedintheorder5′-ε-Gγ-Aγ-ψβ-δ-β-3′.Theεlocusisexpressedinearlyembryoniclife(asa2ε2).Theγgenesareexpressedinfetallife,makingfetalhemoglobin(HbF,α2γ2).AdulthemoglobinconsistsofHbA(α2β2)orHbA2(α2δ2).TheΨβisapseudo-genethathassequencehomologywithβbutcontainsmutationsthatpreventitsexpression.Alocuscontrolregion(LCR)locatedupstream(5′)fromtheεgenecontrolstherateoftranscriptionoftheen-tireβ-globingenecluster.Deletions(solidbar)oftheβlocuscauseβ-thalassemia(deficiencyorab-0sence[β]ofβ-globin).AdeletionofδandβcauseshemoglobinLepore(onlyhemoglobinαispresent).0Aninversion(Aγδβ)inthisregion(coloredbar)disruptsgenefunctionandalsoresultsinthalassemia0(typeIII).Eachtypeofthalassemiatendstobefoundinacertaingroupofpeople,eg,the(Aγδβ)dele-tioninversionoccursinpersonsfromIndia.Manymoredeletionsinthisregionhavebeenmapped,andeachcausessometypeofthalassemia.differentlesionsinandaroundtheβ-globingeneturnresultsinanA-to-UchangeinthemRNAcorre-(Table40–6).spondingtothesixthcodonoftheβ-globingene.Thealteredcodonspecifiesadifferentaminoacid(valineC.POINTMUTATIONSratherthanglutamicacid),andthiscausesastructuralTheclassicexampleissicklecelldisease,whichisabnormalityoftheβ-globinmolecule.Otherpointmu-9causedbymutationofasinglebaseoutofthe3×10tationsinandaroundtheβ-globingeneresultinde-inthegenome,aT-to-ADNAsubstitution,whichincreasedproductionor,insomeinstances,noproduc-5′II123′Figure40–9.Mutationsintheβ-globingenecausingβ-thalassemia.Theβ-globingeneisshowninthe5′to3′orientation.Thecross-hatchedareasindicatethe5′and3′nontranslatedregions.Readingfromthe5′to3′direction,theshadedareasareexons1–3andtheclearspacesareintrons1(I1)and2(I2).Mutationsthataf-fecttranscriptioncontrol(•)arelocatedinthe5′flanking-regionDNA.Examplesofnonsensemutations(),mutationsinRNAprocessing(),andRNAcleavagemutations()havebeenidentifiedandareindicated.Insomeregions,manymutationshavebeenfound.Theseareindicatedbythebrackets.
407MOLECULARGENETICS,RECOMBINANTDNA,&GENOMICTECHNOLOGY/409Table40–6.Structuralalterationsoftheβ-globinE.PEDIGREEANALYSISgene.SicklecelldiseaseagainprovidesanexcellentexampleofhowrecombinantDNAtechnologycanbeappliedAlterationFunctionAffectedDiseasetothestudyofhumandisease.ThesubstitutionofTforAinthetemplatestrandofDNAintheβ-globinPointmutationsProteinfoldingSicklecelldiseasegenechangesthesequenceintheregionthatcorre-Transcriptionalcontrolβ-ThalassemiaspondstothesixthcodonfromFrameshiftandnon-β-Thalassemiasensemutations↓RNAprocessingβ-ThalassemiaCCTGAGGCodingstrand0DeletionmRNAproductionβ-ThalassemiaGGACTCCTemplatestrandHemoglobin↑LeporetoRearrangementmRNAproductionβ-ThalassemiatypeIIICCTGTGGCodingstrandGGACACCTemplatestrandanddestroysarecognitionsitefortherestrictionen-tionofβ-globin;β-thalassemiaistheresultofthesezymeMstII(CCTNAGG;denotedbythesmallverticalmutations.(Thethalassemiasarecharacterizedbyde-arrows;Table40–2).OtherMstIIsites5′and3′fromfectsinthesynthesisofhemoglobinsubunits,andsothissite(Figure40–10)arenotaffectedandsowillbeβ-thalassemiaresultswhenthereisinsufficientproduc-cut.Therefore,incubationofDNAfromnormal(AA),tionofβ-globin.)Figure40–9illustratesthatpointheterozygous(AS),andhomozygous(SS)individualsmutationsaffectingeachofthemanyprocessesin-resultsinthreedifferentpatternsonSouthernblotvolvedingeneratinganormalmRNA(andthereforeatransfer(Figure40–10).ThisillustrateshowaDNAnormalprotein)havebeenimplicatedasacauseofpedigreecanbeestablishedusingtheprinciplesdis-β-thalassemia.cussedinthischapter.Pedigreeanalysishasbeenap-D.DELETIONS,INSERTIONS,&pliedtoanumberofgeneticdiseasesandismostusefulREARRANGEMENTSOFDNAinthosecausedbydeletionsandinsertionsortherarerinstancesinwhicharestrictionendonucleasecleavageStudiesofbacteria,viruses,yeasts,andfruitfliesshowsiteisaffected,asintheexamplecitedinthispara-thatpiecesofDNAcanmovefromoneplacetoan-graph.TheanalysisisfacilitatedbythePCRreaction,otherwithinagenome.ThedeletionofacriticalpiecewhichcanprovidesufficientDNAforanalysisfromofDNA,therearrangementofDNAwithinagene,orjustafewnucleatedredbloodcells.theinsertionofapieceofDNAwithinacodingorreg-ulatoryregioncanallcausechangesingeneexpressionF.PRENATALDIAGNOSISresultingindisease.Again,amolecularanalysisofβ-thalassemiaproducesnumerousexamplesoftheseIfthegeneticlesionisunderstoodandaspecificprobeprocesses—particularlydeletions—ascausesofdiseaseisavailable,prenataldiagnosisispossible.DNAfrom(Figure40–8).Theglobingeneclustersseemparticu-cellscollectedfromaslittleas10mLofamnioticfluidlarlypronetothislesion.Deletionsintheα-globin(orbychorionicvillusbiopsy)canbeanalyzedbycluster,locatedonchromosome16,causeα-thal-Southernblottransfer.Afetuswiththerestrictionpat-assemia.ThereisastrongethnicassociationformanyternAAinFigure40–10doesnothavesicklecelldis-ofthesedeletions,sothatnorthernEuropeans,Fil-ease,norisitacarrier.AfetuswiththeSSpatternwillipinos,blacks,andMediterraneanpeopleshavediffer-developthedisease.ProbesarenowavailableforthisentlesionsallresultingintheabsenceofhemoglobinAtypeofanalysisofmanygeneticdiseases.andα-thalassemia.AsimilaranalysiscouldbemadeforanumberofG.RESTRICTIONFRAGMENTLENGTHotherdiseases.PointmutationsareusuallydefinedbyPOLYMORPHISM(RFLP)sequencingthegeneinquestion,thoughoccasionally,ifThedifferencesinDNAsequencecitedabovecanre-themutationdestroysorcreatesarestrictionenzymesultinvariationsofrestrictionsitesandthusinthesite,thetechniqueofrestrictionfragmentanalysiscanlengthofrestrictionfragments.Aninheriteddifferencebeusedtopinpointthelesion.Deletionsorinsertionsinthepatternofrestriction(eg,aDNAvariationoccur-ofDNAlargerthan50bpcanoftenbedetectedbytheringinmorethan1%ofthegeneralpopulation)isSouthernblottingprocedure.knownasarestrictionfragmentlengthpolymorphism,
408410/CHAPTER40A.MstIIrestrictionsitesaroundandintheβ-globingeneNormal(A)5′3′1.15kb0.2kbSickle(S)5′3′1.35kbB.PedigreeanalysisFragmentsize1.35kb1.15kbASASSSAAASASPhenotypeFigure40–10.Pedigreeanalysisofsicklecelldisease.Thetoppartofthefig-ure(A)showsthefirstpartoftheβ-globingeneandtheMstIIrestrictionen-zymesitesinthenormal(A)andsicklecell(S)β-globingenes.DigestionwiththerestrictionenzymeMstIIresultsinDNAfragments1.15kband0.2kblonginnormalindividuals.TheT-to-AchangeinindividualswithsicklecelldiseaseabolishesoneofthethreeMstIIsitesaroundtheβ-globingene;hence,asinglerestrictionfragment1.35kbinlengthisgeneratedinresponsetoMstII.ThissizedifferenceiseasilydetectedonaSouthernblot.(The0.2-kbfragmentwouldrunoffthegelinthisillustration.)(B)Pedigreeanalysisshowsthreepossibili-ties:AA=normal(opencircle);AS=heterozygous(half-solidcircles,half-solidsquare);SS=homozygous(solidsquare).Thisapproachallowsforprenataldi-agnosisofsicklecelldisease(dash-sidedsquare).
409MOLECULARGENETICS,RECOMBINANTDNA,&GENOMICTECHNOLOGY/411orRFLP.AnextensiveRFLPmapofthehumanH.MICROSATELLITEDNAPOLYMORPHISMSgenomehasbeenconstructed.ThisisprovingusefulinShort(2–6bp),inherited,tandemrepeatunitsofDNAthehumangenomesequencingprojectandisanimpor-occurabout50,000–100,000timesinthehumantantcomponentoftheefforttounderstandvarioussin-genome(Chapter36).Becausetheyoccurmorefre-gle-geneandmultigenicdiseases.RFLPsresultfromquently—andinviewoftheroutineapplicationofsen-single-basechanges(eg,sicklecelldisease)orfromdele-sitivePCRmethods—theyarereplacingRFLPsasthetionsorinsertionsofDNAintoarestrictionfragmentmarkerlociforvariousgenomesearches.(eg,thethalassemias)andhaveprovedtobeusefuldi-agnostictools.TheyhavebeenfoundatknowngeneI.RFLPS&VNTRSINFORENSICMEDICINElociandinsequencesthathavenoknownfunction;Variablenumbersoftandemlyrepeated(VNTR)unitsthus,RFLPsmaydisruptthefunctionofthegeneorareonecommontypeof“insertion”thatresultsinanmayhavenobiologicconsequences.RFLP.TheVNTRscanbeinherited,inwhichcaseRFLPsareinherited,andtheysegregateinatheyareusefulinestablishinggeneticassociationwithamendelianfashion.AmajoruseofRFLPs(thousandsdiseaseinafamilyorkindred;ortheycanbeuniquetoarenowknown)isinthedefinitionofinheriteddis-anindividualandthusserveasamolecularfingerprinteasesinwhichthefunctionaldeficitisunknown.ofthatperson.RFLPscanbeusedtoestablishlinkagegroups,whichinturn,bytheprocessofchromosomewalking,willJ.GENETHERAPYeventuallydefinethediseaselocus.InchromosomeDiseasescausedbydeficiencyofageneproduct(Tablewalking(Figure40–11),afragmentrepresentingone40–5)areamenabletoreplacementtherapy.Thestrat-endofalongpieceofDNAisusedtoisolateanotheregyistocloneagene(eg,thegenethatcodesforthatoverlapsbutextendsthefirst.Thedirectionofex-adenosinedeaminase)intoavectorthatwillreadilybetensionisdeterminedbyrestrictionmapping,andthetakenupandincorporatedintothegenomeofahostprocedureisrepeatedsequentiallyuntilthedesiredse-cell.Bonemarrowprecursorcellsarebeinginvestigatedquenceisobtained.TheXchromosome-linkeddisor-forthispurposebecausetheypresumablywillresettleindersareparticularlyamenabletothisapproach,sincethemarrowandreplicatethere.Theintroducedgeneonlyasinglealleleisexpressed.Hence,20%ofthede-wouldbegintodirecttheexpressionofitsproteinprod-finedRFLPsareontheXchromosome,andareason-uct,andthiswouldcorrectthedeficiencyinthehostablycompletelinkagemapofthischromosomeexists.cell.ThegenefortheX-linkeddisorder,Duchenne-typemusculardystrophy,wasfoundusingRFLPs.Likewise,K.TRANSGENICANIMALSthedefectinHuntington’sdiseasewaslocalizedtotheThesomaticcellgenereplacementdescribedaboveterminalregionoftheshortarmofchromosome4,andwouldobviouslynotbepassedontooffspring.Otherthedefectthatcausespolycystickidneydiseaseislinkedstrategiestoaltergermcelllineshavebeendevisedbuttotheα-globinlocusonchromosome16.havebeentestedonlyinexperimentalanimals.AcertainIntactDNA5′GeneX3′Fragments12345Initial*probeFigure40–11.Thetechniqueofchromosomewalking.GeneXistobeisolatedfromalargepieceofDNA.Theexactlocationofthisgeneisnotknown,butaprobe(*——)directedagainstafrag-mentofDNA(shownatthe5′endinthisrepresentation)isavailable,asisalibrarycontainingase-riesofoverlappingDNAfragments.Forthesakeofsimplicity,onlyfiveoftheseareshown.Theinitialprobewillhybridizeonlywithclonescontainingfragment1,whichcanthenbeisolatedandusedasaprobetodetectfragment2.Thisprocedureisrepeateduntilfragment4hybridizeswithfragment5,whichcontainstheentiresequenceofgeneX.
410412/CHAPTER40percentageofgenesinjectedintoafertilizedmouseovumsquarecentimeters.BycouplingsuchDNAmicroarrayswillbeincorporatedintothegenomeandfoundinbothwithhighlysensitivedetectionofhybridizedfluores-somaticandgermcells.HundredsoftransgenicanimalscentlylabelednucleicacidprobesderivedfrommRNA,havebeenestablished,andtheseareusefulforanalysisofinvestigatorscanrapidlyandaccuratelygenerateprofilestissue-specificeffectsongeneexpressionandeffectsofofgeneexpression(eg,specificcellularmRNAcontent)overproductionofgeneproducts(eg,thosefromthefromcellandtissuesamplesassmallas1gramorless.growthhormonegeneoroncogenes)andindiscoveringThusentiretranscriptomeinformation(theentirecol-genesinvolvedindevelopment—aprocessthathereto-lectionofcellularmRNAs)forsuchcellortissuesourcesforehasbeendifficulttostudy.Thetransgenicapproachcanreadilybeobtainedinonlyafewdays.Transcrip-hasrecentlybeenusedtocorrectageneticdeficiencyintomeinformationallowsonetopredictthecollectionofmice.Fertilizedovaobtainedfrommicewithgenetichy-proteinsthatmightbeexpressedinaparticularcell,tis-pogonadismwereinjectedwithDNAcontainingthesue,ororganinnormalanddiseasestatesbaseduponthecodingsequenceforthegonadotropin-releasinghormonemRNAspresentinthosecells.Complementingthishigh-(GnRH)precursorprotein.Thisgenewasexpressedandthroughput,transcript-profilingmethodistherecentde-regulatednormallyinthehypothalamusofacertainvelopmentofhigh-sensitivity,high-throughputmassnumberoftheresultantmice,andtheseanimalswereinspectrometryofcomplexproteinsamples.Newermassallrespectsnormal.Theiroffspringalsoshowednoevi-spectrometrymethodsallowonetoidentifyhundredstodenceofGnRHdeficiency.Thisis,therefore,evidencethousandsofproteinsinproteinsextractedfromveryofsomaticcellexpressionofthetransgeneandofitssmallnumbersofcells(<1g).Thiscriticalinformationmaintenanceingermcells.tellsinvestigatorswhichofthemanymRNAsdetectedintranscriptmicroarraymappingstudiesareactuallytrans-latedintoprotein,generallytheultimatedictatorofphe-TargetedGeneDisruptionorKnockoutnotype.MicroarraytechniquesandmassspectrometricIntransgenicanimals,oneisaddingoneormorecopiesproteinidentificationexperimentsbothleadtothegen-ofagenetothegenome,andthereisnowaytocontrolerationofhugeamountsofdata.Appropriatedataman-wherethatgeneeventuallyresides.Acomplementary—agementandinterpretationofthedelugeofinformationandmuchmoredifficult—approachinvolvestheselec-forthcomingfromsuchstudieshasrelieduponstatisticaltiveremovalofagenefromthegenome.Geneknock-methods;andthisnewtechnology,coupledwiththeoutanimals(usuallymice)aremadebycreatingafloodofDNAsequenceinformation,hasledtothede-mutationthattotallydisruptsthefunctionofagene.velopmentofthefieldofbioinformatics,anewdisci-Thisisthenusedtoreplaceoneofthetwogenesinanplinewhosegoalistohelpmanage,analyze,andinte-embryonicstemcellthatcanbeusedtocreateahet-gratethisfloodofbiologicallyimportantinformation.erozygoustransgenicanimal.ThematingoftwosuchFutureworkattheintersectionofbioinformaticsandanimalswill,bymendeliangenetics,resultinaho-transcript-proteinprofilingwillrevolutionizeourunder-mozygousmutationin25%ofoffspring.Severalhun-standingofbiologyandmedicine.dredstrainsofmicewithknockoutsofspecificgeneshavebeendeveloped.SUMMARYRNATranscript&ProteinProfiling•Avarietyofverysensitivetechniquescannowbeap-pliedtotheisolationandcharacterizationofgenesThe“-omic”revolutionofthelastseveralyearshascul-andtothequantitationofgeneproducts.minatedinthedeterminationofthenucleotidese-quencesofentiregenomes,includingthoseofbudding•InDNAcloning,aparticularsegmentofDNAisre-andfissionyeasts,variousbacteria,thefruitfly,thewormmovedfromitsnormalenvironmentusingoneofCaenorhabditiselegans,themouseand,mostnotably,hu-manyrestrictionendonucleases.Thisisthenligatedmans.Additionalgenomesarebeingsequencedatanac-intooneofseveralvectorsinwhichtheDNAseg-celeratingpace.TheavailabilityofallofthisDNAse-mentcanbeamplifiedandproducedinabundance.quenceinformation,coupledwithengineeringadvances,•TheclonedDNAcanbeusedasaprobeinoneofhasleadtothedevelopmentofseveralrevolutionaryseveraltypesofhybridizationreactionstodetectmethodologies,mostofwhicharebaseduponhigh-den-otherrelatedoradjacentpiecesofDNA,oritcanbesitymicroarraytechnology.WenowhavetheabilitytousedtoquantitategeneproductssuchasmRNA.depositthousandsofspecific,known,definableDNAse-•ManipulationoftheDNAtochangeitsstructure,so-quences(moretypicallynowsyntheticoligonucleotides)calledgeneticengineering,isakeyelementincloningonaglassmicroscope-styleslideinthespaceofafew(eg,theconstructionofchimericmolecules)andcan
411MOLECULARGENETICS,RECOMBINANTDNA,&GENOMICTECHNOLOGY/413alsobeusedtostudythefunctionofacertainfrag-quencesofasinglestrandofDNAorRNA.mentofDNAandtoanalyzehowgenesareregulated.Hybridization:Thespecificreassociationofcom-•ChimericDNAmoleculesareintroducedintocellsplementarystrandsofnucleicacids(DNAwithtomaketransfectedcellsorintothefertilizedoocyteDNA,DNAwithRNA,orRNAwithRNA).tomaketransgenicanimals.Insert:AnadditionallengthofbasepairsinDNA,generallyintroducedbythetechniquesofrecom-•TechniquesinvolvingclonedDNAareusedtolocatebinantDNAtechnology.genestospecificregionsofchromosomes,toidentifyIntron:Thesequenceofagenethatistranscribedthegenesresponsiblefordiseases,tostudyhowfaultybutexcisedbeforetranslation.generegulationcausesdisease,todiagnosegeneticLibrary:Acollectionofclonedfragmentsthatrep-diseases,andincreasinglytotreatgeneticdiseases.resentstheentiregenome.LibrariesmaybeeithergenomicDNA(inwhichbothintronsandexonsGLOSSARYarerepresented)orcDNA(inwhichonlyexonsARS:Autonomouslyreplicatingsequence;theori-arerepresented).ginofreplicationinyeast.Ligation:Theenzyme-catalyzedjoininginphos-Autoradiography:ThedetectionofradioactivephodiesterlinkageoftwostretchesofDNAormolecules(eg,DNA,RNA,protein)byvisualiza-RNAintoone;therespectiveenzymesareDNAtionoftheireffectsonphotographicfilm.andRNAligases.Bacteriophage:Avirusthatinfectsabacterium.Lines:Longinterspersedrepeatsequences.Blunt-endedDNA:TwostrandsofaDNAduplexMicrosatellitepolymorphism:Heterozygosityofahavingendsthatareflushwitheachother.certainmicrosatelliterepeatinanindividual.cDNA:Asingle-strandedDNAmoleculethatisMicrosatelliterepeatsequences:DispersedorcomplementarytoanmRNAmoleculeandissyn-grouprepeatsequencesof2–5bprepeateduptothesizedfromitbytheactionofreversetranscrip-50times.Mayoccurat50–100thousandloca-tase.tionsinthegenome.Chimericmolecule:Amolecule(eg,DNA,RNA,Nicktranslation:AtechniqueforlabelingDNAprotein)containingsequencesderivedfromtwobasedontheabilityoftheDNApolymerasefromdifferentspecies.EcolitodegradeastrandofDNAthathasbeenClone:Alargenumberoforganisms,cellsormole-nickedandthentoresynthesizethestrand;ifara-culesthatareidenticalwithasingleparentalor-dioactivenucleosidetriphosphateisemployed,theganismcellormolecule.rebuiltstrandbecomeslabeledandcanbeusedasCosmid:AplasmidintowhichtheDNAsequencesaradioactiveprobe.frombacteriophagelambdathatarenecessaryforNorthernblot:AmethodfortransferringRNAthepackagingofDNA(cossites)havebeenin-fromanagarosegeltoanitrocellulosefilter,onserted;thispermitstheplasmidDNAtobepack-whichtheRNAcanbedetectedbyasuitableagedinvitro.probe.Endonuclease:AnenzymethatcleavesinternalOligonucleotide:Ashort,definedsequenceofnu-bondsinDNAorRNA.cleotidesjoinedtogetherinthetypicalphosphodi-Excinuclease:Theexcisionnucleaseinvolvedinnu-esterlinkage.cleotideexchangerepairofDNA.Ori:TheoriginofDNAreplication.Exon:ThesequenceofagenethatisrepresentedPAC:Ahighcapacity(70–95kb)cloningvector(expressed)asmRNA.baseduponthelyticE.colibacteriophageP1thatExonuclease:Anenzymethatcleavesnucleotidesreplicatesinbacteriaasanextrachromosomalele-fromeitherthe3′or5′endsofDNAorRNA.ment.Fingerprinting:TheuseofRFLPsorrepeatse-Palindrome:AsequenceofduplexDNAthatisthequenceDNAtoestablishauniquepatternofsamewhenthetwostrandsarereadinoppositedi-DNAfragmentsforanindividual.rections.Footprinting:DNAwithproteinboundisresistantPlasmid:Asmall,extrachromosomal,circularmole-todigestionbyDNaseenzymes.Whenasequenc-culeofDNAthatreplicatesindependentlyoftheingreactionisperformedusingsuchDNA,apro-hostDNA.tectedarea,representingthe“footprint”ofthePolymerasechainreaction(PCR):Anenzymaticboundprotein,willbedetected.methodfortherepeatedcopying(andthusampli-Hairpin:Adouble-helicalstretchformedbybasefication)ofthetwostrandsofDNAthatmakeuppairingbetweenneighboringcomplementaryse-aparticulargenesequence.
412414/CHAPTER40Primosome:ThemobilecomplexofhelicaseandSpliceosome:Themacromolecularcomplexrespon-primasethatisinvolvedinDNAreplication.sibleforprecursormRNAsplicing.Thespliceo-Probe:AmoleculeusedtodetectthepresenceofasomeconsistsofatleastfivesmallnuclearRNAsspecificfragmentofDNAorRNAin,forin-(snRNA;U1,U2,U4,U5,andU6)andmanystance,abacterialcolonythatisformedfromage-proteins.neticlibraryorduringanalysisbyblottransferSplicing:TheremovalofintronsfromRNAac-techniques;commonprobesarecDNAmolecules,companiedbythejoiningofitsexons.syntheticoligodeoxynucleotidesofdefinedse-Sticky-endedDNA:Complementarysinglestrandsquence,orantibodiestospecificproteins.ofDNAthatprotrudefromoppositeendsofaProteome:Theentirecollectionofexpressedpro-DNAduplexorfromtheendsofdifferentduplexteinsinanorganism.molecules(seealsoBlunt-endedDNA,above).Pseudogene:AninactivesegmentofDNAarisingTandem:Usedtodescribemultiplecopiesofthebymutationofaparentalactivegene.samesequence(eg,DNA)thatlieadjacenttooneRecombinantDNA:ThealteredDNAthatresultsanother.fromtheinsertionofasequenceofdeoxynu-Terminaltransferase:Anenzymethataddsnu-cleotidesnotpreviouslypresentintoanexistingcleotidesofonetype(eg,deoxyadenonucleotidylmoleculeofDNAbyenzymaticorchemicalresidues)tothe3′endofDNAstrands.means.Transcription:TemplateDNA-directedsynthesisRestrictionenzyme:Anendodeoxynucleasethatofnucleicacids;typicallyDNA-directedsynthesiscausescleavageofbothstrandsofDNAathighlyofRNA.specificsitesdictatedbythebasesequence.Transcriptome:TheentirecollectionofexpressedReversetranscription:RNA-directedsynthesisofmRNAsinanorganism.DNA,catalyzedbyreversetranscriptase.Transgenic:DescribingtheintroductionofnewRT-PCR:AmethodusedtoquantitatemRNAlev-DNAintogermcellsbyitsinjectionintothenu-elsthatreliesuponafirststepofcDNAcopyingofcleusoftheovum.mRNAspriortoPCRamplificationandquantita-Translation:SynthesisofproteinusingmRNAastion.template.Signal:TheendproductobservedwhenaspecificVector:Aplasmidorbacteriophageintowhichfor-sequenceofDNAorRNAisdetectedbyautoradi-eignDNAcanbeintroducedforthepurposesofographyorsomeothermethod.Hybridizationcloning.withacomplementaryradioactivepolynucleotideWesternblot:Amethodfortransferringproteinto(eg,bySouthernorNorthernblotting)iscom-anitrocellulosefilter,onwhichtheproteincanbemonlyusedtogeneratethesignal.detectedbyasuitableprobe(eg,anantibody).Sines:Shortinterspersedrepeatsequences.SNP:Singlenucleotidepolymorphism.ReferstothefactthatsinglenucleotidegeneticvariationinREFERENCESgenomesequenceexistsatdiscretelocithroughoutLewinB:GenesVII.OxfordUnivPress,1999.thechromosomes.MeasurementofallelicSNPMartinJB,GusellaJF:Huntington’sdisease:pathogenesisanddifferencesisusefulforgenemappingstudies.management.NEnglJMed1986:315:1267.snRNA:SmallnuclearRNA.ThisfamilyofRNAsSambrookJ,FritschEF,ManiatisT:MolecularCloning:ALabora-isbestknownforitsroleinmRNAprocessing.toryManual.ColdSpringHarborLaboratoryPress,1989.Southernblot:AmethodfortransferringDNASpectorDL,GoldmanRD,LeinwandLA:Cells:ALaboratoryfromanagarosegeltonitrocellulosefilter,onManual.ColdSpringHarborLaboratoryPress,1998.whichtheDNAcanbedetectedbyasuitableWatsonJDetal:RecombinantDNA,2nded.ScientificAmericanprobe(eg,complementaryDNAorRNA).Books.Freeman,1992.Southwesternblot:Amethodfordetectingpro-WeatherallDJ:TheNewGeneticsandClinicalPractice,3rded.Ox-tein-DNAinteractionsbyapplyingalabeledDNAfordUnivPress,1991.probetoatransfermembranethatcontainsarena-turedprotein.
413SECTIONVBiochemistryofExtracellular&IntracellularCommunicationMembranes:Structure&Function41RobertK.Murray,MD,PhD,&DarylK.Granner,MDBIOMEDICALIMPORTANCEMAINTENANCEOFANORMALINTRA-Membranesarehighlyviscous,plasticstructures.&EXTRACELLULARENVIRONMENTPlasmamembranesformclosedcompartmentsaroundISFUNDAMENTALTOLIFEcellularprotoplasmtoseparateonecellfromanotherLifeoriginatedinanaqueousenvironment;enzymere-andthuspermitcellularindividuality.Theplasmaactions,cellularandsubcellularprocesses,andsoforthmembranehasselectivepermeabilitiesandactsasahavethereforeevolvedtoworkinthismilieu.Sincebarrier,therebymaintainingdifferencesincompositionmammalsliveinagaseousenvironment,howisthebetweentheinsideandoutsideofthecell.Theselectiveaqueousstatemaintained?Membranesaccomplishthispermeabilitiesareprovidedmainlybychannelsandbyinternalizingandcompartmentalizingbodywater.pumpsforionsandsubstrates.Theplasmamembranealsoexchangesmaterialwiththeextracellularenviron-TheBody’sInternalWatermentbyexocytosisandendocytosis,andtherearespe-cialareasofmembranestructure—thegapjunctions—IsCompartmentalizedthroughwhichadjacentcellsexchangematerial.InWatermakesupabout60%oftheleanbodymassofaddition,theplasmamembraneplayskeyrolesincell-thehumanbodyandisdistributedintwolargecom-cellinteractionsandintransmembranesignaling.partments.Membranesalsoformspecializedcompartmentswithinthecell.SuchintracellularmembraneshelpA.INTRACELLULARFLUID(ICF)shapemanyofthemorphologicallydistinguishableThiscompartmentconstitutestwo-thirdsoftotalbodystructures(organelles),eg,mitochondria,ER,sarcoplas-waterandprovidestheenvironmentforthecell(1)tomicreticulum,Golgicomplexes,secretorygranules,make,store,andutilizeenergy;(2)torepairitself;lysosomes,andthenuclearmembrane.Membraneslo-(3)toreplicate;and(4)toperformspecialfunctions.calizeenzymes,functionasintegralelementsinexcita-tion-responsecoupling,andprovidesitesofenergyB.EXTRACELLULARFLUID(ECF)transduction,suchasinphotosynthesisandoxidativeThiscompartmentcontainsaboutone-thirdoftotalphosphorylation.bodywaterandisdistributedbetweentheplasmaandChangesinmembranestructure(egcausedbyis-interstitialcompartments.Theextracellularfluidisachemia)canaffectwaterbalanceandionfluxandthere-deliverysystem.Itbringstothecellsnutrients(eg,glu-foreeveryprocesswithinthecell.Specificdeficienciescose,fattyacids,aminoacids),oxygen,variousionsandoralterationsofcertainmembranecomponentsleadtotraceminerals,andavarietyofregulatorymoleculesavarietyofdiseases(seeTable41–5).Inshort,normal(hormones)thatcoordinatethefunctionsofwidelysep-cellularfunctiondependsonnormalmembranes.aratedcells.ExtracellularfluidremovesCO2,waste415
414416/CHAPTER41products,andtoxicordetoxifiedmaterialsfromtheim-mediatecellularenvironment.Myelin0.23TheIonicCompositionsofIntracellularMouse0.85&ExtracellularFluidsDifferGreatlylivercellsAsillustratedinTable41–1,theinternalenvironmentRetinalrodsisrichinK+andMg2+,andphosphateisitsmajor(bovine)1.0anion.ExtracellularfluidischaracterizedbyhighNa+andCa2+content,andCl−isthemajoranion.NoteHumanerythrocyte1.1alsothattheconcentrationofglucoseishigherinextra-Plasmamembranecellularfluidthaninthecell,whereastheoppositeistrueforproteins.Whyistheresuchadifference?ItisAmeba1.3thoughtthattheprimordialseainwhichlifeoriginatedwasrichinK+andMg2+.Itthereforefollowsthaten-zymereactionsandotherbiologicprocessesevolvedtoHeLacells1.5functionbestinthatenvironment—hencethehighconcentrationoftheseionswithincells.CellswereMitochondrial1.1facedwithstrongselectionpressureastheseagraduallyoutermembrane+2+changedtoacompositionrichinNaandCa.VastchangeswouldhavebeenrequiredforevolutionofaSarcoplasmicreticulum2.0completelynewsetofbiochemicalandphysiologicma-chinery;instead,asithappened,cellsdevelopedbarri-Mitochondrialers—membraneswithassociated“pumps”—tomain-innermembrane3.2taintheinternalmicroenvironment.01234MEMBRANESARECOMPLEXRatioofproteintolipidSTRUCTURESCOMPOSEDOFLIPIDS,Figure41–1.RatioofproteintolipidindifferentPROTEINS,&CARBOHYDRATESmembranes.ProteinsequalorexceedthequantityofWeshallmainlydiscussthemembranespresentineu-lipidinnearlyallmembranes.Theoutstandingexcep-karyoticcells,althoughmanyoftheprinciplesde-tionismyelin,anelectricalinsulatorfoundonmanyscribedalsoapplytothemembranesofprokaryotes.nervefibers.Thevariouscellularmembraneshavedifferentcompo-sitions,asreflectedintheratioofproteintolipid(Fig-ure41–1).Thisisnotsurprising,giventheirdivergentThesesheet-likestructuresarenoncovalentassembliesfunctions.Membranesareasymmetricsheet-likeen-thatarethermodynamicallystableandmetabolicallyac-closedstructureswithdistinctinnerandoutersurfaces.tive.Numerousproteinsarelocatedinmembranes,wheretheycarryoutspecificfunctionsoftheorganelle,thecell,ortheorganism.Table41–1.ComparisonofthemeanconcentrationsofvariousmoleculesoutsideandTheMajorLipidsinMammalianinsideamammaliancell.MembranesArePhospholipids,Glycosphingolipids,&CholesterolSubstanceExtracellularFluidIntracellularFluidA.PHOSPHOLIPIDSNa+140mmol/L10mmol/LOfthetwomajorphospholipidclassespresentinmem-K+4mmol/L140mmol/Lbranes,phosphoglyceridesarethemorecommonand2+Ca(free)2.5mmol/L0.1μmol/Lconsistofaglycerolbackbonetowhichareattached2+Mg1.5mmol/L30mmol/Ltwofattyacidsinesterlinkageandaphosphorylatedal-−CI100mmol/L4mmol/Lcohol(Figure41–2).Thefattyacidconstituentsare−HCO327mmol/L10mmol/Lusuallyeven-numberedcarbonmolecules,mostcom-3−PO42mmol/L60mmol/Lmonlycontaining16or18carbons.Theyareun-Glucose5.5mmol/L0–1mmol/Lbranchedandcanbesaturatedorunsaturated.Thesim-Protein2g/dL16g/dLplestphosphoglycerideisphosphatidicacid,whichis
415MEMBRANES:STRUCTURE&FUNCTION/417FattyacidsEacheukaryoticcellmembranehasasomewhatdif-ferentlipidcomposition,thoughphospholipidsaretheOmajorclassinall.RCO1CH12MembraneLipidsAreAmphipathicRCO2CHO–2Allmajorlipidsinmembranescontainbothhydropho-3CHRO2OPO3bicandhydrophilicregionsandarethereforetermedO“amphipathic.”Membranesthemselvesarethusam-phipathic.IfthehydrophobicregionswereseparatedGlycerolAlcoholfromtherestofthemolecule,itwouldbeinsolubleinwaterbutsolubleinoil.Conversely,ifthehydrophilicFigure41–2.Aphosphoglycerideshowingthefattyregionwereseparatedfromtherestofthemolecule,itacids(R1andR2),glycerol,andphosphorylatedalcoholwouldbeinsolubleinoilbutsolubleinwater.Theam-components.Inphosphatidicacid,R3ishydrogen.phipathicnatureofaphospholipidisrepresentedinFigure41–3.Thus,thepolarheadgroupsofthephos-pholipidsandthehydroxylgroupofcholesterolinter-1,2-diacylglycerol3-phosphate,akeyintermediateinfacewiththeaqueousenvironment;asimilarsituationtheformationofallotherphosphoglycerides(ChapterappliestothesugarmoietiesoftheGSLs(seebelow).24).Inotherphosphoglycerides,the3-phosphateises-Saturatedfattyacidshavestraighttails,whereasterifiedtoanalcoholsuchasethanolamine,choline,unsaturatedfattyacids,whichgenerallyexistinthecisserine,glycerol,orinositol(Chapter14).forminmembranes,makekinkedtails(Figure41–3).Thesecondmajorclassofphospholipidsiscom-Asmorekinksareinsertedinthetails,themembraneposedofsphingomyelin,whichcontainsasphingosinebecomeslesstightlypackedandthereforemorefluid.backboneratherthanglycerol.AfattyacidisattachedDetergentsareamphipathicmoleculesthatareimpor-byanamidelinkagetotheaminogroupofsphingosine,tantinbiochemistryaswellasinthehousehold.Theformingceramide.Theprimaryhydroxylgroupofmolecularstructureofadetergentisnotunlikethatofasphingosineisesterifiedtophosphorylcholine.Sphin-phospholipid.Certaindetergentsarewidelyusedtosol-gomyelin,asthenameimplies,isprominentinmyelinubilizemembraneproteinsasafirststepintheirpurifi-sheaths.cation.ThehydrophobicendofthedetergentbindstoTheamountsandfattyacidcompositionsofthevar-iousphospholipidsvaryamongthedifferentcellularmembranes.B.GLYCOSPHINGOLIPIDSPolarheadgroupTheglycosphingolipids(GSLs)aresugar-containinglipidsbuiltonabackboneofceramide;theyincludegalactosyl-andglucosylceramide(cerebrosides)andthegangliosides.TheirstructuresaredescribedinChapter14.Theyaremainlylocatedintheplasmamembranesofcells.Apolar,hydrocarbontailsC.STEROLSThemostcommonsterolinmembranesischolesterol(Chapter14),whichresidesmainlyintheplasmamem-branesofmammaliancellsbutcanalsobefoundinSSUSlesserquantitiesinmitochondria,Golgicomplexes,andFigure41–3.Diagrammaticrepresentationofanuclearmembranes.Cholesterolintercalatesamongthephospholipidsofthemembrane,withitshydroxylphospholipidorothermembranelipid.Thepolarheadgroupattheaqueousinterfaceandtheremainderofthegroupishydrophilic,andthehydrocarbontailsarehy-moleculewithintheleaflet.Itseffectonthefluidityofdrophobicorlipophilic.Thefattyacidsinthetailsaremembranesisdiscussedsubsequently.saturated(S)orunsaturated(U);theformerareusuallyAlloftheabovelipidscanbeseparatedfromonean-attachedtocarbon1ofglycerolandthelattertocar-otherbytechniquessuchascolumn,thinlayer,andbon2.Notethekinkinthetailoftheunsaturatedfattygas-liquidchromatographyandtheirstructuresestab-acid(U),whichisimportantinconferringincreasedlishedbymassspectrometry.membranefluidity.
416418/CHAPTER41hydrophobicregionsoftheproteins,displacingmostofAqueoustheirboundlipids.ThepolarendofthedetergentisHydro-free,bringingtheproteinsintosolutionasdetergent-philicproteincomplexes,usuallyalsocontainingsomeresid-uallipids.Hydro-phobicMembraneLipidsFormBilayersTheamphipathiccharacterofphospholipidssuggestsHydro-thatthetworegionsofthemoleculehaveincompatiblephilicsolubilities;however,inasolventsuchaswater,phos-Aqueouspholipidsorganizethemselvesintoaformthatthermo-dynamicallyservesthesolubilityrequirementsofbothFigure41–5.Diagramofasectionofabilayermem-regions.Amicelle(Figure41–4)issuchastructure;braneformedfromphospholipidmolecules.Theunsat-thehydrophobicregionsareshieldedfromwater,whileuratedfattyacidtailsarekinkedandleadtomorespac-thehydrophilicpolargroupsareimmersedintheaque-ingbetweenthepolarheadgroups,hencetomoreousenvironment.However,micellesareusuallyrela-roomformovement.Thisinturnresultsinincreasedtivelysmallinsize(eg,approximately200nm)andmembranefluidity.(Slightlymodifiedandreproduced,thusarelimitedintheirpotentialtoformmembranes.withpermission,fromStryerL:Biochemistry,2nded.Free-Aswasrecognizedin1925byGorterandGrendel,aman,1981.)bimolecularlayer,orlipidbilayer,canalsosatisfythethermodynamicrequirementsofamphipathicmole-culesinanaqueousenvironment.Bilayers,notmi-favorableenvironment,buteventheseexposededgescelles,areindeedthekeystructuresinbiologicmem-canbeeliminatedbyfoldingthesheetbackuponitselfbranes.Abilayerexistsasasheetinwhichthetoformanenclosedvesiclewithnoedges.Abilayercanhydrophobicregionsofthephospholipidsareprotectedextendoverrelativelylargedistances(eg,1mm).Thefromtheaqueousenvironment,whilethehydrophilicclosedbilayerprovidesoneofthemostessentialproper-regionsareimmersedinwater(Figure41–5).Onlythetiesofmembranes.Itisimpermeabletomostwater-endsoredgesofthebilayersheetareexposedtoanun-solublemolecules,sincetheywouldbeinsolubleinthehydrophobiccoreofthebilayer.Lipidbilayersareformedbyself-assembly,drivenbythehydrophobiceffect.Whenlipidmoleculescometogetherinabilayer,theentropyofthesurround-ingsolventmoleculesincreases.Twoquestionsarisefromconsiderationoftheabove.First,howmanybiologicmaterialsarelipid-solubleandcanthereforereadilyenterthecell?Gasessuchasoxygen,CO2,andnitrogen—smallmoleculeswithlittleinteractionwithsolvents—readilydiffusethroughthehydrophobicregionsofthemembrane.ThepermeabilitycoefficientsofseveralionsandofanumberofothermoleculesinalipidbilayerareshowninFigure41–6.Thethreeelectrolytesshown(Na+,K+,−andCl)crossthebilayermuchmoreslowlythanwater.Ingeneral,thepermeabilitycoefficientsofsmallmoleculesinalipidbilayercorrelatewiththeirsolubili-tiesinnonpolarsolvents.Forinstance,steroidsmorereadilytraversethelipidbilayercomparedwithelec-trolytes.Thehighpermeabilitycoefficientofwaterit-Figure41–4.Diagrammaticcross-sectionofami-selfissurprisingbutispartlyexplainedbyitssmallsizecelle.Thepolarheadgroupsarebathedinwater,andrelativelackofcharge.whereasthehydrophobichydrocarbontailsaresur-Thesecondquestionconcernsmoleculesthatareroundedbyotherhydrocarbonsandtherebypro-notlipid-soluble:Howarethetransmembraneconcen-tectedfromwater.Micellesarerelativelysmall(com-trationgradientsfornon-lipid-solublemoleculesmain-paredwithlipidbilayers)sphericalstructures.tained?Theansweristhatmembranescontainproteins,
417MEMBRANES:STRUCTURE&FUNCTION/419K+TryptophanIndoleuesfortheirtransferfromtheinteriorofamembraneNa+Cl–GlucoseUrea,H2Otowater.Hydrophobicaminoacidshavepositiveval-glycerolues;polaraminoacidshavenegativevalues.Thetotalfreeenergyvaluesfortransferringsuccessivesequencesof20aminoacidsintheproteinareplotted,yieldinga−1so-calledhydropathyplot.Valuesofover20kcal⋅mol10–1410–1210–1010–810–610–410–2areconsistentwith—butdonotprove—atransmem-Permeabilitycoefficient(cm/s)branelocation.LowHighAnotheraspectoftheinteractionoflipidsandpro-PermeabilityteinsisthatsomeproteinsareanchoredtooneleafletoranotherofthebilayerbycovalentlinkagestocertainFigure41–6.Permeabilitycoefficientsofwater,lipids.Palmitateandmyristatearefattyacidsinvolvedsomeions,andothersmallmoleculesinlipidbilayerinsuchlinkagestospecificproteins.Anumberofothermembranes.Moleculesthatmoverapidlythroughaproteins(seeChapter47)arelinkedtoglycophos-givenmembranearesaidtohaveahighpermeabilityphatidylinositol(GPI)structures.coefficient.(Slightlymodifiedandreproduced,withper-mission,fromStryerL:Biochemistry,2nded.Freeman,DifferentMembranesHaveDifferent1981.)ProteinCompositionsThenumberofdifferentproteinsinamembraneandproteinsarealsoamphipathicmoleculesthatcanbevariesfromlessthanadozeninthesarcoplasmicreticu-insertedintothecorrespondinglyamphipathiclipidbi-lumtoover100intheplasmamembrane.Mostmem-layer.Proteinsformchannelsforthemovementofionsbraneproteinscanbeseparatedfromoneanotherusingandsmallmoleculesandserveastransportersforlargersodiumdodecylsulfatepolyacrylamidegelelectro-moleculesthatotherwisecouldnotpassthebilayer.phoresis(SDS-PAGE),atechniquethathasrevolution-Theseprocessesaredescribedbelow.izedtheirstudy.IntheabsenceofSDS,fewmembraneproteinswouldremainsolubleduringelectrophoresis.MembraneProteinsAreAssociatedProteinsarethemajorfunctionalmoleculesofmem-WiththeLipidBilayerbranesandconsistofenzymes,pumpsandchannels,structuralcomponents,antigens(eg,forhistocompati-Membranephospholipidsactasasolventformem-bility),andreceptorsforvariousmolecules.Becausebraneproteins,creatinganenvironmentinwhichtheeverymembranepossessesadifferentcomplementoflattercanfunction.Ofthe20aminoacidscontributingproteins,thereisnosuchthingasatypicalmembranetotheprimarystructureofproteins,thefunctionalstructure.Theenzymaticpropertiesofseveraldifferentgroupsattachedtotheαcarbonarestronglyhydropho-membranesareshowninTable41–2.bicinsix,weaklyhydrophobicinafew,andhy-drophilicintheremainder.AsdescribedinChapter5,MembranesAreDynamicStructurestheα-helicalstructureofproteinsminimizesthehy-drophiliccharacterofthepeptidebondsthemselves.Membranesandtheircomponentsaredynamicstruc-Thus,proteinscanbeamphipathicandformaninte-tures.Thelipidsandproteinsinmembranesundergogralpartofthemembranebyhavinghydrophilicre-turnovertherejustastheydoinothercompartmentsofgionsprotrudingattheinsideandoutsidefacesofthethecell.Differentlipidshavedifferentturnoverrates,membranebutconnectedbyahydrophobicregiontra-andtheturnoverratesofindividualspeciesofmem-versingthehydrophobiccoreofthebilayer.Infact,braneproteinsmayvarywidely.Themembraneitselfthoseportionsofmembraneproteinsthattraversecanturnoverevenmorerapidlythananyofitscon-membranesdocontainsubstantialnumbersofhy-stituents.Thisisdiscussedinmoredetailinthesectiondrophobicaminoacidsandalmostinvariablyhaveei-onendocytosis.therahighα-helicalorβ-pleatedsheetcontent.Formanymembranes,astretchofapproximately20aminoMembranesAreAsymmetricStructuresacidsinanαhelixwillspanthebilayer.Itispossibletocalculatewhetheraparticularse-Thisasymmetrycanbepartiallyattributedtotheirreg-quenceofaminoacidspresentinaproteinisconsistentulardistributionofproteinswithinthemembranes.Anwithatransmembranelocation.Thiscanbedonebyinside-outsideasymmetryisalsoprovidedbytheex-consultingatablethatliststhehydrophobicitiesofeachternallocationofthecarbohydratesattachedtomem-ofthe20commonaminoacidsandthefreeenergyval-braneproteins.Inaddition,specificenzymesarelo-
418420/CHAPTER41presentinthetwoleaflets,contributingtotheasym-Table41–2.Enzymaticmarkersofdifferentmetricdistributionoftheselipidmolecules.Inaddi-1membranes.tion,phospholipidexchangeproteinsrecognizespecificphospholipidsandtransferthemfromonemembraneMembraneEnzyme(eg,theendoplasmicreticulum[ER])toothers(eg,mi-tochondrialandperoxisomal).Thereisfurtherasym-Plasma5’-NucleotidaseAdenylylcyclasemetrywithregardtoGSLsandalsoglycoproteins;theNa+-K+ATPasesugarmoietiesofthesemoleculesallprotrudeoutwardfromtheplasmamembraneandareabsentfromitsEndoplasmicreticulumGlucose-6-phosphataseinnerface.GolgiapparatusCisGlcNActransferaseIMembranesContainIntegralMedialGolgimannosidaseII&PeripheralProteinsTransGalactosyltransferase(Figure41–7)TGNSialyltransferaseItisusefultoclassifymembraneproteinsintotwoInnermitochondrialmembraneATPsynthasetypes:integralandperipheral.Mostmembranepro-1Membranescontainmanyproteins,someofwhichhaveenzy-teinsfallintotheintegralclass,meaningthattheyinter-maticactivity.Someoftheseenzymesarelocatedonlyincertainactextensivelywiththephospholipidsandrequirethemembranesandcanthereforebeusedasmarkerstofollowtheuseofdetergentsfortheirsolubilization.Also,theygen-purificationofthesemembranes.erallyspanthebilayer.IntegralproteinsareusuallyTGN,transgolginetwork.globularandarethemselvesamphipathic.Theyconsistoftwohydrophilicendsseparatedbyaninterveninghy-catedexclusivelyontheoutsideorinsideofmem-drophobicregionthattraversesthehydrophobiccoreofbranes,asinthemitochondrialandplasmamembranes.thebilayer.Asthestructuresofintegralmembranepro-Thereareregionalasymmetriesinmembranes.teinswerebeingelucidated,itbecameapparentthatSome,suchasoccuratthevillousbordersofmucosalcertainones(eg,transportermolecules,variousrecep-cells,arealmostmacroscopicallyvisible.Others,suchastors,andGproteins)spanthebilayermanytimes(seethoseatgapjunctions,tightjunctions,andsynapses,Figure46–5).Integralproteinsarealsoasymmetricallyoccupymuchsmallerregionsofthemembraneanddistributedacrossthemembranebilayer.Thisasym-generatecorrespondinglysmallerlocalasymmetries.metricorientationisconferredatthetimeoftheirin-Thereisalsoinside-outside(transverse)asymmetrysertioninthelipidbilayer.Thehydrophilicexternalre-ofthephospholipids.Thecholine-containingphos-gionofanamphipathicprotein,whichissynthesizedpholipids(phosphatidylcholineandsphingomyelin)onpolyribosomes,musttraversethehydrophobiccorearelocatedmainlyintheoutermolecularlayer;theofitstargetmembraneandeventuallybefoundontheaminophospholipids(phosphatidylserineandphos-outsideofthatmembrane.Themolecularmechanismsphatidylethanolamine)arepreferentiallylocatedintheinvolvedininsertionofproteinsintomembranesandinnerleaflet.Obviously,ifthisasymmetryistoexistatthetopicofmembraneassemblyarediscussedinChap-all,theremustbelimitedtransversemobility(flip-flop)ter46.ofthemembranephospholipids.Infact,phospholipidsPeripheralproteinsdonotinteractdirectlywithinsyntheticbilayersexhibitanextraordinarilyslowthephospholipidsinthebilayerandthusdonotrequirerateofflip-flop;thehalf-lifeoftheasymmetrycanbeuseofdetergentsfortheirrelease.Theyareweaklymeasuredinseveralweeks.However,whencertainboundtothehydrophilicregionsofspecificintegralmembraneproteinssuchastheerythrocyteproteingly-proteinsandcanbereleasedfromthembytreatmentcophorinareinsertedartificiallyintosyntheticbilayers,withsaltsolutionsofhighionicstrength.Forexample,thefrequencyofphospholipidflip-flopmayincreaseasankyrin,aperipheralprotein,isboundtotheintegralmuchas100-fold.protein“band3”oferythrocytemembrane.Spectrin,aThemechanismsinvolvedintheestablishmentofcytoskeletalstructurewithintheerythrocyte,isinturnlipidasymmetryarenotwellunderstood.Theenzymesboundtoankyrinandtherebyplaysanimportantroleinvolvedinthesynthesisofphospholipidsarelocatedinmaintenanceofthebiconcaveshapeoftheerythro-onthecytoplasmicsideofmicrosomalmembranevesi-cyte.Manyhormonereceptormoleculesareintegralcles.Translocases(flippases)existthattransfercertainproteins,andthespecificpolypeptidehormonesthatphospholipids(eg,phosphatidylcholine)fromtheinnerbindtothesereceptormoleculesmaythereforebecon-totheouterleaflet.Specificproteinsthatpreferen-sideredperipheralproteins.Peripheralproteins,suchastiallybindindividualphospholipidsalsoappeartobepolypeptidehormones,mayhelporganizethedistribu-
419MEMBRANES:STRUCTURE&FUNCTION/421Figure41–7.Thefluidmosaicmodelofmembranestructure.Themembraneconsistsofabimolecu-larlipidlayerwithproteinsinsertedinitorboundtoeithersurface.Integralmembraneproteinsarefirmlyembeddedinthelipidlayers.Someoftheseproteinscompletelyspanthebilayerandarecalledtransmembraneproteins,whileothersareembeddedineithertheouterorinnerleafletofthelipidbi-layer.Looselyboundtotheouterorinnersurfaceofthemembranearetheperipheralproteins.Manyoftheproteinsandlipidshaveexternallyexposedoligosaccharidechains.(Reproduced,withpermission,fromJunqueiraLC,CarneiroJ:BasicHistology:Text&Atlas,10thed.McGraw-Hill,2003.)tionofintegralproteins,suchastheirreceptors,withincompositiontopermitsystematicexaminationofthetheplaneofthebilayer(seebelow).effectsoffattyacidcompositiononcertainmembranefunctions(eg,transport).(2)PurifiedmembraneproteinsorenzymescanbeARTIFICIALMEMBRANESMODELincorporatedintothesevesiclesinordertoassesswhatMEMBRANEFUNCTIONfactors(eg,specificlipidsorancillaryproteins)thepro-teinsrequiretoreconstitutetheirfunction.Investiga-Artificialmembranesystemscanbepreparedbyappro-2+tionsofpurifiedproteins,eg,theCaATPaseofthepriatetechniques.Thesesystemsgenerallyconsistofsarcoplasmicreticulum,haveincertaincasessuggestedmixturesofoneormorephospholipidsofnaturalorthatonlyasingleproteinandasinglelipidarerequiredsyntheticoriginthatcanbetreated(eg,byusingmildtoreconstituteanionpump.sonication)toformsphericalvesiclesinwhichthelipids(3)Theenvironmentofthesesystemscanberigidlyformabilayer.Suchvesicles,surroundedbyalipidbi-controlledandsystematicallyvaried(eg,ionconcentra-layer,aretermedliposomes.tions).Thesystemscanalsobeexposedtoknownlig-Someoftheadvantagesandusesofartificialmem-andsif,forexample,theliposomescontainspecificre-branesystemsinthestudyofmembranefunctioncanceptorproteins.bebrieflyexplained.(4)Whenliposomesareformed,theycanbemade(1)Thelipidcontentofthemembranescanbevar-toentrapcertaincompoundsinsidethemselves,eg,ied,allowingsystematicexaminationoftheeffectsofdrugsandisolatedgenes.Thereisinterestinusinglipo-varyinglipidcompositiononcertainfunctions.Forin-somestodistributedrugstocertaintissues,andifcom-stance,vesiclescanbemadethatarecomposedsolelyofponents(eg,antibodiestocertaincellsurfacemole-phosphatidylcholineor,alternatively,ofknownmix-cules)couldbeincorporatedintoliposomessothattheyturesofdifferentphospholipids,glycolipids,andcho-wouldbetargetedtospecifictissuesortumors,thelesterol.Thefattyacidmoietiesofthelipidsusedcantherapeuticimpactwouldbeconsiderable.DNAen-alsobevariedbyemployingsyntheticlipidsofknowntrappedinsideliposomesappearstobelesssensitiveto
420422/CHAPTER41attackbynucleases;thisapproachmayproveusefulinhighcholesterol:phospholipidratios,transitiontemper-attemptsatgenetherapy.aturesarealtogetherindistinguishable.Thefluidityofamembranesignificantlyaffectsitsfunctions.Asmembranefluidityincreases,sodoesitsTHEFLUIDMOSAICMODELpermeabilitytowaterandothersmallhydrophilicmol-OFMEMBRANESTRUCTUREecules.Thelateralmobilityofintegralproteinsin-ISWIDELYACCEPTEDcreasesasthefluidityofthemembraneincreases.IftheactivesiteofanintegralproteininvolvedinagivenThefluidmosaicmodelofmembranestructurepro-functionisexclusivelyinitshydrophilicregions,chang-posedin1972bySingerandNicolson(Figure41–7)isinglipidfluiditywillprobablyhavelittleeffectonthenowwidelyaccepted.Themodelisoftenlikenedtoice-activityoftheprotein;however,iftheproteinisin-bergs(membraneproteins)floatinginaseaofpredomi-volvedinatransportfunctioninwhichtransportcom-nantlyphospholipidmolecules.Earlyevidencefortheponentsspanthemembrane,lipidphaseeffectsmaymodelwasthefindingthatcertainspecies-specificinte-significantlyalterthetransportrate.Theinsulinrecep-gralproteins(detectedbyfluorescentlabelingtech-torisanexcellentexampleofalteredfunctionwithniques)rapidlyandrandomlyredistributedinthechangesinfluidity.Astheconcentrationofunsaturatedplasmamembraneofaninterspecieshybridcellformedfattyacidsinthemembraneisincreased(bygrowingbytheartificiallyinducedfusionoftwodifferentparentculturedcellsinamediumrichinsuchmolecules),flu-cells.Ithassubsequentlybeendemonstratedthatphos-idityincreases.Thisaltersthereceptorsothatitbindspholipidsalsoundergorapidredistributionintheplanemoreinsulin.ofthemembrane.ThisdiffusionwithintheplaneofAstateoffluidityandthusoftranslationalmobilitythemembrane,termedtranslationaldiffusion,canbeinamembranemaybeconfinedtocertainregionsofquiterapidforaphospholipid;infact,withintheplanemembranesundercertainconditions.Forexample,ofthemembrane,onemoleculeofphospholipidcanprotein-proteininteractionsmaytakeplacewithinthemoveseveralmicrometerspersecond.planeofthemembrane,suchthattheintegralproteinsThephasechanges—andthusthefluidityofmem-formarigidmatrix—incontrasttothemoreusualsitu-branes—arelargelydependentuponthelipidcomposi-ation,wherethelipidactsasthematrix.Suchregionstionofthemembrane.Inalipidbilayer,thehydropho-ofrigidproteinmatrixcanexistsidebysideinthesamebicchainsofthefattyacidscanbehighlyalignedormembranewiththeusuallipidmatrix.Gapjunctionsorderedtoprovidearatherstiffstructure.Asthetem-andtightjunctionsareclearexamplesofsuchside-by-peratureincreases,thehydrophobicsidechainsundergosidecoexistenceofdifferentmatrices.atransitionfromtheorderedstate(moregel-likeorcrystallinephase)toadisorderedone,takingonamoreLipidRafts&CaveolaeAreSpecialliquid-likeorfluidarrangement.ThetemperatureatFeaturesofSomeMembraneswhichthestructureundergoesthetransitionfromor-deredtodisordered(ie,melts)iscalledthe“transitionWhilethefluidmosaicmodelofmembranestructuretemperature”(Tm).Thelongerandmoresaturatedhasstoodupwelltodetailedscrutiny,additionalfea-fattyacidchainsinteractmorestronglywitheachotherturesofmembranestructureandfunctionarecon-viatheirlongerhydrocarbonchainsandthuscausestantlyemerging.TwostructuresofparticularcurrenthighervaluesofTm—ie,highertemperaturesarere-interest,locatedinsurfacemembranes,arelipidraftsquiredtoincreasethefluidityofthebilayer.Ontheandcaveolae.Theformeraredynamicareasoftheexo-otherhand,unsaturatedbondsthatexistintheciscon-plasmicleafletofthelipidbilayerenrichedincholes-figurationtendtoincreasethefluidityofabilayerbyde-terolandsphingolipids;theyareinvolvedinsignalcreasingthecompactnessofthesidechainpackingwith-transductionandpossiblyotherprocesses.Caveolaeoutdiminishinghydrophobicity(Figure41–3).Themayderivefromlipidrafts.Manyifnotallofthemphospholipidsofcellularmembranesgenerallycontaincontaintheproteincaveolin-1,whichmaybeinvolvedatleastoneunsaturatedfattyacidwithatleastonecisintheirformationfromrafts.Caveolaeareobservabledoublebond.byelectronmicroscopyasflask-shapedindentationsofCholesterolmodifiesthefluidityofmembranes.Atthecellmembrane.Proteinsdetectedincaveolaein-temperaturesbelowtheTm,itinterfereswiththeinter-cludevariouscomponentsofthesignal-transductionactionofthehydrocarbontailsoffattyacidsandthussystem(eg,theinsulinreceptorandsomeGproteins),increasesfluidity.AttemperaturesabovetheTm,itlim-thefolatereceptor,andendothelialnitricoxidesyn-itsdisorderbecauseitismorerigidthanthehydrocar-thase(eNOS).Caveolaeandlipidraftsareactiveareasbontailsofthefattyacidsandcannotmoveintheofresearch,andideasconcerningthemandtheirpossi-membranetothesameextent,thuslimitingfluidity.Atblerolesinvariousdiseasesarerapidlyevolving.
421MEMBRANES:STRUCTURE&FUNCTION/423MEMBRANESELECTIVITYALLOWSinverselyproportionatetothenumberofhydrogenSPECIALIZEDFUNCTIONSbondsthatmustbebrokeninorderforasoluteintheexternalaqueousphasetobecomeincorporatedintheIftheplasmamembraneisrelativelyimpermeable,howhydrophobicbilayer.Electrolytes,poorlysolubleindomostmoleculesenteracell?Howisselectivityoflipid,donotformhydrogenbondswithwater,buttheythismovementestablished?Answerstosuchquestionsdoacquireashellofwaterfromhydrationbyelectrosta-areimportantinunderstandinghowcellsadjusttoaticinteraction.Thesizeoftheshellisdirectlypropor-constantlychangingextracellularenvironment.Meta-tionatetothechargedensityoftheelectrolyte.Elec-zoanorganismsalsomusthavemeansofcommunicat-trolyteswithalargechargedensityhavealargershellofingbetweenadjacentanddistantcells,sothatcomplexhydrationandthusaslowerdiffusionrate.Na+,forex-biologicprocessescanbecoordinated.Thesesignalsample,hasahigherchargedensitythanK+.Hydratedmustarriveatandbetransmittedbythemembrane,orNa+isthereforelargerthanhydratedK+;hence,thelat-theymustbegeneratedasaconsequenceofsomeinter-tertendstomovemoreeasilythroughthemembrane.actionwiththemembrane.Someofthemajormecha-Thefollowingfactorsaffectnetdiffusionofasub-nismsusedtoaccomplishthesedifferentobjectivesarestance:(1)Itsconcentrationgradientacrossthemem-listedinTable41–3.brane.Solutesmovefromhightolowconcentration.(2)Theelectricalpotentialacrossthemembrane.PassiveMechanismsMoveSomeSmallSolutesmovetowardthesolutionthathastheoppositeMoleculesAcrossMembranescharge.Theinsideofthecellusuallyhasanegativecharge.(3)Thepermeabilitycoefficientofthesub-Moleculescanpassivelytraversethebilayerdownelec-stanceforthemembrane.(4)Thehydrostaticpressuretrochemicalgradientsbysimplediffusionorbyfacili-gradientacrossthemembrane.Increasedpressurewilltateddiffusion.Thisspontaneousmovementtowardincreasetherateandforceofthecollisionbetweentheequilibriumcontrastswithactivetransport,whichre-moleculesandthemembrane.(5)Temperature.In-quiresenergybecauseitconstitutesmovementagainstcreasedtemperaturewillincreaseparticlemotionandanelectrochemicalgradient.Figure41–8providesathusincreasethefrequencyofcollisionsbetweenexter-schematicrepresentationofthesemechanisms.nalparticlesandthemembrane.Inaddition,amulti-Asdescribedabove,somesolutessuchasgasescantudeofchannelsexistinmembranesthatroutetheenterthecellbydiffusingdownanelectrochemicalgra-entryofionsintocells.dientacrossthemembraneanddonotrequiremeta-bolicenergy.ThesimplepassivediffusionofasoluteacrossthemembraneislimitedbythethermalagitationIonChannelsAreTransmembraneofthatspecificmolecule,bytheconcentrationgradientProteinsThatAllowtheSelectiveacrossthemembrane,andbythesolubilityofthatEntryofVariousIonssolute(thepermeabilitycoefficient,Figure41–6)intheInnaturalmembranes,asopposedtosyntheticmem-hydrophobiccoreofthemembranebilayer.Solubilityisbranebilayers,therearetransmembranechannels,pore-likestructurescomposedofproteinsthatconstitutese-lectiveionchannels.Cation-conductivechannelshaveTable41–3.Transferofmaterialandinformationanaveragediameterofabout5–8nmandarenegativelyacrossmembranes.chargedwithinthechannel.Thepermeabilityofachan-neldependsuponthesize,theextentofhydration,andCross-membranemovementofsmallmoleculestheextentofchargedensityontheion.Specificchan-nelsforNa+,K+,Ca2+,andCl−havebeenidentified;twoDiffusion(passiveandfacilitated)ActivetransportsuchchannelsareillustratedinFigure41–9.BothareCross-membranemovementoflargemoleculesseentoconsistoffoursubunits.EachsubunitconsistsofEndocytosissixα-helicaltransmembranedomains.TheaminoandExocytosiscarboxylterminalsofbothionchannelsarelocatedinSignaltransmissionacrossmembranesthecytoplasm,withbothextracellularandintracellularCellsurfacereceptorsloopsbeingpresent.Theactualporesinthechannels1.Signaltransduction(eg,glucagon→cAMP)throughwhichtheionspassarenotshowninthefigure.2.Signalinternalization(coupledwithendocytosis,eg,Theyformthecenter(diameterabout5–8nm)ofatheLDLreceptor)structureformedbyappositionofthesubunits.TheMovementtointracellularreceptors(steroidhormones;achannelsareveryselective,inmostcasespermittingtheformofdiffusion)passageofonlyonetypeofion(Na+,Ca2+,etc).ManyIntercellularcontactandcommunicationvariationsontheabovestructuralthemesarefound,but
422424/CHAPTER41TransportedmoleculeCarrierChannelproteinproteinLipidElectrochemicalbilayergradientEnergySimpleFacilitateddiffusiondiffusionPassivetransportActivetransportFigure41–8.Manysmallunchargedmoleculespassfreelythroughthelipidbilayer.Chargedmolecules,largerunchargedmolecules,andsomesmallun-chargedmoleculesaretransferredthroughchannelsorporesorbyspecificcarrierproteins.Passivetransportisalwaysdownanelectrochemicalgradient,towardequilibrium.Activetransportisagainstanelectrochemicalgradientandrequiresaninputofenergy,whereaspassivetransportdoesnot.(Redrawnandreproduced,withpermission,fromAlbertsBetal:MolecularBiologyoftheCell.Garland,1983.)allionchannelsarebasicallymadeupoftransmembraneTable41–4;otheraspectsofionchannelsarediscussedsubunitsthatcometogethertoformacentralporebrieflyinChapter49.throughwhichionspassselectively.Thecombinationofx-raycrystallography(wherepossible)andsite-directedIonophoresAreMoleculesThatActasmutagenesisaffordsapowerfulapproachtodelineatingMembraneShuttlesforVariousIonsthestructure-functionrelationshipsofionchannels.Themembranesofnervecellscontainwell-studiedCertainmicrobessynthesizesmallorganicmolecules,ionchannelsthatareresponsiblefortheactionpoten-ionophores,thatfunctionasshuttlesforthemovementtialsgeneratedacrossthemembrane.Theactivityofofionsacrossmembranes.Theseionophorescontainhy-someofthesechannelsiscontrolledbyneurotransmit-drophiliccentersthatbindspecificionsandaresur-ters;hence,channelactivitycanberegulated.Oneionroundedbyperipheralhydrophobicregions;thisarrange-canregulatetheactivityofthechannelofanotherion.mentallowsthemoleculestodissolveeffectivelyinthe2+Forexample,adecreaseofCaconcentrationinthemembraneanddiffusetransverselytherein.Others,likeextracellularfluidincreasesmembranepermeabilityandthewell-studiedpolypeptidegramicidin,formchannels.increasesthediffusionofNa+.ThisdepolarizestheMicrobialtoxinssuchasdiphtheriatoxinandacti-membraneandtriggersnervedischarge,whichmayex-vatedserumcomplementcomponentscanproducelargeplainthenumbness,tingling,andmusclecrampssymp-poresincellularmembranesandtherebyprovidemacro-2+tomaticofalowlevelofplasmaCa.moleculeswithdirectaccesstotheinternalmilieu.Channelsareopentransientlyandthusare“gated.”Gatescanbecontrolledbyopeningorclosing.Inlig-AquaporinsAreProteinsThatFormWaterand-gatedchannels,aspecificmoleculebindstoare-ChannelsinCertainMembranesceptorandopensthechannel.Voltage-gatedchannelsopen(orclose)inresponsetochangesinmembranepo-Incertaincells(eg,redcells,cellsofthecollectingduc-tential.Somepropertiesofionchannelsarelistedintulesofthekidney),themovementofwaterbysimple
423MEMBRANES:STRUCTURE&FUNCTION/425RatbrainNa+channelIIIIIIIVOutside123456123456123456123456InsideCNRabbitskeletalmuscleCa2+channelIIIIIIIV123456123456123456123456Figure41–9.Diagrammaticrepresentationofthestructuresoftwoionchannels.TheRomannumer-alsindicatethefoursubunitsofeachchannelandtheArabicnumeralstheα-helicaltransmembranedo-mainsofeachsubunit.Theactualporesthroughwhichtheionspassarenotshownbutareformedbyappositionofthevarioussubunits.Thespecificareasofthesubunitsinvolvedintheopeningandclos-ingofthechannelsarealsonotindicated.(AfterWKCatterall.ModifiedandreproducedfromHallZW:AnIntroductiontoMolecularNeurobiology.Sinauer,1992.)
424426/CHAPTER41Table41–4.Somepropertiesofionchannels.•Theyarecomposedoftransmembraneproteinsubunits.•Mostarehighlyselectiveforoneion;afewarenonselec-Lipidbilayertive.•Theyallowimpermeableionstocrossmembranesatratesapproachingdiffusionlimits.67•Theycanpermitionfluxesof10–10/s.•Theiractivitiesareregulated.UniportSymportAntiport•Thetwomaintypesarevoltage-gatedandligand-gated.•Theyareusuallyhighlyconservedacrossspecies.++2+−Cotransport•MostcellshaveavarietyofNa,K,Ca,andCIchannels.•MutationsingenesencodingthemcancausespecificFigure41–10.Schematicrepresentationoftypesof1diseases.transportsystems.Transporterscanbeclassifiedwith•Theiractivitiesareaffectedbycertaindrugs.regardtothedirectionofmovementandwhetherone1ormoreuniquemoleculesaremoved.(Redrawnandre-SomediseasescausedbymutationsofionchannelsarebrieflydiscussedinChapter49.produced,withpermission,fromAlbertsBetal:MolecularBiologyoftheCell.Garland,1983.)diffusionisaugmentedbymovementthroughwaterMutationsinbacteriaandmammaliancells(includingchannels.Thesechannelsarecomposedoftetramericsomethatresultinhumandisease)havesupportedtransmembraneproteinsnamedaquaporins.Atleasttheseconclusions.Facilitateddiffusionandactivetrans-fivedistinctaquaporins(AP-1toAP-5)havebeeniden-portresembleasubstrate-enzymereactionexcepttified.MutationsinthegeneencodingAP-2havebeenthatnocovalentinteractionoccurs.Thesepointsofre-showntobethecauseofonetypeofnephrogenicdia-semblanceareasfollows:(1)Thereisaspecificbindingbetesinsipidus.siteforthesolute.(2)Thecarrierissaturable,soithasamaximumrateoftransport(Vmax;Figure41–11).(3)Thereisabindingconstant(Km)forthesolute,andPLASMAMEMBRANESAREINVOLVEDINFACILITATEDDIFFUSION,ACTIVETRANSPORT,&OTHERPROCESSESPassiveTransportsystemscanbedescribedinafunctionaldiffusionVmaxsenseaccordingtothenumberofmoleculesmovedandthedirectionofmovement(Figure41–10)oraccordingtowhethermovementistowardorawayfromequilib-rium.AuniportsystemmovesonetypeofmoleculeCarrier-mediateddiffusionbidirectionally.Incotransportsystems,thetransferofRateonesolutedependsuponthestoichiometricsimultane-ousorsequentialtransferofanothersolute.Asymportmovesthesesolutesinthesamedirection.Examplesaretheproton-sugartransporterinbacteriaandtheNa+-sugartransporters(forglucoseandcertainothersugars)andNa+-aminoacidtransportersinmammaliancells.KmAntiportsystemsmovetwomoleculesinoppositedi-Soluteconcentrationrections(eg,Na+inandCa2+out).MoleculesthatcannotpassfreelythroughthelipidFigure41–11.Acomparisonofthekineticsofcar-bilayermembranebythemselvesdosoinassociationrier-mediated(facilitated)diffusionwithpassivediffu-withcarrierproteins.Thisinvolvestwoprocesses—sion.Therateofmovementinthelatterisdirectlypro-facilitateddiffusionandactivetransport—andhighlyportionatetosoluteconcentration,whereasthespecifictransportsystems.processissaturablewhencarriersareinvolved.TheFacilitateddiffusionandactivetransportsharemanyconcentrationathalf-maximalvelocityisequaltothefeatures.Bothappeartoinvolvecarrierproteins,andbindingconstant(Km)ofthecarrierforthesolute.(Vmax,bothshowspecificityforions,sugars,andaminoacids.maximalrate.)
425MEMBRANES:STRUCTURE&FUNCTION/427sothewholesystemhasaKm(Figure41–11).(4)Struc-terminedbythefollowingfactors:(1)Theconcentra-turallysimilarcompetitiveinhibitorsblocktransport.tiongradientacrossthemembrane.(2)TheamountofMajordifferencesarethefollowing:(1)Facilitatedcarrieravailable(thisisakeycontrolstep).(3)Thera-diffusioncanoperatebidirectionally,whereasactivepidityofthesolute-carrierinteraction.(4)Therapiditytransportisusuallyunidirectional.(2)Activetransportoftheconformationalchangeforboththeloadedandalwaysoccursagainstanelectricalorchemicalgradient,theunloadedcarrier.andsoitrequiresenergy.Hormonesregulatefacilitateddiffusionbychangingthenumberoftransportersavailable.InsulinincreasesFacilitatedDiffusionglucosetransportinfatandmusclebyrecruitingtrans-portersfromanintracellularreservoir.Insulinalsoen-Somespecificsolutesdiffusedownelectrochemicalgra-hancesaminoacidtransportinliverandothertissues.dientsacrossmembranesmorerapidlythanmightbeOneofthecoordinatedactionsofglucocorticoidhor-expectedfromtheirsize,charge,orpartitioncoeffi-monesistoenhancetransportofaminoacidsintoliver,cients.Thisfacilitateddiffusionexhibitspropertieswheretheaminoacidsthenserveasasubstrateforglu-distinctfromthoseofsimplediffusion.Therateoffa-coneogenesis.Growthhormoneincreasesaminoacidcilitateddiffusion,auniportsystem,canbesaturated;transportinallcells,andestrogensdothisintheuterus.ie,thenumberofsitesinvolvedindiffusionofthespe-Thereareatleastfivedifferentcarriersystemsforcificsolutesappearsfinite.Manyfacilitateddiffusionaminoacidsinanimalcells.Eachisspecificforagroupsystemsarestereospecificbut,likesimplediffusion,re-ofcloselyrelatedaminoacids,andmostoperateasNa+-quirenometabolicenergy.symportsystems(Figure41–10).Asdescribedearlier,theinside-outsideasymmetryofmembraneproteinsisstable,andmobilityofproteinsActiveTransportacross(ratherthanin)themembraneisrare;therefore,transversemobilityofspecificcarrierproteinsisnotTheprocessofactivetransportdiffersfromdiffusioninlikelytoaccountforfacilitateddiffusionprocessesex-thatmoleculesaretransportedawayfromthermody-ceptinafewunusualcases.namicequilibrium;hence,energyisrequired.Thisen-A“Ping-Pong”mechanism(Figure41–12)ex-ergycancomefromthehydrolysisofATP,fromelec-plainsfacilitateddiffusion.Inthismodel,thecarriertronmovement,orfromlight.Themaintenanceofproteinexistsintwoprincipalconformations.Intheelectrochemicalgradientsinbiologicsystemsissoim-“pong”state,itisexposedtohighconcentrationsofportantthatitconsumesperhaps30–40%ofthetotalsolute,andmoleculesofthesolutebindtospecificsitesenergyexpenditureinacell.onthecarrierprotein.Transportoccurswhenaconfor-Ingeneral,cellsmaintainalowintracellularNa+mationalchangeexposesthecarriertoalowerconcen-concentrationandahighintracellularK+concentrationtrationofsolute(“ping”state).Thisprocessiscom-(Table41–1),alongwithanetnegativeelectricalpo-pletelyreversible,andnetfluxacrossthemembranetentialinside.Thepumpthatmaintainsthesegradientsdependsupontheconcentrationgradient.TherateatisanATPasethatisactivatedbyNa+andK+(Na+-K+whichsolutesenteracellbyfacilitateddiffusionisde-ATPase;seeFigure41–13).TheATPaseisanintegralPongPingFigure41–12.The“Ping-Pong”modeloffacilitateddiffusion.Aproteincarrier(graystructure)inthelipidbi-layerassociateswithasoluteinhighconcentrationononesideofthemembrane.Aconformationalchangeen-sues(“pong”to“ping”),andthesoluteisdischargedonthesidefavoringthenewequilibrium.Theemptycarrierthenrevertstotheoriginalconformation(“ping”to“pong”)tocompletethecycle.
426428/CHAPTER41onlywherethemembraneisfreeoftheinsulation.TheINSIDEOUTSIDEmyelinmembraneiscomposedofphospholipids,cho-Membranelesterol,proteins,andGSLs.Relativelyfewproteinsare3Na+foundinthemyelinmembrane;thosepresentappeartoATP3Na+holdtogethermultiplemembranebilayerstoformthehydrophobicinsulatingstructurethatisimpermeable2+toionsandwater.Certaindiseases,eg,multiplesclero-MgsisandtheGuillain-Barrésyndrome,arecharacterized2K+bydemyelinationandimpairednerveconduction.ADP+Pi+GlucoseTransportInvolves2KSeveralMechanismsFigure41–13.StoichiometryoftheNa+-K+ATPase+Adiscussionofthetransportofglucosesummarizespump.ThispumpmovesthreeNaionsfrominsidethe+manyofthepointsmadeinthischapter.GlucosemustcelltotheoutsideandbringstwoKionsfromtheout-entercellsasthefirststepinenergyutilization.InsidetotheinsideforeverymoleculeofATPhydrolyzedadipocytesandmuscle,glucoseentersbyaspecifictoADPbythemembrane-associatedATPase.Ouabaintransportsystemthatisenhancedbyinsulin.Changesandothercardiacglycosidesinhibitthispumpbyact-intransportareprimarilyduetoalterationsofVmaxingontheextracellularsurfaceofthemembrane.(presumablyfrommoreorfeweractivetransporters),(CourtesyofRPost.)butchangesinKmmayalsobeinvolved.Glucosetrans-portinvolvesdifferentaspectsoftheprinciplesoftrans-+portdiscussedabove.GlucoseandNabindtodifferent+membraneproteinandrequiresphospholipidsforac-sitesontheglucosetransporter.Namovesintothecelltivity.TheATPasehascatalyticcentersforbothATPdownitselectrochemicalgradientand“drags”glucose+withit(Figure41–14).Therefore,thegreatertheNa+andNaonthecytoplasmicsideofthemembrane,but+gradient,themoreglucoseenters;andifNa+inextra-theKbindingsiteislocatedontheextracellularsideofthemembrane.OuabainordigitalisinhibitsthisATP-cellularfluidislow,glucosetransportstops.Tomain-asebybindingtotheextracellulardomain.InhibitiontainasteepNa+gradient,thisNa+-glucosesymportisoftheATPasebyouabaincanbeantagonizedbyextra-dependentongradientsgeneratedbyanNa+-K+pumpcellularK+.thatmaintainsalowintracellularNa+concentration.SimilarmechanismsareusedtotransportothersugarsNerveImpulsesAreTransmittedaswellasaminoacids.ThetranscellularmovementofsugarsinvolvesoneUp&DownMembranesadditionalcomponent:auniportthatallowstheglucoseThemembraneformingthesurfaceofneuronalcellsaccumulatedwithinthecelltomoveacrossadifferentmaintainsanasymmetryofinside-outsidevoltage(elec-surfacetowardanewequilibrium;thisoccursinintesti-tricalpotential)andiselectricallyexcitable.Whenap-nalandrenalcells,forexample.propriatelystimulatedbyachemicalsignalmediatedbyaspecificsynapticmembranereceptor(seediscussionofCellsTransportCertainMacromoleculesthetransmissionofbiochemicalsignals,below),gatesinAcrossthePlasmaMembranethemembraneareopenedtoallowtherapidinfluxofNa+orCa2+(withorwithouttheeffluxofK+),sothatTheprocessbywhichcellstakeuplargemoleculesisthevoltagedifferencerapidlycollapsesandthatseg-called“endocytosis.”Someofthesemolecules(eg,mentofthemembraneisdepolarized.However,asare-polysaccharides,proteins,andpolynucleotides),whensultoftheactionoftheionpumpsinthemembrane,hydrolyzedinsidethecell,yieldnutrients.Endocytosisthegradientisquicklyrestored.providesamechanismforregulatingthecontentofcer-Whenlargeareasofthemembranearedepolarizedtainmembranecomponents,hormonereceptorsbeinginthismanner,theelectrochemicaldisturbancepropa-acaseinpoint.Endocytosiscanbeusedtolearnmoregatesinwave-likeformdownthemembrane,generat-abouthowcellsfunction.DNAfromonecelltypecaninganerveimpulse.Myelinsheets,formedbybeusedtotransfectadifferentcellandalterthelatter’sSchwanncells,wraparoundnervefibersandprovideanfunctionorphenotype.Aspecificgeneisoftenem-electricalinsulatorthatsurroundsmostofthenerveandployedintheseexperiments,andthisprovidesauniquegreatlyspeedsupthepropagationofthewave(signal)waytostudyandanalyzetheregulationofthatgene.byallowingionstoflowinandoutofthemembraneDNAtransfectiondependsuponendocytosis;endocy-
427MEMBRANES:STRUCTURE&FUNCTION/429LUMENAB+GlucoseNa(Symport)CYTOSOLGlucoseNa++KCP++NaKGlucoseEXTRACELLULARFLUIDFigure41–14.Thetranscellularmovementofglu-+Vcoseinanintestinalcell.GlucosefollowsNaacrosstheluminalepithelialmembrane.TheNa+gradientthat++CVdrivesthissymportisestablishedbyNa-Kexchange,whichoccursatthebasalmembranefacingtheextra-Figure41–15.Twotypesofendocytosis.Anendo-cellularfluidcompartment.Glucoseathighconcentra-cytoticvesicle(V)formsasaresultofinvaginationofationwithinthecellmoves“downhill”intotheextracel-portionoftheplasmamembrane.Fluid-phaseendocy-lularfluidbyfacilitateddiffusion(auniportmechanism).tosis(A)israndomandnondirected.Receptor-medi-atedendocytosis(B)isselectiveandoccursincoatedpits(CP)linedwiththeproteinclathrin(thefuzzymate-rial).Targetingisprovidedbyreceptors(blacksymbols)tosisisresponsiblefortheentryofDNAintothecell.specificforavarietyofmolecules.Thisresultsinthefor-Suchexperimentscommonlyusecalciumphosphate,2+mationofacoatedvesicle(CV).sinceCastimulatesendocytosisandprecipitatesDNA,whichmakestheDNAabetterobjectforendo-cytosis.Cellsalsoreleasemacromoleculesbyexocyto-sis.Endocytosisandexocytosisbothinvolvevesiclefor-reusedinthecytoplasm.Endocytosisrequires(1)en-2+mationwithorfromtheplasmamembrane.ergy,usuallyfromthehydrolysisofATP;(2)Cainextracellularfluid;and(3)contractileelementsintheA.ENDOCYTOSIScell(probablythemicrofilamentsystem)(Chapter49).AlleukaryoticcellsarecontinuouslyingestingpartsofTherearetwogeneraltypesofendocytosis.Phago-theirplasmamembranes.Endocytoticvesiclesaregen-cytosisoccursonlyinspecializedcellssuchaseratedwhensegmentsoftheplasmamembraneinvagi-macrophagesandgranulocytes.Phagocytosisinvolvesnate,enclosingaminutevolumeofextracellularfluidtheingestionoflargeparticlessuchasviruses,bacteria,anditscontents.Thevesiclethenpinchesoffasthefu-cells,ordebris.Macrophagesareextremelyactiveinsionofplasmamembranessealstheneckofthevesiclethisregardandmayingest25%oftheirvolumeperattheoriginalsiteofinvagination(Figure41–15).Thishour.Insodoing,amacrophagemayinternalize3%ofvesiclefuseswithothermembranestructuresandthusitsplasmamembraneeachminuteortheentiremem-achievesthetransportofitscontentstoothercellularbraneevery30minutes.compartmentsorevenbacktothecellexterior.MostPinocytosisisapropertyofallcellsandleadstotheendocytoticvesiclesfusewithprimarylysosomestocellularuptakeoffluidandfluidcontents.Thereareformsecondarylysosomes,whichcontainhydrolytictwotypes.Fluid-phasepinocytosisisanonselectiveenzymesandarethereforespecializedorganellesforin-processinwhichtheuptakeofasolutebyformationoftracellulardisposal.Themacromolecularcontentsaresmallvesiclesissimplyproportionatetoitsconcentra-digestedtoyieldaminoacids,simplesugars,ornu-tioninthesurroundingextracellularfluid.Theforma-cleotides,andtheydiffuseoutofthevesiclestobetionofthesevesiclesisanextremelyactiveprocess.Fi-
428430/CHAPTER41broblasts,forexample,internalizetheirplasmamem-terol—releasedduringthedegradationofLDL.Disor-braneataboutone-thirdtherateofmacrophages.ThisdersoftheLDLreceptoranditsinternalizationareprocessoccursmorerapidlythanmembranesaremade.medicallyimportantandarediscussedinChapter25.ThesurfaceareaandvolumeofacelldonotchangeAbsorptivepinocytosisofextracellularglycopro-much,somembranesmustbereplacedbyexocytosisorteinsrequiresthattheglycoproteinscarryspecificcar-bybeingrecycledasfastastheyareremovedbyendocy-bohydraterecognitionsignals.Theserecognitionsignalstosis.areboundbymembranereceptormolecules,whichTheothertypeofpinocytosis,absorptivepinocyto-playaroleanalogoustothatoftheLDLreceptor.Asis,isareceptor-mediatedselectiveprocessprimarilyre-galactosylreceptoronthesurfaceofhepatocytesisin-sponsiblefortheuptakeofmacromoleculesforwhichstrumentalintheabsorptivepinocytosisofasialoglyco-thereareafinitenumberofbindingsitesontheplasmaproteinsfromthecirculation(Chapter47).Acidhydro-membrane.Thesehigh-affinityreceptorspermitthese-lasestakenupbyabsorptivepinocytosisinfibroblastslectiveconcentrationofligandsfromthemedium,min-arerecognizedbytheirmannose6-phosphatemoieties.imizetheuptakeoffluidorsolubleunboundmacro-Interestingly,themannose6-phosphatemoietyalsomolecules,andmarkedlyincreasetherateatwhichseemstoplayanimportantroleintheintracellulartar-specificmoleculesenterthecell.Thevesiclesformedgetingoftheacidhydrolasestothelysosomesoftheduringabsorptivepinocytosisarederivedfrominvagi-cellsinwhichtheyaresynthesized(Chapter47).nations(pits)thatarecoatedonthecytoplasmicsideThereisadarksidetoreceptor-mediatedendocyto-withafilamentousmaterial.Inmanysystems,thepro-sisinthatviruseswhichcausesuchdiseasesashepatitisteinclathrinisthefilamentousmaterial.Ithasathree-(affectinglivercells),poliomyelitis(affectingmotorlimbedstructure(calledatriskelion),witheachlimbneurons),andAIDS(affectingTcells)initiatetheirbeingmadeupofonelightandoneheavychainofdamagebythismechanism.Irontoxicityalsobeginsclathrin.Thepolymerizationofclathrinintoavesicleiswithexcessiveuptakeduetoendocytosis.directedbyassemblyparticles,composedoffouradapterproteins.TheseinteractwithcertainaminoB.EXOCYTOSISacidsequencesinthereceptorsthatbecomecargo,en-Mostcellsreleasemacromoleculestotheexteriorbyex-suringselectivityofuptake.ThelipidPIP2alsoplaysanocytosis.Thisprocessisalsoinvolvedinmembranere-importantroleinvesicleassembly.Inaddition,thepro-modeling,whenthecomponentssynthesizedintheteindynamin,whichbothbindsandhydrolyzesGTP,Golgiapparatusarecarriedinvesiclestotheplasmaisnecessaryforthepinchingoffofclathrin-coatedvesi-membrane.Thesignalforexocytosisisoftenahor-clesfromthecellsurface.Coatedpitsmayconstituteasmonewhich,whenitbindstoacell-surfacereceptor,2+muchas2%ofthesurfaceofsomecells.inducesalocalandtransientchangeinCaconcentra-2+Asanexample,thelow-densitylipoprotein(LDL)tion.Catriggersexocytosis.Figure41–16providesamoleculeanditsreceptor(Chapter25)areinternalizedcomparisonofthemechanismsofexocytosisandendo-bymeansofcoatedpitscontainingtheLDLreceptor.cytosis.TheseendocytoticvesiclescontainingLDLanditsre-Moleculesreleasedbyexocytosisfallintothreecate-ceptorfusetolysosomesinthecell.Thereceptorisre-gories:(1)Theycanattachtothecellsurfaceandbe-leasedandrecycledbacktothecellsurfacemembrane,comeperipheralproteins,eg,antigens.(2)Theycanbe-buttheapoproteinofLDLisdegradedandthecholes-comepartoftheextracellularmatrix,eg,collagenandterylestersmetabolized.SynthesisoftheLDLreceptorglycosaminoglycans.(3)Theycanenterextracellularisregulatedbysecondaryortertiaryconsequencesoffluidandsignalothercells.Insulin,parathyroidhor-pinocytosis,eg,bymetabolicproducts—suchascholes-mone,andthecatecholaminesareallpackagedingran-ExocytosisEndocytosisFigure41–16.Acomparisonofthemechanismsofendocytosisandexocyto-sis.Exocytosisinvolvesthecontactoftwoinsidesurface(cytoplasmicside)mono-layers,whereasendocytosisresultsfromthecontactoftwooutersurfacemono-layers.
429MEMBRANES:STRUCTURE&FUNCTION/431ulesandprocessedwithincells,tobereleaseduponap-aminoacids,sugars,lipids,urate,anions,cations,water,propriatestimulation.andvitaminsacrosstheplasmamembrane.MutationsingenesencodingproteinsinothermembranescanSomeSignalsAreTransmittedalsohaveharmfulconsequences.Forexample,muta-tionsingenesencodingmitochondrialmembraneAcrossMembranesproteinsinvolvedinoxidativephosphorylationcanSpecificbiochemicalsignalssuchasneurotransmitters,causeneurologicandotherproblems(eg,Leber’shered-hormones,andimmunoglobulinsbindtospecificre-itaryopticneuropathy;LHON).Membraneproteinsceptors(integralproteins)exposedtotheoutsideofcanalsobeaffectedbyconditionsotherthanmuta-cellularmembranesandtransmitinformationthroughtions.Formationofautoantibodiestotheacetyl-thesemembranestothecytoplasm.Thisprocess,calledcholinereceptorinskeletalmusclecausesmyastheniatransmembranesignaling,involvesthegenerationofagravis.Ischemiacanquicklyaffecttheintegrityofvari-numberofsignals,includingcyclicnucleotides,cal-ousionchannelsinmembranes.Abnormalitiesofcium,phosphoinositides,anddiacylglycerol.Itisdis-membraneconstituentsotherthanproteinscanalsobecussedindetailinChapter43.harmful.Withregardtolipids,excessofcholesterol(eg,infamilialhypercholesterolemia),oflysophospho-InformationCanBeCommunicatedlipid(eg,afterbitesbycertainsnakes,whosevenombyIntercellularContactcontainsphospholipases),orofglycosphingolipids(eg,inasphingolipidosis)canallaffectmembranefunction.Therearemanyareasofintercellularcontactinameta-zoanorganism.ThisnecessitatescontactbetweentheCysticFibrosisIsDuetoMutationsintheplasmamembranesoftheindividualcells.CellshaveGeneEncodingaChlorideChanneldevelopedspecializedregionsontheirmembranesforintercellularcommunicationincloseproximity.GapCysticfibrosis(CF)isarecessivegeneticdisorderpreva-junctionsmediateandregulatethepassageofionsandlentamongwhitesinNorthAmericaandcertainpartssmallmolecules(upto1000–2000MW)throughaofnorthernEurope.Itischaracterizedbychronicbac-narrowhydrophiliccoreconnectingthecytosolofadja-terialinfectionsoftheairwaysandsinuses,fatmaldiges-centcells.Thesestructuresareprimarilycomposedoftionduetopancreaticexocrineinsufficiency,infertilitytheproteinconnexin,whichcontainsfourmembrane-inmalesduetoabnormaldevelopmentofthevasdefer-spanningαhelices.Aboutadozengenesencodingdif-ens,andelevatedlevelsofchlorideinsweat(>60ferentconnexinshavebeencloned.Anassemblyof12mmol/L).connexinmoleculesformsastructure(aconnexon)AfteraHerculeanlandmarkendeavor,thegeneforwithacentralchannelthatformsbridgesbetweenadja-CFwasidentifiedin1989onchromosome7.Itwascentcells.Ionsandsmallmoleculespassfromthecy-foundtoencodeaproteinof1480aminoacids,namedtosolofonecelltothatofanotherthroughthechan-cysticfibrosistransmembraneregulator(CFTR),anels,whichopenandcloseinaregulatedfashion.cyclicAMP-regulatedCl−channel(seeFigure41–17).−AnabnormalityofmembraneClpermeabilityisbe-MUTATIONSAFFECTINGMEMBRANElievedtoresultintheincreasedviscosityofmanybodilyPROTEINSCAUSEDISEASESsecretions,thoughtheprecisemechanismsarestillunderinvestigation.Thecommonestmutation(~70%InviewofthefactthatmembranesarelocatedinsoincertainCaucasianpopulations)isdeletionofthreemanyorganellesandareinvolvedinsomanyprocesses,bases,resultinginlossofresidue508,aphenylalanineitisnotsurprisingthatmutationsaffectingtheirpro-(ΔF508).However,morethan900othermutationshaveteinconstituentsshouldresultinmanydiseasesordis-beenidentified.ThesemutationsaffectCFTRinatorders.Proteinsinmembranescanbeclassifiedasre-leastfourways:(1)itsamountisreduced;(2)depend-ceptors,transporters,ionchannels,enzymes,andingupontheparticularmutation,itmaybesusceptiblestructuralcomponents.MembersofalloftheseclassestomisfoldingandretentionwithintheERorGolgiap-areoftenglycosylated,sothatmutationsaffectingthisparatus;(3)mutationsinthenucleotide-bindingdo-−processmayaltertheirfunction.ExamplesofdiseasesmainsmayaffecttheabilityoftheClchanneltoopen,ordisordersduetoabnormalitiesinmembraneproteinsaneventaffectedbyATP;(4)themutationsmayalsoarelistedinTable41–5;thesemainlyreflectmutationsreducetherateofionflowthroughachannel,generat-−inproteinsoftheplasmamembrane,withoneaffect-inglessofaClcurrent.inglysosomalfunction(I-celldisease).Over30geneticThemostseriousandlife-threateningcomplicationdiseasesordisordershavebeenascribedtomutationsisrecurrentpulmonaryinfectionsduetoovergrowthofaffectingvariousproteinsinvolvedinthetransportofvariouspathogensintheviscoussecretionsoftherespi-
430432/CHAPTER41Table41–5.Somediseasesorpathologicstatesresultingfromorattributedtoabnormalities1ofmembranes.DiseaseAbnormalityAchondroplasiaMutationsinthegeneencodingthefibroblastgrowthfactorreceptor3(MIM100800)Familialhypercholester-MutationsinthegeneencodingtheLDLreceptorolemia(MIM143890)−CysticfibrosisMutationsinthegeneencodingtheCFTRprotein,aCltransporter(MIM219700)CongenitallongQTsyn-Mutationsingenesencodingionchannelsintheheartdrome(MIM192500)WilsondiseaseMutationsinthegeneencodingacopper-dependentATPase(MIM277900)I-celldiseaseMutationsinthegeneencodingGIcNAcphosphotransferase,resultinginabsenceoftheMan6-P(MIM252500)signalforlysosomallocalizationofcertainhydrolasesHereditaryspherocytosisMutationsinthegenesencodingspectrinorotherstructuralproteinsintheredcellmembrane(MIM182900)MetastasisAbnormalitiesintheoligosaccharidechainsofmembraneglycoproteinsandglycolipidsarethoughttobeofimportanceParoxysmalnocturnalMutationresultingindeficientattachmentoftheGPIanchortocertainproteinsoftheredcellhemoglobinuriamembrane(MIM311770)1Thedisorderslistedarediscussedfurtherinotherchapters.Thetablelistsexamplesofmutationsaffectingreceptors,atransporter,anionchannel,anenzyme,andastructuralprotein.Examplesofalteredordefectiveglycosylationofglycoproteinsarealsopresented.Mostoftheconditionslistedaffecttheplasmamembrane.ratorytract.Poornutritionasaresultofpancreaticin-sufficiencyworsensthesituation.ThetreatmentofCFthusrequiresacomprehensiveefforttomaintainnutri-tionalstatus,topreventandcombatpulmonaryinfec-tions,andtomaintainphysicalandpsychologichealth.AdvancesinmoleculargeneticsmeanthatmutationAminoanalysiscanbeperformedforprenataldiagnosisandforterminalNBF1carriertestinginfamiliesinwhichonechildalreadyhasRdomainNBF2thecondition.Effortsareinprogresstousegenether-apytorestoretheactivityofCFTR.AnaerosolizedCarboxylpreparationofhumanDNasethatdigeststheDNAofterminalmicroorganismsintherespiratorytracthasprovedhelpfulintherapy.Figure41–17.DiagramofthestructureoftheCFTRprotein(nottoscale).TheproteincontainstwelveSUMMARYtransmembranesegments(probablyhelical),twonu-cleotide-bindingfoldsordomains(NBF1andNBF2),•Membranesarecomplexstructurescomposedofandoneregulatory(R)domain.NBF1andNBF2proba-lipids,carbohydrates,andproteins.blybindATPandcoupleitshydrolysistotransportof•Thebasicstructureofallmembranesisthelipidbi-−Cl.Phe508,themajorlocusofmutationsincysticfi-layer.Thisbilayerisformedbytwosheetsofphos-brosis,islocatedinNBF1.pholipidsinwhichthehydrophilicpolarheadgroups
431MEMBRANES:STRUCTURE&FUNCTION/433aredirectedawayfromeachotherandareexposedto•Receptorsmaybeintegralcomponentsofmem-theaqueousenvironmentontheouterandinnersur-branes(particularlytheplasmamembrane).Thein-facesofthemembrane.Thehydrophobicnonpolarteractionofaligandwithitsreceptormaynotin-tailsofthesemoleculesareorientedtowardeachvolvethemovementofeitherintothecell,buttheother,inthedirectionofthecenterofthemem-interactionresultsinthegenerationofasignalthatbrane.influencesintracellularprocesses(transmembrane•Membraneproteinsareclassifiedasintegraliftheysignaling).arefirmlyembeddedinthebilayerandasperipheral•Mutationsthataffectthestructureofmembranepro-iftheyarelooselyattachedtotheouterorinnersur-teins(receptors,transporters,ionchannels,enzymes,face.andstructuralproteins)maycausediseases;examples•The20orsodifferentmembranesinamammalianincludecysticfibrosisandfamilialhypercholes-cellhaveintrinsicfunctions(eg,enzymaticactivity),terolemia.andtheydefinecompartments,orspecializedenvi-ronments,withinthecellthathavespecificfunctions(eg,lysosomes).REFERENCES•Certainmoleculesfreelydiffuseacrossmembranes,butthemovementofothersisrestrictedbecauseofDoyleDAetal:Thestructureofthepotassiumchannel:molecularbasisofK+conductanceandselectivity.Science1998;280:size,charge,orsolubility.69.•VariouspassiveandactivemechanismsareemployedFelixR:Channelopathies:ionchanneldefectslinkedtoheritabletomaintaingradientsofsuchmoleculesacrossdiffer-clinicaldisorders.JMedGenet2000;37:729.entmembranes.GaravitoRM,Ferguson-MillerS:Detergentsastoolsinmembrane•Certainsolutes,eg,glucose,entercellsbyfacilitatedbiochemistry.JBiolChem2001;276:32403.diffusion,alongadownhillgradientfromhightolowGilloolyDJ,StenmarkH:Alipidoilstheendocytosismachine.Sci-concentration.Specificcarriermolecules,ortrans-ence2001;291;993.porters,areinvolvedinsuchprocesses.KnowlesMR,DuriePR:Whatiscysticfibrosis?NEnglJMed•Ligand-orvoltage-gatedionchannelsareoftenem-2002;347:439.ployedtomovechargedmolecules(Na+,K+,Ca2+,LongoN:Inheriteddefectsofmembranetransport.In:Harrison’sPrinciplesofInternalMedicine,15thed.BraunwaldEetaletc)acrossmembranes.(editors).McGraw-Hill,2001.•LargemoleculescanenterorleavecellsthroughMarxJ:Caveolae:aonce-elusivestructuregetssomerespect.Sci-mechanismssuchasendocytosisorexocytosis.Theseence2001;294;1862.processesoftenrequirebindingofthemoleculetoaWhiteSHetal:Howmembranesshapeproteinstructure.JBiolreceptor,whichaffordsspecificitytotheprocess.Chem2001:276:32395.
432TheDiversityoftheEndocrineSystem42DarylK.Granner,MDACTHAdrenocorticotropichormoneGHGrowthhormoneANFAtrialnatriureticfactorIGF-IInsulin-likegrowthfactor-IcAMPCyclicadenosinemonophosphateLHLuteotropichormoneCBGCorticosteroid-bindingglobulinLPHLipotropinCGChorionicgonadotropinMITMonoiodotyrosinecGMPCyclicguanosinemonophosphateMSHMelanocyte-stimulatinghormoneCLIPCorticotropin-likeintermediatelobeOHSDHydroxysteroiddehydrogenasepeptidePNMTPhenylethanolamine-N-methyltransferaseDBHDopamineβ-hydroxylasePOMCPro-opiomelanocortinDHEADehydroepiandrosteroneSHBGSexhormone-bindingglobulinDHTDihydrotestosteroneStARSteroidogenicacuteregulatory(protein)DITDiiodotyrosineTBGThyroxine-bindingglobulinDOCDeoxycorticosteroneTEBGTestosterone-estrogen-bindingglobulinEGFEpidermalgrowthfactorTRHThyrotropin-releasinghormoneFSHFollicle-stimulatinghormoneTSHThyrotropin-stimulatinghormoneBIOMEDICALIMPORTANCEtivebecausehormonescanactonadjacentcells(paracrineaction)andonthecellinwhichtheywereThesurvivalofmulticellularorganismsdependsontheirsynthesized(autocrineaction)withoutenteringthesys-abilitytoadapttoaconstantlychangingenvironment.temiccirculation.Adiversearrayofhormones—eachIntercellularcommunicationmechanismsarenecessarywithdistinctivemechanismsofactionandpropertiesofrequirementsforthisadaptation.Thenervoussystembiosynthesis,storage,secretion,transport,andmetabo-andtheendocrinesystemprovidethisintercellular,or-lism—hasevolvedtoprovidehomeostaticresponses.ganism-widecommunication.ThenervoussystemwasThisbiochemicaldiversityisthetopicofthischapter.originallyviewedasprovidingafixedcommunicationsystem,whereastheendocrinesystemsuppliedhor-THETARGETCELLCONCEPTmones,whicharemobilemessages.Infact,thereisare-markableconvergenceoftheseregulatorysystems.ForThereareabout200typesofdifferentiatedcellsinhu-example,neuralregulationoftheendocrinesystemismans.Onlyafewproducehormones,butvirtuallyallofimportantintheproductionandsecretionofsomehor-the75trillioncellsinahumanaretargetsofoneormones;manyneurotransmittersresemblehormonesinmoreoftheover50knownhormones.Theconceptoftheirsynthesis,transport,andmechanismofaction;andthetargetcellisausefulwayoflookingathormoneac-manyhormonesaresynthesizedinthenervoussystem.tion.ItwasthoughtthathormonesaffectedasinglecellTheword“hormone”isderivedfromaGreektermthattype—oronlyafewkindsofcells—andthatahormonemeanstoarousetoactivity.Asclassicallydefined,ahor-elicitedauniquebiochemicalorphysiologicaction.Wemoneisasubstancethatissynthesizedinoneorganandnowknowthatagivenhormonecanaffectseveraldif-transportedbythecirculatorysystemtoactonanotherferentcelltypes;thatmorethanonehormonecanaffecttissue.However,thisoriginaldescriptionistoorestric-agivencelltype;andthathormonescanexertmanydif-434
433THEDIVERSITYOFTHEENDOCRINESYSTEM/435ferenteffectsinonecellorindifferentcells.WiththeTable42–2.Determinantsofthetargetdiscoveryofspecificcell-surfaceandintracellularhor-cellresponse.monereceptors,thedefinitionofatargethasbeenex-pandedtoincludeanycellinwhichthehormone(lig-Thenumber,relativeactivity,andstateofoccupancyoftheand)bindstoitsreceptor,whetherornotabiochemicalspecificreceptorsontheplasmamembraneorintheorphysiologicresponsehasyetbeendetermined.cytoplasmornucleus.SeveralfactorsdeterminetheresponseofatargetcellThemetabolism(activationorinactivation)ofthehormoneintoahormone.Thesecanbethoughtofintwogeneralthetargetcell.ways:(1)asfactorsthataffecttheconcentrationoftheThepresenceofotherfactorswithinthecellthatareneces-hormoneatthetargetcell(seeTable42–1)and(2)assaryforthehormoneresponse.factorsthataffecttheactualresponseofthetargetcellUp-ordown-regulationofthereceptorconsequenttothetothehormone(seeTable42–2).interactionwiththeligand.Postreceptordesensitzationofthecell,includingdown-HORMONERECEPTORSAREregulationofthereceptor.OFCENTRALIMPORTANCEReceptorsDiscriminatePreciselylogicallyrelevant:(1)bindingshouldbespecific,ie,dis-placeablebyagonistorantagonist;(2)bindingshouldOneofthemajorchallengesfacedinmakingthehor-besaturable;and(3)bindingshouldoccurwithinthemone-basedcommunicationsystemworkisillustratedconcentrationrangeoftheexpectedbiologicresponse.inFigure42–1.Hormonesarepresentatverylowcon-centrationsintheextracellularfluid,generallyinthe–15–9rangeof10to10mol/L.ThisconcentrationisBothRecognition&CouplingmuchlowerthanthatofthemanystructurallysimilarDomainsOccuronReceptorsmolecules(sterols,aminoacids,peptides,proteins)andAllreceptorshaveatleasttwofunctionaldomains.Aothermoleculesthatcirculateatconcentrationsinthe10–5to10–3mol/Lrange.Targetcells,therefore,mustrecognitiondomainbindsthehormoneligandandasecondregiongeneratesasignalthatcoupleshormonedistinguishnotonlybetweendifferenthormonespre-recognitiontosomeintracellularfunction.Couplingsentinsmallamountsbutalsobetweenagivenhor-moneandthe106-to109-foldexcessofothersimilar(signaltransduction)occursintwogeneralways.Polypeptideandproteinhormonesandthecate-molecules.Thishighdegreeofdiscriminationispro-cholaminesbindtoreceptorslocatedintheplasmavidedbycell-associatedrecognitionmoleculescalledre-membraneandtherebygenerateasignalthatregulatesceptors.Hormonesinitiatetheirbiologiceffectsbyvariousintracellularfunctions,oftenbychangingthebindingtospecificreceptors,andsinceanyeffectiveactivityofanenzyme.Incontrast,steroid,retinoid,andcontrolsystemalsomustprovideameansofstoppingathyroidhormonesinteractwithintracellularreceptors,response,hormone-inducedactionsgenerallyterminateanditisthisligand-receptorcomplexthatdirectlypro-whentheeffectordissociatesfromthereceptor.videsthesignal,generallytospecificgeneswhoserateofAtargetcellisdefinedbyitsabilitytoselectivelytranscriptionistherebyaffected.bindagivenhormonetoitscognatereceptor.SeveralThedomainsresponsibleforhormonerecognitionbiochemicalfeaturesofthisinteractionareimportantinandsignalgenerationhavebeenidentifiedinthepro-orderforhormone-receptorinteractionstobephysio-teinpolypeptideandcatecholaminehormonereceptors.Steroid,thyroid,andretinoidhormonereceptorshaveseveralfunctionaldomains:onesitebindsthehormone;Table42–1.DeterminantsoftheconcentrationanotherbindstospecificDNAregions;athirdisin-ofahormoneatthetargetcell.volvedintheinteractionwithothercoregulatorpro-teinsthatresultintheactivation(orrepression)ofgeneTherateofsynthesisandsecretionofthehormones.transcription;andafourthmayspecifybindingtooneTheproximityofthetargetcelltothehormonesource(dilu-ormoreotherproteinsthatinfluencetheintracellulartioneffect).traffickingofthereceptor.ThedissociationconstantsofthehormonewithspecificThedualfunctionsofbindingandcouplingulti-plasmatransportproteins(ifany).matelydefineareceptor,anditisthecouplingofhor-Theconversionofinactiveorsuboptimallyactiveformsofthemonebindingtosignaltransduction—so-calledrecep-hormoneintothefullyactiveform.tor-effectorcoupling—thatprovidesthefirststepinTherateofclearancefromplasmabyothertissuesorbyamplificationofthehormonalresponse.Thisdualpur-digestion,metabolism,orexcretion.posealsodistinguishesthetargetcellreceptorfromthe
434436/CHAPTER42◆❁❁✴❖✪❁❉◗Figure42–1.Specificityandselectivityof✧❙◆✪hormonereceptors.Manydifferentmolecules✴✧❙ECFcirculateintheextracellularfluid(ECF),but❃❃❍contentonlyafewarerecognizedbyhormonerecep-✧❍◗tors.Receptorsmustselectthesemolecules❉❉❖❁✪❁fromamonghighconcentrationsoftheothermolecules.ThissimplifieddrawingshowsthatHormoneReceptoracellmayhavenohormonereceptors(1),haveonereceptor(2+5+6),havereceptorsforseveralhormones(3),orhaveareceptorbut123456Celltypesnohormoneinthevicinity(4).plasmacarrierproteinsthatbindhormonebutdonotHORMONESCANBECLASSIFIEDgenerateasignal(seeTable42–6).INSEVERALWAYSHormonescanbeclassifiedaccordingtochemicalcom-ReceptorsAreProteinsposition,solubilityproperties,locationofreceptors,Severalclassesofpeptidehormonereceptorshavebeenandthenatureofthesignalusedtomediatehormonaldefined.Forexample,theinsulinreceptorisahet-actionwithinthecell.Aclassificationbasedonthelasterotetramer(α2β2)linkedbymultipledisulfidebondstwopropertiesisillustratedinTable42–3,andgeneralinwhichtheextracellularαsubunitbindsinsulinandfeaturesofeachgroupareillustratedinTable42–4.themembrane-spanningβsubunittransducesthesig-ThehormonesingroupIarelipophilic.Aftersecre-nalthroughthetyrosineproteinkinasedomainlocatedtion,thesehormonesassociatewithplasmatransportorinthecytoplasmicportionofthispolypeptide.There-carrierproteins,aprocessthatcircumventstheproblemceptorsforinsulin-likegrowthfactorI(IGF-I)andofsolubilitywhileprolongingtheplasmahalf-lifeoftheepidermalgrowthfactor(EGF)aregenerallysimilarinhormone.Therelativepercentagesofboundandfreestructuretotheinsulinreceptor.Thegrowthhormonehormonearedeterminedbythebindingaffinityandandprolactinreceptorsalsospantheplasmamem-bindingcapacityofthetransportprotein.Thefreehor-braneoftargetcellsbutdonotcontainintrinsicpro-mone,whichisthebiologicallyactiveform,readilytra-teinkinaseactivity.Ligandbindingtothesereceptors,versesthelipophilicplasmamembraneofallcellsandhowever,resultsintheassociationandactivationofaencountersreceptorsineitherthecytosolornucleusofcompletelydifferentproteinkinasepathway,theJak-targetcells.Theligand-receptorcomplexisassumedtoStatpathway.Polypeptidehormoneandcatecho-betheintracellularmessengerinthisgroup.laminereceptors,whichtransducesignalsbyalteringThesecondmajorgroupconsistsofwater-solubletherateofproductionofcAMPthroughG-proteins,hormonesthatbindtotheplasmamembraneofthetar-arecharacterizedbythepresenceofsevendomainsthatgetcell.Hormonesthatbindtothesurfacesofcellsspantheplasmamembrane.ProteinkinaseactivationcommunicatewithintracellularmetabolicprocessesandthegenerationofcyclicAMP,(cAMP,3′5′-throughintermediarymoleculescalledsecondmessen-adenylicacid;seeFigure20–5)isadownstreamactiongers(thehormoneitselfisthefirstmessenger),whichofthisclassofreceptor(seeChapter43forfurtherde-aregeneratedasaconsequenceoftheligand-receptortails).interaction.ThesecondmessengerconceptarosefromAcomparisonofseveraldifferentsteroidreceptorsanobservationthatepinephrinebindstotheplasmawiththyroidhormonereceptorsrevealedaremarkablemembraneofcertaincellsandincreasesintracellularconservationoftheaminoacidsequenceincertainre-cAMP.Thiswasfollowedbyaseriesofexperimentsingions,particularlyintheDNA-bindingdomains.ThiswhichcAMPwasfoundtomediatetheeffectsofmanyledtotherealizationthatreceptorsofthesteroidorhormones.Hormonesthatclearlyemploythismecha-thyroidtypearemembersofalargesuperfamilyofnu-nismareshowningroupII.AofTable42–3.Todate,clearreceptors.Manyrelatedmembersofthisfamilyonlyonehormone,atrialnatriureticfactor(ANF),useshavenoknownligandatpresentandthusarecalledor-cGMPasitssecondmessenger,butotherhormonesphanreceptors.ThenuclearreceptorsuperfamilyplayswillprobablybeaddedtogroupII.B.Severalhor-acriticalroleintheregulationofgenetranscriptionbymones,manyofwhichwerepreviouslythoughttoaf-2+hormones,asdescribedinChapter43.fectcAMP,appeartouseioniccalcium(Ca)or
435THEDIVERSITYOFTHEENDOCRINESYSTEM/437Table42–3.ClassificationofhormonesbyTable42–4.Generalfeaturesofhormoneclasses.mechanismofaction.GroupIGroupIII.HormonesthatbindtointracellularreceptorsTypesSteroids,iodothyro-Polypeptides,proteins,Androgensnines,calcitriol,glycoproteins,cate-Calcitriol(1,25[OH]2-D3)retinoidscholaminesEstrogensGlucocorticoidsSolubilityLipophilicHydrophilicMineralocorticoidsTransportYesNoProgestinsproteinsRetinoicacidThyroidhormones(T3andT4)Plasmahalf-Long(hourstoShort(minutes)II.Hormonesthatbindtocellsurfacereceptorslifedays)A.ThesecondmessengeriscAMP:ReceptorIntracellularPlasmamembraneα2-Adrenergiccatecholamines2+β-AdrenergiccatecholaminesMediatorReceptor-hormonecAMP,cGMP,Ca,AdrenocorticotropichormonecomplexmetabolitesofcomplexAntidiuretichormonephosphoinositols,CalcitoninkinasecascadesChorionicgonadotropin,humanCorticotropin-releasinghormoneFollicle-stimulatinghormonemetabolitesofcomplexphosphoinositides(orboth)asGlucagontheintracellularsignal.TheseareshowningroupII.CLipotropinLuteinizinghormoneofthetable.TheintracellularmessengerforgroupII.DMelanocyte-stimulatinghormoneisaproteinkinase-phosphatasecascade.SeveraloftheseParathyroidhormonehavebeenidentified,andagivenhormonemayuseSomatostatinmorethanonekinasecascade.AfewhormonesfitintoThyroid-stimulatinghormonemorethanonecategory,andassignmentschangeasB.ThesecondmessengeriscGMP:newinformationisbroughtforward.AtrialnatriureticfactorNitricoxideC.Thesecondmessengeriscalciumorphosphatidyl-DIVERSITYOFTHEENDOCRINESYSTEMinositols(orboth):Acetylcholine(muscarinic)HormonesAreSynthesizedinaα1-AdrenergiccatecholaminesVarietyofCellularArrangementsAngiotensinIIAntidiuretichormone(vasopressin)HormonesaresynthesizedindiscreteorgansdesignedCholecystokininsolelyforthisspecificpurpose,suchasthethyroid(tri-Gastriniodothyronine),adrenal(glucocorticoidsandmineralo-Gonadotropin-releasinghormonecorticoids),andthepituitary(TSH,FSH,LH,growthOxytocinhormone,prolactin,ACTH).SomeorgansaredesignedPlatelet-derivedgrowthfactortoperformtwodistinctbutcloselyrelatedfunctions.SubstancePForexample,theovariesproducematureoocytesandThyrotropin-releasinghormonethereproductivehormonesestradiolandprogesterone.D.ThesecondmessengerisakinaseorphosphataseThetestesproducematurespermatozoaandtestos-cascade:terone.HormonesarealsoproducedinspecializedcellsChorionicsomatomammotropinwithinotherorganssuchasthesmallintestineEpidermalgrowthfactor(glucagon-likepeptide),thyroid(calcitonin),andkid-Erythropoietinney(angiotensinII).Finally,thesynthesisofsomehor-FibroblastgrowthfactormonesrequirestheparenchymalcellsofmorethanoneGrowthhormoneorgan—eg,theskin,liver,andkidneyarerequiredforInsulinInsulin-likegrowthfactorsIandIItheproductionof1,25(OH)2-D3(calcitriol).ExamplesNervegrowthfactorofthisdiversityintheapproachtohormonesynthesis,Platelet-derivedgrowthfactoreachofwhichhasevolvedtofulfillaspecificpurpose,Prolactinarediscussedbelow.
436438/CHAPTER42HormonesAreChemicallyDiverseMANYHORMONESAREMADEHormonesaresynthesizedfromawidevarietyofchem-FROMCHOLESTEROLicalbuildingblocks.Alargeseriesisderivedfromcho-AdrenalSteroidogenesislesterol.Theseincludetheglucocorticoids,mineralo-corticoids,estrogens,progestins,and1,25(OH)2-D3Theadrenalsteroidhormonesaresynthesizedfrom(seeFigure42–2).Insomecases,asteroidhormoneischolesterol.Cholesterolismostlyderivedfromthetheprecursormoleculeforanotherhormone.Forex-plasma,butasmallportionissynthesizedinsitufromample,progesteroneisahormoneinitsownrightbutacetyl-CoAviamevalonateandsqualene.Muchoftheisalsoaprecursorintheformationofglucocorticoids,cholesterolintheadrenalisesterifiedandstoredincy-mineralocorticoids,testosterone,andestrogens.Testos-toplasmiclipiddroplets.UponstimulationoftheteroneisanobligatoryintermediateinthebiosynthesisadrenalbyACTH,anesteraseisactivated,andthefreeofestradiolandintheformationofdihydrotestosteronecholesterolformedistransportedintothemitochon-(DHT).Intheseexamples,describedindetailbelow,drion,whereacytochromeP450sidechaincleav-thefinalproductisdeterminedbythecelltypeandtheageenzyme(P450scc)convertscholesteroltopreg-associatedsetofenzymesinwhichtheprecursorexists.nenolone.CleavageofthesidechaininvolvessequentialTheaminoacidtyrosineisthestartingpointinthehydroxylations,firstatC22andthenatC20,followedbysynthesisofthecatecholaminesandofthethyroidhor-sidechaincleavage(removalofthesix-carbonfragmentmonestetraiodothyronine(thyroxine;T4)andtriiodo-isocaproaldehyde)togivethe21-carbonsteroid(Figurethyronine(T3)(Figure42–2).T3andT4areuniquein42–3,top).AnACTH-dependentsteroidogenicacutethattheyrequiretheadditionofiodine(asI−)forbioac-regulatory(StAR)proteinisessentialforthetransporttivity.BecausedietaryiodineisveryscarceinmanyofcholesteroltoP450sccintheinnermitochondrialpartsoftheworld,anintricatemechanismforaccumu-membrane.latingandretainingI−hasevolved.AllmammaliansteroidhormonesareformedfromManyhormonesarepolypeptidesorglycoproteins.cholesterolviapregnenolonethroughaseriesofreac-Theserangeinsizefromthyrotropin-releasinghor-tionsthatoccurineitherthemitochondriaorendoplas-mone(TRH),atripeptide,tosingle-chainpolypeptidesmicreticulumoftheadrenalcell.Hydroxylasesthatre-likeadrenocorticotropichormone(ACTH;39aminoquiremolecularoxygenandNADPHareessential,andacids),parathyroidhormone(PTH;84aminoacids),dehydrogenases,anisomerase,andalyasereactionareandgrowthhormone(GH;191aminoacids)(Figurealsonecessaryforcertainsteps.Thereiscellularspeci-42–2).InsulinisanABchainheterodimerof21and30ficityinadrenalsteroidogenesis.Forinstance,18-aminoacids,respectively.Follicle-stimulatinghormonehydroxylaseand19-hydroxysteroiddehydrogenase,(FSH),luteinizinghormone(LH),thyroid-stimulatingwhicharerequiredforaldosteronesynthesis,arefoundhormone(TSH),andchorionicgonadotropin(CG)areonlyinthezonaglomerulosacells(theouterregionofglycoproteinhormonesofαβheterodimericstructure.theadrenalcortex),sothatthebiosynthesisofthismin-Theαchainisidenticalinallofthesehormones,anderalocorticoidisconfinedtothisregion.Aschematicdistinctβchainsimparthormoneuniqueness.Theserepresentationofthepathwaysinvolvedinthesynthesishormoneshaveamolecularmassintherangeof25–30ofthethreemajorclassesofadrenalsteroidsispre-kDadependingonthedegreeofglycosylationandthesentedinFigure42–4.Theenzymesareshowninthelengthoftheβchain.rectangularboxes,andthemodificationsateachstepareshaded.HormonesAreSynthesized&ModifiedA.MINERALOCORTICOIDSYNTHESISForFullActivityinaVarietyofWaysSynthesisofaldosteronefollowsthemineralocorticoidSomehormonesaresynthesizedinfinalformandse-pathwayandoccursinthezonaglomerulosa.Preg-cretedimmediately.Includedinthisclassarethehor-nenoloneisconvertedtoprogesteronebytheactionofmonesderivedfromcholesterol.Otherssuchasthecat-twosmoothendoplasmicreticulumenzymes,3-5,4echolaminesaresynthesizedinfinalformandstoredinhydroxysteroiddehydrogenase(3-OHSD)and-theproducingcells.Othersaresynthesizedfrompre-isomerase.ProgesteroneishydroxylatedattheC21posi-cursormoleculesintheproducingcell,thenaretiontoform11-deoxycorticosterone(DOC),whichisanprocessedandsecreteduponaphysiologiccue(insulin).active(Na+-retaining)mineralocorticoid.Thenexthy-Finally,stillothersareconvertedtoactiveformsfromdroxylation,atC11,producescorticosterone,whichhasprecursormoleculesintheperiphery(T3andDHT).glucocorticoidactivityandisaweakmineralocorticoid(itAlloftheseexamplesarediscussedinmoredetailhaslessthan5%ofthepotencyofaldosterone).Insomebelow.species(eg,rodents),itisthemostpotentglucocorticoid.
437THEDIVERSITYOFTHEENDOCRINESYSTEM/439A.CHOLESTEROLDERIVATIVESCH2OHCH3OHOHCOCOOHOHCH2HOOHHOOOHO17ß-EstradiolTestosteroneCortisolProgesterone1,25(OH)2-D3B.TYROSINEDERIVATIVESHOHIIOHOHOCH2CHCOOHHOCCNH2INH2HHT3NorepinephrineHOHIIOHCH3OHOCHCHCOOHHOCCNH2IINH2HHT4EpinephrineC.PEPTIDESOFVARIOUSSIZES123456789101112SerserTyrserSerserMetserGluserHlsserPheserArgserTrpserGlyserLysserProser12313serValConservedregion;requiredforfullbiologicactivityGlyser(pyro)GluHlsProNH242322212019181716214Lysser25ProTyrValLysValserProserArgserArgserLysser15Asp26Ala27GlyVariableregion;notrequiredforbiologicactivityTRHGluAspserGlnserSerserAlaserGluserAlaserPheserProserLeuserGluserPheser282930313233343536373839StructureofhumanACTH.D.GLYCOPROTEINS(TSH,FSH,LH)ACTHcommonαsubunitsuniqueβsubunitsFigure42–2.Chemicaldiversityofhormones.A.Cholesterolderivatives.B.Tyrosinederivatives.C.PeptidesofvarioussizesD.Glycoproteins(TSH,FSH,LH)withcommonαsubunitsanduniqueβsubunits.
438440/CHAPTER42Cholesterolsidechaincleavage21CH3CCCCCCC20COC18H1217CACTH111316CD(cAMP)+CCCC19141519P450scc2108OCAB357HOHO46CholesterolPregnenolone+isocaproaldehydeBasicsteroidhormonestructuresCH2OHCH3OHOHCOCOHOOHHOOOO17β—EstradiolTestosteroneCortisolProgesteroneEstranegroup(C18)Androstanegroup(C19)Pregnanegroup(C21)Figure42–3.Cholesterolside-chaincleavageandbasicsteroidhormonestructures.Thebasicsterolringsareiden-tifiedbythelettersA–D.Thecarbonatomsarenumbered1–21startingwiththeAring.Notethattheestranegrouphas18carbons(C18),etc.C21hydroxylationisnecessaryforbothmineralocorticoidcosteroneoraldosterone,dependingonthecelltype).andglucocorticoidactivity,butmoststeroidswithaC1717α-Hydroxylaseisasmoothendoplasmicreticulumhydroxylgrouphavemoreglucocorticoidandlessminer-enzymethatactsuponeitherprogesteroneor,morealocorticoidaction.Inthezonaglomerulosa,whichdoescommonly,pregnenolone.17α-HydroxyprogesteroneisnothavethesmoothendoplasmicreticulumenzymehydroxylatedatC21toform11-deoxycortisol,whichis17α-hydroxylase,amitochondrial18-hydroxylaseispres-thenhydroxylatedatC11toformcortisol,themostpo-ent.The18-hydroxylase(aldosteronesynthase)actsontentnaturalglucocorticoidhormoneinhumans.21-Hy-corticosteronetoform18-hydroxycorticosterone,whichdroxylaseisasmoothendoplasmicreticulumenzyme,ischangedtoaldosteronebyconversionofthe18-alcoholwhereas11β-hydroxylaseisamitochondrialenzyme.toanaldehyde.ThisuniquedistributionofenzymesandSteroidogenesisthusinvolvestherepeatedshuttlingofthespecialregulationofthezonaglomerulosabyK+andsubstratesintoandoutofthemitochondria.angiotensinIIhaveledsomeinvestigatorstosuggestthat,inadditiontotheadrenalbeingtwoglands,theadrenalC.ANDROGENSYNTHESIScortexisactuallytwoseparateorgans.ThemajorandrogenorandrogenprecursorproducedbyB.GLUCOCORTICOIDSYNTHESIStheadrenalcortexisdehydroepiandrosterone(DHEA).CortisolsynthesisrequiresthreehydroxylaseslocatedinMost17-hydroxypregnenolonefollowstheglucocorticoidthefasciculataandreticulariszonesoftheadrenalcortexpathway,butasmallfractionissubjectedtooxidativefis-thatactsequentiallyontheC17,C21,andC11positions.sionandremovalofthetwo-carbonsidechainthroughThefirsttworeactionsarerapid,whileC11hydroxyla-theactionof17,20-lyase.Thelyaseactivityisactuallytionisrelativelyslow.IftheC11positionishydroxylatedpartofthesameenzyme(P450c17)thatcatalyzes17α-first,theactionof17-hydroxylaseisimpededandthehydroxylation.Thisisthereforeadualfunctionprotein.mineralocorticoidpathwayisfollowed(formingcorti-Thelyaseactivityisimportantinboththeadrenalsand
439THEDIVERSITYOFTHEENDOCRINESYSTEM/441CholesterolSCCCH3CH3COCOO—OH-HYDROXYLASE17,20-LYASEαHO17HOHOPregnenolone17-HydroxypregnenoloneDehydroepiandrosterone3β-HYDROXYSTEROIDDEHYDROGENASE:Δ5,4ISOMERASECH3CH3COCOO17—OH17P450cP450cOOOProgesterone17-HydroxyprogesteroneΔ4ANDROSTENE-3,17-DION21-HYDROXYLASECH2OHCH2OHCOCO—OHOO11-Deoxycorticosterone11-Deoxycortisol11β-HYDROXYLASECH2OHCH2OHCOCOHOHO—OHOOCorticosteroneCORTISOL18-HYDROXYLASE18-HYDROXYDEHYDROGENASECH2OHOCOHCHOOALDOSTERONEFigure42–4.Pathwaysinvolvedinthesynthesisofthethreemajorclassesofadrenalsteroids(mineralocorticoids,glucocorticoids,andandrogens).Enzymesareshownintherectangularboxes,andthemodificationsateachstepareshaded.Notethatthe17α-hydroxylaseand17,20-lyaseactivitiesarebothpartofoneenzyme,designatedP450c17.(Slightlymodifiedandreproduced,withpermis-sion,fromHardingBW:In:Endocrinology,vol2.DeGrootLJ[editor].Grune&Stratton,1979.)
440442/CHAPTER42thegonadsandactsexclusivelyon17α-hydroxy-contain-aregenerallyinactiveorlessactivethantheparentcom-ingmolecules.Adrenalandrogenproductionincreasespound.Metabolismbythesecondpathway,whichislessmarkedlyifglucocorticoidbiosynthesisisimpededbytheefficient,occursprimarilyintargettissuesandproduceslackofoneofthehydroxylases(adrenogenitalsyn-thepotentmetabolitedihydrotestosterone(DHT).drome).DHEAisreallyaprohormone,sincetheactionsThemostsignificantmetabolicproductoftestos-5,4of3β-OHSDandΔ-isomeraseconverttheweakandro-teroneisDHT,sinceinmanytissues,includinggenDHEAintothemorepotentandrostenedione.prostate,externalgenitalia,andsomeareasoftheskin,Smallamountsofandrostenedionearealsoformedinthethisistheactiveformofthehormone.Theplasmacon-adrenalbytheactionofthelyaseon17α-hydroxyproges-tentofDHTintheadultmaleisaboutone-tenththatterone.ReductionofandrostenedioneattheC17positionoftestosterone,andapproximately400μgofDHTisresultsintheformationoftestosterone,themostpotentproduceddailyascomparedwithabout5mgoftestos-adrenalandrogen.Smallamountsoftestosteronearepro-terone.About50–100μgofDHTaresecretedbytheducedintheadrenalbythismechanism,butmostofthistestes.Therestisproducedperipherallyfromtestos-conversionoccursinthetestes.teroneinareactioncatalyzedbytheNADPH-depen-dent5-reductase(Figure42–6).TestosteronecanTesticularSteroidogenesisthusbeconsideredaprohormone,sinceitisconvertedintoamuchmorepotentcompound(dihydrotestos-Testicularandrogensaresynthesizedintheinterstitialterone)andsincemostofthisconversionoccursoutsidetissuebytheLeydigcells.Theimmediateprecursorofthetestes.Someestradiolisformedfromtheperipheralthegonadalsteroids,asfortheadrenalsteroids,ischo-aromatizationoftestosterone,particularlyinmales.lesterol.Therate-limitingstep,asintheadrenal,isde-liveryofcholesteroltotheinnermembraneofthemito-OvarianSteroidogenesischondriabythetransportproteinStAR.Onceintheproperlocation,cholesterolisacteduponbythesideTheestrogensareafamilyofhormonessynthesizedinachaincleavageenzymeP450scc.Theconversionofcho-varietyoftissues.17β-Estradiolistheprimaryestrogenlesteroltopregnenoloneisidenticalinadrenal,ovary,ofovarianorigin.Insomespecies,estrone,synthesizedandtestis.Inthelattertwotissues,however,thereac-innumeroustissues,ismoreabundant.Inpregnancy,tionispromotedbyLHratherthanACTH.relativelymoreestriolisproduced,andthiscomesfromTheconversionofpregnenolonetotestosteronere-theplacenta.Thegeneralpathwayandthesubcellularquirestheactionoffiveenzymeactivitiescontainedinlocalizationoftheenzymesinvolvedintheearlystepsthreeproteins:(1)3β-hydroxysteroiddehydrogenase(3β-ofestradiolsynthesisarethesameasthoseinvolvedinOHSD)andΔ5,4-isomerase;(2)17α-hydroxylaseandandrogenbiosynthesis.Featuresuniquetotheovaryare17,20-lyase;and(3)17β-hydroxysteroiddehydrogenaseillustratedinFigure42–7.(17β-OHSD).Thissequence,referredtoastheproges-Estrogensareformedbythearomatizationofandro-terone(or4)pathway,isshownontherightsideofFig-gensinacomplexprocessthatinvolvesthreehydroxyla-ure42–5.Pregnenolonecanalsobeconvertedtotestos-tionsteps,eachofwhichrequiresO2andNADPH.Theteronebythedehydroepiandrosterone(or5)pathway,aromataseenzymecomplexisthoughttoincludeawhichisillustratedontheleftsideofFigure42–5.TheΔ5P450monooxygenase.Estradiolisformedifthesub-routeappearstobemostusedinhumantestes.strateofthisenzymecomplexistestosterone,whereases-Thefiveenzymeactivitiesarelocalizedinthemicro-troneresultsfromthearomatizationofandrostenedione.somalfractioninrattestes,andthereisaclosefunc-Thecellularsourceofthevariousovariansteroidshastionalassociationbetweentheactivitiesof3β-OHSDbeendifficulttounravel,butatransferofsubstratesbe-andΔ5,4-isomeraseandbetweenthoseofa17α-hydrox-tweentwocelltypesisinvolved.Thecacellsarethesourceylaseand17,20-lyase.Theseenzymepairs,bothcon-ofandrostenedioneandtestosterone.Theseareconvertedtainedinasingleprotein,areshowninthegeneralreac-bythearomataseenzymeingranulosacellstoestroneandtionsequenceinFigure42–5.estradiol,respectively.Progesterone,aprecursorforallsteroidhormones,isproducedandsecretedbythecorpusDihydrotestosteroneIsFormedFromluteumasanend-producthormonebecausethesecellsdonotcontaintheenzymesnecessarytoconvertproges-TestosteroneinPeripheralTissuesteronetoothersteroidhormones(Figure42–8).Testosteroneismetabolizedbytwopathways.Onein-Significantamountsofestrogensareproducedbyvolvesoxidationatthe17position,andtheotherin-theperipheralaromatizationofandrogens.InhumanvolvesreductionoftheAringdoublebondandthe3-ke-males,theperipheralaromatizationoftestosteronetotone.Metabolismbythefirstpathwayoccursinmanyestradiol(E2)accountsfor80%oftheproductionoftissues,includingliver,andproduces17-ketosteroidsthatthelatter.Infemales,adrenalandrogensareimportant
441THEDIVERSITYOFTHEENDOCRINESYSTEM/443CH3CH3COCOHOHOPregnenoloneProgesterone17α-HYDROXYLASE*17α-HYDROXYLASE*CH3CH3COCOOHOHISOMERASE5,4ΔHOO17α-Hydroxypregnenolone17α-Hydroxyprogesterone17,20-LYASE*17,20-LYASE*OOHO–HYDROXYSTEROIDDEHYDROGENASEANDOβDehydroepiandrosterone3Androstenedione17β-HYDROXYSTEROID17β-HYDROXYSTEROIDDEHYDROGENASEDEHYDROGENASEFigure42–5.Pathwaysoftestos-teronebiosynthesis.Thepathwayon5theleftsideofthefigureiscalledtheΔOHOHordehydroepiandrosteronepathway;thepathwayontherightsideiscalled4theΔorprogesteronepathway.Theas-teriskindicatesthatthe17α-hydroxy-laseand17,20-lyaseactivitiesresideinaHOOΔ5-AndrostenediolTESTOSTERONEsingleprotein,P450c17.
442444/CHAPTER42OHOH5α-REDUCTASENADPHOOHTestosteroneDIHYDROTESTOSTERONE(DHT)Figure42–6.Dihydrotestosteroneisformedfromtestosteronethroughactionoftheenzyme5α-reductase.CholesterolPregnenolone17α-HydroxypregnenoloneDehydroepiandrosteroneProgesterone17α-HydroxyprogesteroneAndrostenedioneTestosteroneAROMATASEAROMATASEOOHOthermetabolitesHOHOESTRONE(E1)17β-ESTRADIOL(E2)16α-HydroxylaseOthermetabolitesOHOHHOEstriolFigure42–7.Biosynthesisofestrogens.(Slightlymodifiedandreproduced,withpermission,fromGanongWF:ReviewofMedicalPhysiology,20thed.McGraw-Hill,2001.)
443THEDIVERSITYOFTHEENDOCRINESYSTEM/445Acetatemostoftheprecursorfor1,25(OH)2-D3synthesisisproducedinthemalpighianlayeroftheepidermisfromCholesterol7-dehydrocholesterolinanultravioletlight-mediated,CH3nonenzymaticphotolysisreaction.Theextentofthisconversionisrelateddirectlytotheintensityoftheex-COposureandinverselytotheextentofpigmentationintheskin.Thereisanage-relatedlossof7-dehydrocho-lesterolintheepidermisthatmayberelatedtotheneg-ativecalciumbalanceassociatedwitholdage.B.LIVERHOAspecifictransportproteincalledthevitaminD-bind-PregnenoloneingproteinbindsvitaminD3anditsmetabolitesandmovesvitaminD3fromtheskinorintestinetotheCH3liver,whereitundergoes25-hydroxylation,thefirstobligatoryreactionintheproductionof1,25(OH)2-COD3.25-Hydroxylationoccursintheendoplasmicretic-uluminareactionthatrequiresmagnesium,NADPH,molecularoxygen,andanuncharacterizedcytoplasmicfactor.Twoenzymesareinvolved:anNADPH-depen-dentcytochromeP450reductaseandacytochromeP450.Thisreactionisnotregulated,anditalsooccursOwithlowefficiencyinkidneyandintestine.The25(OH)2-D3entersthecirculation,whereitistheProgesteronemajorformofvitaminDfoundinplasma,andistrans-portedtothekidneybythevitaminD-bindingprotein.Figure42–8.Biosynthesisofprogesteroneinthecorpusluteum.C.KIDNEY25(OH)2-D3isaweakagonistandmustbemodifiedbyhydroxylationatpositionC1forfullbiologicactiv-substrates,sinceasmuchas50%oftheE2producedity.Thisisaccomplishedinmitochondriaoftherenalduringpregnancycomesfromthearomatizationofan-proximalconvolutedtubulebyathree-component2+drogens.Finally,conversionofandrostenedionetomonooxygenasereactionthatrequiresNADPH,Mg,estroneisthemajorsourceofestrogensinpost-molecularoxygen,andatleastthreeenzymes:(1)amenopausalwomen.Aromataseactivityispresentinflavoprotein,renalferredoxinreductase;(2)anironsul-adiposecellsandalsoinliver,skin,andothertissues.furprotein,renalferredoxin;and(3)cytochromeP450.IncreasedactivityofthisenzymemaycontributetotheThissystemproduces1,25(OH)2-D3,whichisthemost“estrogenization”thatcharacterizessuchdiseasesascir-potentnaturallyoccurringmetaboliteofvitaminD.rhosisoftheliver,hyperthyroidism,aging,andobesity.CATECHOLAMINES&THYROID1,25(OH)2-D3(Calcitriol)IsSynthesizedHORMONESAREMADEFROMTYROSINEFromaCholesterolDerivativeCatecholaminesAreSynthesizedinFinal1,25(OH)2-D3isproducedbyacomplexseriesofenzy-Form&StoredinSecretionGranulesmaticreactionsthatinvolvetheplasmatransportofpre-cursormoleculestoanumberofdifferenttissues(FigureThreeamines—dopamine,norepinephrine,andepi-42–9).OneoftheseprecursorsisvitaminD—reallynotnephrine—aresynthesizedfromtyrosineinthechro-avitamin,butthiscommonnamepersists.Theactivemaffincellsoftheadrenalmedulla.Themajorproductmolecule,1,25(OH)2-D3,istransportedtootherorgansoftheadrenalmedullaisepinephrine.Thiscompoundwhereitactivatesbiologicprocessesinamannersimilarconstitutesabout80%ofthecatecholaminesinthetothatemployedbythesteroidhormones.medulla,anditisnotmadeinextramedullarytissue.Incontrast,mostofthenorepinephrinepresentinorgansA.SKINinnervatedbysympatheticnervesismadeinsitu(aboutSmallamountsoftheprecursorfor1,25(OH)2-D3syn-80%ofthetotal),andmostoftherestismadeinotherthesisarepresentinfood(fishliveroil,eggyolk),butnerveendingsandreachesthetargetsitesviathecircu-
444446/CHAPTER42Sunlight7-DehydrocholesterolPrevitaminD3VitaminD325-HydroxylaseSKINLIVEROther25-Hydroxycholecalciferol(25[OH]-D3)metabolites24-Hydroxylase1α-Hydroxylase24,25(OH)2-D3KIDNEY1,25(OH)2-D31,24,25(OH)3-D3242725OH26CH2CH2HOHOHOOH7-DehydrocholesterolVitaminD31,25(OH)2-D3Figure42–9.FormationandhydroxylationofvitaminD3.25-Hydroxylationtakesplaceintheliver,andtheotherhydroxylationsoccurinthekidneys.25,26(OH)2-D3and1,25,26(OH)3-D3areprobablyformedaswell.Theformulasof7-dehydrocholesterol,vitaminD3,and1,25(OH)2-D3arealsoshown.(Modifiedandreproduced,withpermission,fromGanongWF:ReviewofMedicalPhysiology,20thed.McGraw-Hill,2001.)lation.Epinephrineandnorepinephrinemaybepro-Astherate-limitingenzyme,tyrosinehydroxylaseisregu-ducedandstoredindifferentcellsintheadrenallatedinavarietyofways.Themostimportantmecha-medullaandotherchromaffintissues.nisminvolvesfeedbackinhibitionbythecatecholamines,Theconversionoftyrosinetoepinephrinerequireswhichcompetewiththeenzymeforthepteridinecofac-foursequentialsteps:(1)ringhydroxylation;(2)decar-tor.Catecholaminescannotcrosstheblood-brainbarrier;boxylation;(3)sidechainhydroxylationtoformnorepi-hence,inthebraintheymustbesynthesizedlocally.Innephrine;and(4)N-methylationtoformepinephrine.certaincentralnervoussystemdiseases(eg,Parkinson’sThebiosyntheticpathwayandtheenzymesinvolvedaredisease),thereisalocaldeficiencyofdopaminesynthesis.illustratedinFigure42–10.L-Dopa,theprecursorofdopamine,readilycrossestheblood-brainbarrierandsoisanimportantagentintheA.TYROSINEHYDROXYLASEISRATE-LIMITINGtreatmentofParkinson’sdisease.FORCATECHOLAMINEBIOSYNTHESISTyrosineistheimmediateprecursorofcatecholamines,B.DOPADECARBOXYLASEISPRESENTINALLTISSUESandtyrosinehydroxylaseistherate-limitingenzymeinThissolubleenzymerequirespyridoxalphosphateforcatecholaminebiosynthesis.TyrosinehydroxylaseistheconversionofL-dopato3,4-dihydroxyphenylethyl-foundinbothsolubleandparticle-boundformsonlyinamine(dopamine).CompoundsthatresembleL-dopa,tissuesthatsynthesizecatecholamines;itfunctionsasansuchasα-methyldopa,arecompetitiveinhibitorsofoxidoreductase,withtetrahydropteridineasacofactor,tothisreaction.α-MethyldopaiseffectiveintreatingconvertL-tyrosinetoL-dihydroxyphenylalanine(L-dopa).somekindsofhypertension.
445THEDIVERSITYOFTHEENDOCRINESYSTEM/447Otheconversionofdopaminetonorepinephrineoccursinthisorganelle.HCOHD.PHENYLETHANOLAMINE-N-METHYLTRANSFERASEHOCCNH2(PNMT)CATALYZESTHEPRODUCTIONHHOFEPINEPHRINETyrosinePNMTcatalyzestheN-methylationofnorepinephrineTYROSINEHYDROXYLASEtoformepinephrineintheepinephrine-formingcellsoftheadrenalmedulla.SincePNMTissoluble,itisas-Osumedthatnorepinephrine-to-epinephrineconversionHOHCOHoccursinthecytoplasm.ThesynthesisofPNMTisin-ducedbyglucocorticoidhormonesthatreachtheHOCCNH2medullaviatheintra-adrenalportalsystem.ThisspecialHHsystemprovidesfora100-foldsteroidconcentrationDopagradientoversystemicarterialblood,andthishighDOPAintra-adrenalconcentrationappearstobenecessaryforDECARBOXYLASEtheinductionofPNMT.HOT3&T4IllustratetheDiversityHHinHormoneSynthesisHOCCNH2Theformationoftriiodothyronine(T3)andtetra-HHiodothyronine(thyroxine;T4)(seeFigure42–2)illus-DOPAMINEtratesmanyoftheprinciplesofdiversitydiscussedinDOPAMINEthischapter.Thesehormonesrequirearareelementβ-HYDROXYLASE(iodine)forbioactivity;theyaresynthesizedaspartofaverylargeprecursormolecule(thyroglobulin);theyarestoredinanintracellularreservoir(colloid);andthereisHOHperipheralconversionofT4toT3,whichisamuchOHmoreactivehormone.HOCCNH2ThethyroidhormonesT3andT4areuniqueinthatiodine(asiodide)isanessentialcomponentofboth.InHHNOREPINEPHRINEmostpartsoftheworld,iodineisascarcecomponentofsoil,andforthatreasonthereislittleinfood.Acom-PNMTplexmechanismhasevolvedtoacquireandretainthiscrucialelementandtoconvertitintoaformsuitableforincorporationintoorganiccompounds.Atthesametime,thethyroidmustsynthesizethyroninefromtyro-HOHOHCH3sine,andthissynthesistakesplaceinthyroglobulin(Figure42–11).HOCCNHThyroglobulinistheprecursorofT4andT3.ItisaHHlargeiodinated,glycosylatedproteinwithamolecularEPINEPHRINEmassof660kDa.Carbohydrateaccountsfor8–10%ofFigure42–10.Biosynthesisofcatecholamines.theweightofthyroglobulinandiodideforabout0.2–1%,dependingupontheiodinecontentinthe(PNMT,phenylethanolamine-N-methyltransferase.)diet.Thyroglobuliniscomposedoftwolargesubunits.Itcontains115tyrosineresidues,eachofwhichisapo-tentialsiteofiodination.About70%oftheiodideinthyroglobulinexistsintheinactiveprecursors,C.DOPAMINE-HYDROXYLASE(DBH)CATALYZESmonoiodotyrosine(MIT)anddiiodotyrosine(DIT),THECONVERSIONOFDOPAMINETONOREPINEPHRINEwhile30%isintheiodothyronylresidues,T4andT3.DBHisamonooxygenaseandusesascorbateasanelec-Wheniodinesuppliesaresufficient,theT4:T3ratioistrondonor,copperattheactivesite,andfumarateasabout7:1.Iniodinedeficiency,thisratiodecreases,asmodulator.DBHisintheparticulatefractionofthedoestheDIT:MITratio.Thyroglobulin,alargemole-medullarycells,probablyinthesecretiongranule;thus,culeofabout5000aminoacids,providestheconfor-
446448/CHAPTER42FOLLICULARSPACEWITHCOLLOIDMITMITTMIT3DITDITDITDITOxidationIodination*Coupling*I++TgbTgbDITTgbT4PEROXIDASEMITMITMITDITH2O2DITDITDITT4I–PhagocytosisO2andTgbNADPHNADP+pinocytosisH+LysosomesTHYROIDCELLTgbSecondaryTgblysosomeTyrosineHydrolysisDeiodination*MITI–DEIODINASEDITI–Concentration*T3,T4Na+-K+ATPaseReleaseTrans-porterEXTRACELLULARSPACEI–T3,T4Figure42–11.Modelofiodidemetabolisminthethyroidfollicle.Afollicularcellisshownfacingthefollicularlumen(top)andtheextracellularspace(atbottom).Iodideentersthethyroidprimarilythroughatransporter(bottomleft).Thyroidhormonesynthesisoccursinthefollicularspacethroughaseriesofreactions,manyofwhichareperoxidase-mediated.Thyroidhormones,storedinthecolloidinthefollicularspace,arereleasedfromthyroglobulinbyhydrolysisinsidethethyroidcell.(Tgb,thyroglobulin;MIT,monoiodotyrosine;DIT,diiodotyro-sine;T3,triiodothyronine;T4,tetraiodothyronine.)Asterisksindicatestepsorprocessesthatareinheritedenzymedeficiencieswhichcausecongenitalgoiterandoftenresultinhypothyroidism.
447THEDIVERSITYOFTHEENDOCRINESYSTEM/449mationrequiredfortyrosylcouplingandiodideorgani-monesremainasintegralpartsofthyroglobulinuntilficationnecessaryintheformationofthediaminoacidthelatterisdegraded,asdescribedabove.−thyroidhormones.ItissynthesizedinthebasalportionAdeiodinaseremovesIfromtheinactivemono-ofthecellandmovestothelumen,whereitisastorageanddiiodothyroninemoleculesinthethyroid.This−formofT3andT4inthecolloid;severalweeks’supplymechanismprovidesasubstantialamountoftheIusedofthesehormonesexistinthenormalthyroid.WithininT3andT4biosynthesis.AperipheraldeiodinaseinminutesafterstimulationofthethyroidbyTSH,col-targettissuessuchaspituitary,kidney,andliverselec-−loidreentersthecellandthereisamarkedincreaseoftivelyremovesIfromthe5′positionofT4tomakeT3phagolysosomeactivity.Variousacidproteasesand(seeFigure42–2),whichisamuchmoreactivemole-peptidaseshydrolyzethethyroglobulinintoitscon-cule.Inthissense,T4canbethoughtofasaprohor-stituentaminoacids,includingT4andT3,whicharemone,thoughitdoeshavesomeintrinsicactivity.dischargedfromthebasalportionofthecell(seeFigure42–11).Thyroglobulinisthusaverylargeprohor-SEVERALHORMONESAREMADEFROMmone.LARGERPEPTIDEPRECURSORSIodideMetabolismInvolvesFormationofthecriticaldisulfidebridgesininsulinre-SeveralDiscreteStepsquiresthatthishormonebefirstsynthesizedaspartofalargerprecursormolecule,proinsulin.Thisisconceptu-−ThethyroidisabletoconcentrateIagainstastrongallysimilartotheexampleofthethyroidhormones,electrochemicalgradient.Thisisanenergy-dependentwhichcanonlybeformedinthecontextofamuchprocessandislinkedtotheNa+-K+ATPase-dependentlargermolecule.Severalotherhormonesaresynthesized−thyroidalItransporter.Theratioofiodideinthyroidaspartsoflargeprecursormolecules,notbecauseoftoiodideinserum(T:Sratio)isareflectionoftheac-somespecialstructuralrequirementbutratherasativityofthistransporter.Thisactivityisprimarilycon-mechanismforcontrollingtheavailableamountofthetrolledbyTSHandrangesfrom500:1inanimalsactivehormone.PTHandangiotensinIIareexampleschronicallystimulatedwithTSHto5:1orlessinhy-ofthistypeofregulation.Anotherinterestingexamplepophysectomizedanimals(noTSH).TheT:SratioinisthePOMCprotein,whichcanbeprocessedintohumansonanormaliodinedietisabout25:1.manydifferenthormonesinatissue-specificmanner.−ThethyroidistheonlytissuethatcanoxidizeItoaTheseexamplesarediscussedindetailbelow.−highervalencestate,anobligatorystepinIorganifica-tionandthyroidhormonebiosynthesis.Thisstepin-InsulinIsSynthesizedasaPreprohormonevolvesaheme-containingperoxidaseandoccursatthe&ModifiedWithintheCellluminalsurfaceofthefollicularcell.Thyroperoxidase,atetramericproteinwithamolecularmassof60kDa,re-InsulinhasanABheterodimericstructurewithonein-quireshydrogenperoxideasanoxidizingagent.Thetrachain(A6–A11)andtwointerchaindisulfidebridgesH2O2isproducedbyanNADPH-dependentenzyme(A7–B7andA20–B19)(Figure42–12).TheAandBresemblingcytochromecreductase.Anumberofcom-chainscouldbesynthesizedinthelaboratory,butat-−temptsatabiochemicalsynthesisofthematureinsulinpoundsinhibitIoxidationandthereforeitssubse-quentincorporationintoMITandDIT.Themostim-moleculeyieldedverypoorresults.Thereasonforthisportantofthesearethethioureadrugs.Theyareusedasbecameapparentwhenitwasdiscoveredthatinsulinisantithyroiddrugsbecauseoftheirabilitytoinhibitthy-synthesizedasapreprohormone(molecularweightap-roidhormonebiosynthesisatthisstep.Onceiodinationproximately11,500),whichistheprototypeforpeptidesoccurs,theiodinedoesnotreadilyleavethethyroid.thatareprocessedfromlargerprecursormolecules.TheFreetyrosinecanbeiodinated,butitisnotincorpo-hydrophobic23-amino-acidpre-,orleader,sequencedi-ratedintoproteinssincenotRNArecognizesiodinatedrectsthemoleculeintothecisternaeoftheendoplasmictyrosine.reticulumandthenisremoved.Thisresultsinthe9000-ThecouplingoftwoDITmoleculestoformT4—orMWproinsulinmolecule,whichprovidestheconforma-ofanMITandDITtoformT3—occurswithinthetionnecessaryfortheproperandefficientformationofthyroglobulinmolecule.Aseparatecouplingenzymethedisulfidebridges.AsshowninFigure42–12,these-hasnotbeenfound,andsincethisisanoxidativequenceofproinsulin,startingfromtheaminoterminal,processitisassumedthatthesamethyroperoxidasecat-isBchain—connecting(C)peptide—Achain.Thealyzesthisreactionbystimulatingfreeradicalforma-proinsulinmoleculeundergoesaseriesofsite-specifictionofiodotyrosine.Thishypothesisissupportedbypeptidecleavagesthatresultintheformationofequimo-−laramountsofmatureinsulinandCpeptide.Theseen-theobservationthatthesamedrugswhichinhibitIox-idationalsoinhibitcoupling.Theformedthyroidhor-zymaticcleavagesaresummarizedinFigure42–12.
448450/CHAPTER4220GlnLeuSerGlyAlaGlyProProLeuGlyGlyAlaGlyLeuConnectingpeptideLeuGluGlu10GlyValSerGlnLeu31GlyGlnValLysGlnArgLeu1GlyAspIleNH–COOHGlu2ValAsn–21GluCysAla1PheSGlnSTyrGlu1ValAsnCysAchainGluArgAsnCysLeuThrSerGlnSArgGlnIleCysSerLeuTyrSMis10Thr30LeuSLysCysInsulinProSThrGlyTyrSerBchainPheMisPheLeuGlyArg10ValGluGluGlyAlaLeuTyrLeuValCys20Figure42–12.Structureofhumanproinsulin.InsulinandC-peptidemoleculesareconnectedattwositesbydipeptidelinks.Aninitialcleavagebyatrypsin-likeenzyme(openarrows)followedbyseveralcleavagesbyacar-boxypeptidase-likeenzyme(solidarrows)resultsintheproductionoftheheterodimeric(AB)insulinmolecule(lightblue)andtheC-peptide.ParathyroidHormone(PTH)IsSecretedasmRNA,andthisisfollowedbyanincreasedrateofan84-Amino-AcidPeptidePTHsynthesisandsecretion.However,about80–90%oftheproPTHsynthesizedcannotbeaccountedforasTheimmediateprecursorofPTHisproPTH,whichintactPTHincellsorintheincubationmediumofex-differsfromthenative84-amino-acidhormonebyhav-perimentalsystems.Thisfindingledtotheconclusioningahighlybasichexapeptideaminoterminalexten-thatmostoftheproPTHsynthesizedisquicklyde-sion.Theprimarygeneproductandtheimmediatepre-graded.Itwaslaterdiscoveredthatthisrateofdegrada-cursorforproPTHisthe115-amino-acidpreproPTH.2+tiondecreaseswhenCaconcentrationsarelow,anditThisdiffersfromproPTHbyhavinganadditional25-2+increaseswhenCaconcentrationsarehigh.Veryspe-amino-acidaminoterminalextensionthat,incommoncificfragmentsofPTHaregeneratedduringitsprote-withtheotherleaderorsignalsequencescharacteristicolyticdigestion(Figure42–13).Anumberofprote-ofsecretedproteins,ishydrophobic.Thecompleteolyticenzymes,includingcathepsinsBandD,havestructureofpreproPTHandthesequencesofproPTHbeenidentifiedinparathyroidtissue.CathepsinBandPTHareillustratedinFigure42–13.PTH1–34hascleavesPTHintotwofragments:PTH1–36andfullbiologicactivity,andtheregion25–34isprimarilyPTH37–84.PTH37–84isnotfurtherdegraded;however,responsibleforreceptorbinding.PTH1–36israpidlyandprogressivelycleavedintodi-ThebiosynthesisofPTHanditssubsequentsecre-andtripeptides.MostoftheproteolysisofPTHoccurs2+tionareregulatedbytheplasmaionizedcalcium(Ca)withinthegland,butanumberofstudiesconfirmthatconcentrationthroughacomplexprocess.Anacutede-PTH,oncesecreted,isproteolyticallydegradedinother2+creaseofCaresultsinamarkedincreaseofPTHtissues,especiallytheliver,bysimilarmechanisms.
449THEDIVERSITYOFTHEENDOCRINESYSTEM/451Leader(pre)sequence–6–10–20–31ProLysGlyAspSerArgAlaLeuPheCysIleAlaLeuMetValIleMetValLysValMetAspLysAlaSerMetMetNH2SersequenceValLys(2)(1)Lys–1Arg(3)1AlaValSer1020GluIleGlnPheMetHisAsnLeuGlyLysHisLeuSerSerMetGluArgValGluTrpLeuArgLysLysLeuFullbiologicactivitysequenceGln30AspValHisAsnC-fragmentsequencePhe5040ValAspGluLysLysArgProArgGlnSerSerGlyAspArgTyrAlaIleSerAlaGlyLeuAlaAsnVal(4)60LeuVal(5)GluSerHisGlnLysO7080SerLeuGlyGluAlaAspLysAlaAspValAspValLeuIleLysAlaLysProGlnCOHFigure42–13.Structureofbovinepreproparathyroidhormone.Arrowsindicatesitescleavedbypro-cessingenzymesintheparathyroidgland(1–5)andintheliveraftersecretionofthehormone(4–5).Thebiologicallyactiveregionofthemoleculeisflankedbysequencenotrequiredforactivityontargetre-ceptors.(Slightlymodifiedandreproduced,withpermission,fromHabenerJF:Recentadvancesinparathy-roidhormoneresearch.ClinBiochem1981;14:223.)AngiotensinIIIsAlsoSynthesizedtorsthatdecreasesfluidvolume(dehydration,decreasedFromaLargePrecursorbloodpressure,fluidorbloodloss)ordecreasesNaClconcentrationstimulatesreninrelease.Renalsympa-Therenin-angiotensinsystemisinvolvedintheregula-theticnervesthatterminateinthejuxtaglomerularcellstionofbloodpressureandelectrolytemetabolismmediatethecentralnervoussystemandposturaleffects(throughproductionofaldosterone).Theprimaryhor-onreninreleaseindependentlyofthebaroreceptorandmoneinvolvedintheseprocessesisangiotensinII,ansalteffects,amechanismthatinvolvestheβ-adrenergicoctapeptidemadefromangiotensinogen(Figurereceptor.Reninactsuponthesubstrateangiotensino-42–14).Angiotensinogen,alargeα2-globulinmadeingentoproducethedecapeptideangiotensinI.liver,isthesubstrateforrenin,anenzymeproducedinAngiotensin-convertingenzyme,aglycoproteinthejuxtaglomerularcellsoftherenalafferentarteriole.foundinlung,endothelialcells,andplasma,removesThepositionofthesecellsmakesthemparticularlysen-twocarboxylterminalaminoacidsfromthedecapep-sitivetobloodpressurechanges,andmanyofthephysi-tideangiotensinItoformangiotensinIIinastepthatologicregulatorsofreninreleaseactthroughrenalisnotthoughttoberate-limiting.Variousnonapeptidebaroreceptors.Thejuxtaglomerularcellsarealsosensi-analogsofangiotensinIandothercompoundsactastivetochangesofNa+andCl−concentrationinthecompetitiveinhibitorsofconvertingenzymeandarerenaltubularfluid;therefore,anycombinationoffac-usedtotreatrenin-dependenthypertension.Theseare
450452/CHAPTER42AngiotensinogenAsp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu(~400moreaminoacids)RENINAngiotensinIAsp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-LeuCONVERTINGENZYMEANGIOTENSINIIAsp-Arg-Val-Tyr-Ile-His-Pro-PheAMINOPEPTIDASEAngiotensinIIIArg-Val-Tyr-Ile-His-Pro-PheANGIOTENSINASESDegradationproductsFigure42–14.Formationandmetabolismofangiotensins.Smallarrowsin-dicatecleavagesites.referredtoasangiotensin-convertingenzyme(ACE)ComplexProcessingGeneratesinhibitors.AngiotensinIIincreasesbloodpressurebythePro-opiomelanocortin(POMC)causingvasoconstrictionofthearterioleandisaveryPeptideFamilypotentvasoactivesubstance.Itinhibitsreninreleasefromthejuxtaglomerularcellsandisapotentstimula-ThePOMCfamilyconsistsofpeptidesthatactashor-torofaldosteroneproduction.ThisresultsinNa+re-mones(ACTH,LPH,MSH)andothersthatmayservetention,volumeexpansion,andincreasedbloodpres-asneurotransmittersorneuromodulators(endorphins)sure.(seeFigure42–15).POMCissynthesizedasaprecur-Insomespecies,angiotensinIIisconvertedtothesormoleculeof285aminoacidsandisprocesseddiffer-heptapeptideangiotensinIII(Figure42–14),anequallyentlyinvariousregionsofthepituitary.potentstimulatorofaldosteroneproduction.Inhu-ThePOMCgeneisexpressedintheanteriorandin-mans,theplasmalevelofangiotensinIIisfourtimestermediatelobesofthepituitary.ThemostconservedgreaterthanthatofangiotensinIII,somosteffectsaresequencesbetweenspeciesarewithintheaminotermi-exertedbytheoctapeptide.AngiotensinsIIandIIIarenalfragment,theACTHregion,andtheβ-endorphinrapidlyinactivatedbyangiotensinases.region.POMCorrelatedproductsarefoundinseveralAngiotensinIIbindstospecificadrenalcortexothervertebratetissues,includingthebrain,placenta,glomerulosacellreceptors.Thehormone-receptorin-gastrointestinaltract,reproductivetract,lung,andlym-teractiondoesnotactivateadenylylcyclase,andcAMPphocytes.doesnotappeartomediatetheactionofthishormone.ThePOMCproteinisprocesseddifferentlyinthean-TheactionsofangiotensinII,whicharetostimulateteriorlobethanintheintermediatelobe.Theintermedi-theconversionofcholesteroltopregnenoloneandofatelobeofthepituitaryisrudimentaryinadulthumans,corticosteroneto18-hydroxycorticosteroneandaldos-butitisactiveinhumanfetusesandinpregnantwomenterone,mayinvolvechangesintheconcentrationofin-duringlategestationandisalsoactiveinmanyanimaltracellularcalciumandofphospholipidmetabolitesbyspecies.ProcessingofthePOMCproteinintheperiph-mechanismssimilartothosedescribedinChapter43.eraltissues(gut,placenta,malereproductivetract)resem-
451THEDIVERSITYOFTHEENDOCRINESYSTEM/453POMC(1–134)ACTH(1–39)β-LPH(42–134)α-MSHCLIPγ-LPHβ-Endorphin(1–13)(18–39)(42–101)(104–134)β-MSHγ-Endorphin(84–101)(104–118)α-Endorphin(104–117)Figure42–15.Productsofpro-opiomelanocortin(POMC)cleavage.(MSH,melanocyte-stimulatinghormone;CLIP,corticotropin-likeinter-mediatelobepeptide;LPH,lipotropin.)blesthatintheintermediatelobe.Therearethreebasicthereisnointracellularreservoirofthesehormones.peptidegroups:(1)ACTH,whichcangiverisetoThecatecholamines,alsosynthesizedinactiveform,areα-MSHandcorticotropin-likeintermediatelobepeptidestoredingranulesinthechromaffincellsintheadrenal(CLIP);(2)β-lipotropin(β-LPH),whichcanyieldmedulla.Inresponsetoappropriateneuralstimulation,γ-LPH,β-MSH,andβ-endorphin(andthusα-andthesegranulesarereleasedfromthecellthroughexocy-γ-endorphins);and(3)alargeaminoterminalpeptide,tosis,andthecatecholaminesarereleasedintothecircu-whichgeneratesγ-MSH.Thediversityoftheseproductslation.Aseveral-hourreservesupplyofcatecholaminesisduetothemanydibasicaminoacidclustersthatareexistsinthechromaffincells.potentialcleavagesitesfortrypsin-likeenzymes.EachofParathyroidhormonealsoexistsinstoragevesicles.thepeptidesmentionedisprecededbyLys-Arg,Arg-Lys,Asmuchas80–90%oftheproPTHsynthesizedisde-Arg-Arg,orLys-Lysresidues.Aftertheprehormoneseg-gradedbeforeitentersthisfinalstoragecompartment,2+mentiscleaved,thenextcleavage,inbothanteriorandespeciallywhenCalevelsarehighintheparathyroid2+intermediatelobes,isbetweenACTHandβ-LPH,re-cell(seeabove).PTHissecretedwhenCaislowinsultinginanaminoterminalpeptidewithACTHandatheparathyroidcells,whichcontainaseveral-hoursup-β-LPHsegment(Figure42–15).ACTH1–39issubse-plyofthehormone.quentlycleavedfromtheaminoterminalpeptide,andinThehumanpancreassecretesabout40–50unitsofin-theanteriorlobeessentiallynofurthercleavagesoccur.Insulindaily,whichrepresentsabout15–20%ofthehor-theintermediatelobe,ACTH1–39iscleavedintoα-MSHmonestoredintheBcells.InsulinandtheC-peptide(see(residues1–13)andCLIP(18–39);β-LPH(42–134)isFigure42–12)arenormallysecretedinequimolarconvertedtoγ-LPH(42–101)andβ-endorphin(104–amounts.Stimulisuchasglucose,whichprovokesinsulin134).β-MSH(84–101)isderivedfromγ-LPH.secretion,thereforetriggertheprocessingofproinsulintoThereareextensiveadditionaltissue-specificmodifi-insulinasanessentialpartofthesecretoryresponse.cationsofthesepeptidesthataffectactivity.TheseAseveral-weeksupplyofT3andT4existsinthethy-modificationsincludephosphorylation,acetylation,roglobulinthatisstoredincolloidinthelumenoftheglycosylation,andamidation.thyroidfollicles.ThesehormonescanbereleaseduponstimulationbyTSH.Thisisthemostexaggeratedex-THEREISVARIATIONINTHESTORAGEampleofaprohormone,asamoleculecontainingap-&SECRETIONOFHORMONESproximately5000aminoacidsmustbefirstsynthe-sized,thendegraded,tosupplyafewmoleculesoftheAsmentionedabove,thesteroidhormonesandactivehormonesT4andT3.1,25(OH)2-D3aresynthesizedintheirfinalactiveThediversityinstorageandsecretionofhormonesform.Theyarealsosecretedastheyaremade,andthusisillustratedinTable42–5.
452454/CHAPTER42Table42–5.Diversityinthestorageofhormones.lives.AnotableexceptionisIGF-I,whichistransportedboundtomembersofafamilyofbindingproteins.HormoneSupplyStoredinCellThyroidHormonesAreTransportedSteroidsand1,25(OH)2-D3NonebyThyroid-BindingGlobulinCatecholaminesandPTHHoursManyoftheprinciplesdiscussedaboveareillustratedinInsulinDaysadiscussionofthyroid-bindingproteins.One-halftoT3andT4Weekstwo-thirdsofT4andT3inthebodyisinanextrathy-roidalreservoir.Mostofthiscirculatesinboundform,ie,boundtoaspecificbindingprotein,thyroxine-SOMEHORMONESHAVEPLASMAbindingglobulin(TBG).TBG,aglycoproteinwithamolecularmassof50kDa,bindsT4andT3andhastheTRANSPORTPROTEINScapacitytobind20μg/dLofplasma.UndernormalTheclassIhormonesarehydrophobicinchemicalna-circumstances,TBGbinds—noncovalently—nearlyalltureandthusarenotverysolubleinplasma.Thesehor-oftheT4andT3inplasma,anditbindsT4withgreatermones,principallythesteroidsandthyroidhormones,affinitythanT3(Table42–7).Theplasmahalf-lifeofhavespecializedplasmatransportproteinsthatservesev-T4iscorrespondinglyfourtofivetimesthatofT3.Theeralpurposes.First,theseproteinscircumventthesolu-small,unbound(free)fractionisresponsibleforthebi-bilityproblemandtherebydeliverthehormonetotheologicactivity.Thus,inspiteofthegreatdifferenceintargetcell.Theyalsoprovideacirculatingreservoiroftotalamount,thefreefractionofT3approximatesthatthehormonethatcanbesubstantial,asinthecaseoftheofT4,andgiventhatT3isintrinsicallymoreactivethanthyroidhormones.Hormones,whenboundtothetrans-T4,mostbiologicactivityisattributedtoT3.TBGdoesportproteins,cannotbemetabolized,therebyprolongingnotbindanyotherhormones.theirplasmahalf-life(t1/2).ThebindingaffinityofagivenhormonetoitstransporterdeterminestheboundGlucocorticoidsAreTransportedversusfreeratioofthehormone.Thisisimportantbe-byCorticosteroid-BindingGlobulincauseonlythefreeformofahormoneisbiologicallyac-Hydrocortisone(cortisol)alsocirculatesinplasmaintive.Ingeneral,theconcentrationoffreehormoneinplasmaisverylow,intherangeof10–15to10–9mol/L.Itprotein-boundandfreeforms.Themainplasmabind-ingproteinisanα-globulincalledtranscortin,orcor-isimportanttodistinguishbetweenplasmatransportticosteroid-bindingglobulin(CBG).CBGispro-proteinsandhormonereceptors.Bothbindhormonesducedintheliver,anditssynthesis,likethatofTBG,isbutwithverydifferentcharacteristics(Table42–6).increasedbyestrogens.CBGbindsmostofthehor-Thehydrophilichormones—generallyclassIIandmonewhenplasmacortisollevelsarewithinthenormalofpeptidestructure—arefreelysolubleinplasmaandrange;muchsmalleramountsofcortisolareboundtodonotrequiretransportproteins.Hormonessuchasalbumin.Theavidityofbindinghelpsdeterminetheinsulin,growthhormone,ACTH,andTSHcirculatebiologichalf-livesofvariousglucocorticoids.Cortisolinthefree,activeformandhaveveryshortplasmahalf-bindstightlytoCBGandhasat1/2of1.5–2hours,whilecorticosterone,whichbindslesstightly,hasat1/2Table42–6.Comparisonofreceptorswithoflessthan1hour(Table42–8).Theunbound(free)transportproteins.cortisolconstitutesabout8%ofthetotalandrepresentsthebiologicallyactivefraction.BindingtoCBGisnotrestrictedtoglucocorticoids.DeoxycorticosteroneandFeatureReceptorsTransportProteinsConcentrationVerylowVeryhigh(thousands/cell)(billions/μL)BindingaffinityHigh(pmol/LtoLow(μmol/Lrange)Table42–7.ComparisonofT4andT3inplasma.nmol/Lrange)FreeHormoneTotalt1BindingspecificityVeryhighLow2HormonePercentinBloodSaturabilityYesNo(μg/dL)ofTotalng/dLMolarity(days)ReversibilityYesYes−11T480.03~2.243.0×106.5−11SignaltransductionYesNoT30.150.3~0.4~0.6×101.5
453THEDIVERSITYOFTHEENDOCRINESYSTEM/455Table42–8.Approximateaffinitiesofsteroidsforbindingcapacitytheyprobablybufferagainstsuddenserum-bindingproteins.changesintheplasmalevel.Becausethemetabolicclearanceratesofthesesteroidsareinverselyrelatedto11theaffinityoftheirbindingtoSHBG,estroneisclearedSHBGCBGmorerapidlythanestradiol,whichinturnisclearedDihydrotestosterone1>100morerapidlythantestosteroneorDHT.Testosterone2>100Estradiol5>10SUMMARYEstrone>10>100Progesterone>100~2•ThepresenceofaspecificreceptordefinesthetargetCortisol>100~3cellsforagivenhormone.Corticosterone>100~5•Receptorsareproteinsthatbindspecifichormones1AffinityexpressedasKd(nmol/L).andgenerateanintracellularsignal(receptor-effectorcoupling).•Somehormoneshaveintracellularreceptors;othersprogesteroneinteractwithCBGwithsufficientaffinitybindtoreceptorsontheplasmamembrane.tocompeteforcortisolbinding.Aldosterone,themost•Hormonesaresynthesizedfromanumberofprecur-potentnaturalmineralocorticoid,doesnothaveaspe-sormolecules,includingcholesterol,tyrosineperse,cificplasmatransportprotein.GonadalsteroidsbindandalltheconstituentaminoacidsofpeptidesandveryweaklytoCBG(Table42–8).proteins.GonadalSteroidsAreTransported•Anumberofmodificationprocessesaltertheactivityofhormones.Forexample,manyhormonesaresyn-bySexHormone-BindingGlobulinthesizedfromlargerprecursormolecules.Mostmammals,humansincluded,haveaplasmaβ-•Thecomplementofenzymesinaparticularcelltypeglobulinthatbindstestosteronewithspecificity,rela-allowsfortheproductionofaspecificclassofsteroidtivelyhighaffinity,andlimitedcapacity(Table42–8).hormone.Thisprotein,usuallycalledsexhormone-binding•Mostofthelipid-solublehormonesareboundtoglobulin(SHBG)ortestosterone-estrogen-bindingratherspecificplasmatransportproteins.globulin(TEBG),isproducedintheliver.Itsproduc-tionisincreasedbyestrogens(womenhavetwicetheserumconcentrationofSHBGasmen),certaintypesofREFERENCESliverdisease,andhyperthyroidism;itisdecreasedbyBartalinaL:Thyroidhormone-bindingproteins:update1994.En-androgens,advancingage,andhypothyroidism.ManydocrRev1994;13:140.oftheseconditionsalsoaffecttheproductionofCBGBeatoMetal:Steroidhormonereceptors:manyactorsinsearchofandTBG.SinceSHBGandalbuminbind97–99%ofaplot.Cell1995;83:851.circulatingtestosterone,onlyasmallfractionoftheDaiG,CarrascoL,CarrascoN:Cloningandcharacterizationofhormoneincirculationisinthefree(biologicallyac-thethyroidiodidetransporter.Nature1996;379:458.tive)form.TheprimaryfunctionofSHBGmaybetoDeLucaHR:ThevitaminDstory:acollaborativeeffortofbasicrestrictthefreeconcentrationoftestosteroneinthescienceandclinicalmedicine.FASEBJ1988;2:224.serum.TestosteronebindstoSHBGwithhigheraffin-DouglassJ,CivelliO,HerbertE:Polyproteingeneexpression:itythandoesestradiol(Table42–8).Therefore,aGenerationofdiversityofneuroendocrinepeptides.AnnuchangeinthelevelofSHBGcausesagreaterchangeinRevBiochem1984;53:665.thefreetestosteronelevelthaninthefreeestradiollevel.MillerWL:Molecularbiologyofsteroidhormonebiosynthesis.EstrogensareboundtoSHBGandprogestinstoEndocrRev1988;9:295.CBG.SHBGbindsestradiolaboutfivetimeslessavidlyNagatsuT:Genesforhumancatecholamine-synthesizingenzymes.thanitbindstestosteroneorDHT,whileprogesteroneNeurosciRes1991;12:315.andcortisolhavelittleaffinityforthisprotein(TableRussellDW,WilsonJD:Steroid5alpha-reductase:twogenes/twoenzymes.AnnuRevBiochem1994;63:25.42–8).Incontrast,progesteroneandcortisolbindwithRussellJetal:Interactionbetweencalciumand1,25-dihydroxy-nearlyequalaffinitytoCBG,whichinturnhaslittlevitaminD3intheregulationofpreproparathyroidhormoneavidityforestradiolandevenlessfortestosterone,andvitaminDreceptormRNAinavianparathyroids.En-DHT,orestrone.docrinology1993;132:2639.ThesebindingproteinsalsoprovideacirculatingSteinerDFetal:Thenewenzymologyofprecursorprocessingen-reservoirofhormone,andbecauseoftherelativelylargedoproteases.JBiolChem1992;267:23435.
454HormoneAction&SignalTransduction43DarylK.Granner,MDBIOMEDICALIMPORTANCEasdescribedinChapter42,ie,basedonthelocationoftheirspecificcellularreceptorsandthetypeofsignalsThehomeostaticadaptationsanorganismmakestoagenerated.GroupIhormonesinteractwithanintracel-constantlychangingenvironmentareinlargepartac-lularreceptorandgroupIIhormoneswithreceptorcomplishedthroughalterationsoftheactivityandrecognitionsiteslocatedontheextracellularsurfaceofamountofproteins.Hormonesprovideamajormeanstheplasmamembraneoftargetcells.Thecytokines,in-offacilitatingthesechanges.Ahormone-receptorinter-terleukins,andgrowthfactorsshouldalsobeconsideredactionresultsingenerationofanintracellularsignalinthislattercategory.Thesemolecules,ofcriticalim-thatcaneitherregulatetheactivityofaselectsetofportanceinhomeostaticadaptation,arehormonesingenes,therebyalteringtheamountofcertainproteinsthesensethattheyareproducedinspecificcells,haveinthetargetcell,oraffecttheactivityofspecificpro-theequivalentofautocrine,paracrine,andendocrineteins,includingenzymesandtransporterorchannelactions,bindtocellsurfacereceptors,andactivateproteins.Thesignalcaninfluencethelocationofpro-manyofthesamesignaltransductionpathwaysem-teinsinthecellandcanaffectgeneralprocessessuchasployedbythemoretraditionalgroupIIhormones.proteinsynthesis,cellgrowth,andreplication,perhapsthrougheffectsongeneexpression.Othersignalingmolecules—includingcytokines,interleukins,growthSIGNALGENERATIONfactors,andmetabolites—usesomeofthesamegeneralmechanismsandsignaltransductionpathways.Exces-TheLigand-ReceptorComplexIsthesive,deficient,orinappropriateproductionandreleaseSignalforGroupIHormonesofhormonesandoftheseotherregulatorymoleculesThelipophilicgroupIhormonesdiffusethroughthearemajorcausesofdisease.Manypharmacotherapeuticplasmamembraneofallcellsbutonlyencountertheiragentsareaimedatcorrectingorotherwiseinfluencingspecific,high-affinityintracellularreceptorsintargetthepathwaysdiscussedinthischapter.cells.Thesereceptorscanbelocatedinthecytoplasmorinthenucleusoftargetcells.Thehormone-receptorHORMONESTRANSDUCESIGNALSTOcomplexfirstundergoesanactivationreaction.AsAFFECTHOMEOSTATICMECHANISMSshowninFigure43–2,receptoractivationoccursbyatleasttwomechanisms.Forexample,glucocorticoidsThegeneralstepsinvolvedinproducingacoordinateddiffuseacrosstheplasmamembraneandencounterresponsetoaparticularstimulusareillustratedintheircognatereceptorinthecytoplasmoftargetcells.Figure43–1.ThestimuluscanbeachallengeoraLigand-receptorbindingresultsinthedissociationofthreattotheorganism,toanorgan,ortotheintegrityheatshockprotein90(hsp90)fromthereceptor.Thisofasinglecellwithinthatorganism.Recognitionofthestepappearstobenecessaryforsubsequentnuclearlo-stimulusisthefirststepintheadaptiveresponse.Atthecalizationoftheglucocorticoidreceptor.Thisreceptororganismiclevel,thisgenerallyinvolvesthenervoussys-alsocontainsnuclearlocalizationsequencesthatassisttemandthespecialsenses(sight,hearing,pain,smell,inthetranslocationfromcytoplasmtonucleus.Thetouch).Attheorganismicorcellularlevel,recognitionnowactivatedreceptormovesintothenucleus(FigureinvolvesphysicochemicalfactorssuchaspH,O2ten-43–2)andbindswithhighaffinitytoaspecificDNAsion,temperature,nutrientsupply,noxiousmetabo-sequencecalledthehormoneresponseelementlites,andosmolarity.Appropriaterecognitionresultsin(HRE).Inthecaseillustrated,thisisaglucocorticoidthereleaseofoneormorehormonesthatwillgovernresponseelement,orGRE.Consensussequencesforgenerationofthenecessaryadaptiveresponse.Forpur-HREsareshowninTable43–1.TheDNA-bound,lig-posesofthisdiscussion,thehormonesarecategorizedandedreceptorservesasahigh-affinitybindingsitefor456
455HORMONEACTION&SIGNALTRANSDUCTION/457STIMULUSRecognitionGroupIhormonesGroupIIhormonesHormonereleaseHormone•receptorcomplexManydifferentsignalsSignalgenerationEffectsGeneTransportersProteinProteintranscriptionChannelstranslocationmodificationCOORDINATEDRESPONSETOSTIMULUSFigure43–1.Hormonalinvolvementinresponsestoastimulus.Achallengetothein-tegrityoftheorganismelicitsaresponsethatincludesthereleaseofoneormorehormones.Thesehormonesgeneratesignalsatorwithintargetcells,andthesesignalsregulateavari-etyofbiologicprocesseswhichprovideforacoordinatedresponsetothestimulusorchal-lenge.SeeFigure43–8foraspecificexample.oneormorecoactivatorproteins,andacceleratedgenegeststhatthesehormonesexerttheirdominanteffecttranscriptiontypicallyensueswhenthisoccurs.Bycon-onmodulatinggenetranscription,butthey—andmanytrast,certainhormonessuchasthethyroidhormonesofthehormonesintheotherclassesdiscussedbelow—andretinoidsdiffusefromtheextracellularfluidacrosscanactatanystepofthe“informationpathway”illus-theplasmamembraneandgodirectlyintothenucleus.tratedinFigure43–3.DirectactionsofsteroidsintheInthiscase,thecognatereceptorisalreadyboundtocytoplasmandonvariousorganellesandmembranestheHRE(thethyroidhormoneresponseelementhavealsobeendescribed.[TRE],inthisexample).However,thisDNA-boundreceptorfailstoactivatetranscriptionbecauseitiscom-plexedwithacorepressor.Indeed,thisreceptor-GROUPII(PEPTIDE&corepressorcomplexservesasanactiverepressorofgeneCATECHOLAMINE)HORMONEStranscription.Theassociationofligandwiththesere-HAVEMEMBRANERECEPTORSceptorsresultsindissociationofthecorepressor.The&USEINTRACELLULARMESSENGERSligandedreceptorisnowcapableofbindingoneormorecoactivatorswithhighaffinity,resultingintheac-Manyhormonesarewater-soluble,havenotransporttivationofgenetranscription.Therelationshipofhor-proteins(andthereforehaveashortplasmahalf-life),monereceptorstoothernuclearreceptorsandtocoreg-andinitiatearesponsebybindingtoareceptorlocatedulatorsisdiscussedinmoredetailbelow.intheplasmamembrane(seeTables42–3and42–4).ByselectivelyaffectinggenetranscriptionandtheThemechanismofactionofthisgroupofhormonesconsequentproductionofappropriatetargetmRNAs,canbestbediscussedintermsoftheintracellularsig-theamountsofspecificproteinsarechangedandmeta-nalstheygenerate.ThesesignalsincludecAMP(cyclicbolicprocessesareinfluenced.TheinfluenceofeachofAMP;3′,5′-adenylicacid;seeFigure18–5),anu-thesehormonesisquitespecific;generally,thehor-cleotidederivedfromATPthroughtheactionofmoneaffectslessthan1%ofthegenes,mRNA,orpro-adenylylcyclase;cGMP,anucleotideformedbygua-2+teinsinatargetcell;sometimesonlyafewareaffected.nylylcyclase;Ca;andphosphatidylinositides.ManyThenuclearactionsofsteroid,thyroid,andretinoidofthesesecondmessengersaffectgenetranscription,ashormonesarequitewelldefined.Mostevidencesug-describedinthepreviousparagraph;buttheyalsoinflu-
456458/CHAPTER43−Cytoplasm++−TRETRETREGREGREGRE+hsp+hspNucleusFigure43–2.RegulationofgeneexpressionbyclassIhormones.Steroidhormonesreadilygainaccesstothecytoplasmiccompartmentoftargetcells.Glucocorticoidhormones(solidtriangles)encountertheircognatereceptorinthecytoplasm,whereitexistsinacomplexwithheatshockprotein90(hsp).Ligandbindingcausesdissociationofhspandaconformationalchangeofthereceptor.Thereceptor•ligandcomplexthentraversesthenuclearmembraneandbindstoDNAwithspecificityandhighaffinityataglucocorticoidresponseelement(GRE).Thiseventtriggerstheassemblyofanumberoftranscriptioncoregula-tors(+),andenhancedtranscriptionensues.Bycontrast,thyroidhor-monesandretinoicacid()directlyenterthenucleus,wheretheircognatereceptorsarealreadyboundtotheappropriateresponseele-mentswithanassociatedtranscriptionrepressorcomplex(−).Thiscomplex,whichconsistsofmoleculessuchasN-CoRorSMRT(seeTable43–6)intheabsenceofligand,activelyinhibitstranscription.Lig-andbindingresultsindissociationoftherepressorcomplexfromthereceptor,allowinganactivatorcomplextoassemble.Thegeneisthenactivelytranscribed.enceavarietyofotherbiologicprocesses,asshowninclonedfromvariousmammalianspecies.AwidevarietyFigure43–1.ofresponsesaremediatedbytheGPCRs.GProtein-CoupledReceptors(GPCR)cAMPIstheIntracellularSignalManyofthegroupIIhormonesbindtoreceptorsthatforManyResponsescoupletoeffectorsthroughaGTP-bindingproteinin-CyclicAMPwasthefirstintracellularsignalidentifiedtermediary.Thesereceptorstypicallyhavesevenhy-inmammaliancells.Severalcomponentscompriseadrophobicplasmamembrane-spanningdomains.Thissystemforthegeneration,degradation,andactionofisillustratedbytheseveninterconnectedcylindersex-cAMP.tendingthroughthelipidbilayerinFigure43–4.Re-ceptorsofthisclass,whichsignalthroughguaninenu-cleotide-boundproteinintermediates,areknownasA.ADENYLYLCYCLASEGprotein-coupledreceptors,orGPCRs.Todate,Differentpeptidehormonescaneitherstimulate(s)orover130Gprotein-linkedreceptorgeneshavebeeninhibit(i)theproductionofcAMPfromadenylylcy-
457HORMONEACTION&SIGNALTRANSDUCTION/459Table43–1.TheDNAsequencesofseveralGene1hormoneresponseelements(HREs).TRANSCRIPTIONHormoneorEffectorHREDNASequencePrimarytranscriptDegradationGlucocorticoidsGREProgestinsPREGGTACANNNTGTTCT←MODIFICATION/PROCESSINGMineralocorticoidsMRE←NUCLEUSAndrogensAREmRNADegradationEstrogensEREAGGTCA–––TGA/TCCT←←ThyroidhormoneTRETransportRetinoicacidRAREAGGTCA←N3,4,5,AGGTCA←VitaminDVDREmRNAActiveinactivedegradationcAMPCRETGACGTCATRANSLATIONCYTOPLASM1Lettersindicatenucleotide;Nmeansanyoneofthefourcanbeusedinthatposition.ThearrowspointinginoppositedirectionsProteinModificationdegradationillustratetheslightlyimperfectinvertedpalindromespresentinmanyHREs;insomecasesthesearecalled“halfbindingsites”be-Figure43–3.The“informationpathway.”Informa-causeeachbindsonemonomerofthereceptor.TheGRE,PRE,tionflowsfromthegenetotheprimarytranscripttoMRE,andAREconsistofthesameDNAsequence.SpecificitymaybeconferredbytheintracellularconcentrationoftheligandormRNAtoprotein.Hormonescanaffectanyofthestepshormonereceptor,byflankingDNAsequencesnotincludedininvolvedandcanaffecttheratesofprocessing,degra-theconsensus,orbyotheraccessoryelements.Asecondgroupofdation,ormodificationofthevariousproducts.HREsincludesthoseforthyroidhormones,estrogens,retinoicacid,andvitaminD.TheseHREsaresimilarexceptfortheorienta-tionandspacingbetweenthehalfpalindromes.Spacingdeter-minesthehormonespecificity.VDRE(N=3),TRE(N=4),andRAREtively.Inthecaseofαs,thismodificationdisruptsthe(N=5)bindtodirectrepeatsratherthantoinvertedrepeats.An-othermemberofthesteroidreceptorsuperfamily,theretinoidXintrinsicGTP-aseactivity;thus,αscannotreassociatereceptor(RXR),formsheterodimerswithVDR,TR,andRARE,andwithβγandisthereforeirreversiblyactivated.ADP-theseconstitutethetrans-actingfactors.cAMPaffectsgenetran-ribosylationofαi-2preventsthedissociationofαi-2scriptionthroughtheCRE.fromβγ,andfreeαi-2thuscannotbeformed.αsactiv-ityinsuchcellsisthereforeunopposed.ThereisalargefamilyofGproteins,andtheseareclase,whichisencodedbyatleastninedifferentgenespartofthesuperfamilyofGTPases.TheGprotein(Table43–2).Twoparallelsystems,astimulatory(s)familyisclassifiedaccordingtosequencehomologyoneandaninhibitory(i)one,convergeuponasingleintofoursubfamilies,asillustratedinTable43–3.catalyticmolecule(C).Eachconsistsofareceptor,RsorThereare21α,5β,and8γsubunitgenes.VariousRi,andaregulatorycomplex,GsandGi.GsandGiarecombinationsofthesesubunitsprovidealargenumbereachtrimerscomposedofα,β,andγsubunits.Becauseofpossibleαβγandcyclasecomplexes.theαsubunitinGsdiffersfromthatinGi,thepro-Theαsubunitsandtheβγcomplexhaveactionsin-teins,whicharedistinctgeneproducts,aredesignateddependentofthoseonadenylylcyclase(seeFigureαsandαi.Theαsubunitsbindguaninenucleotides.43–4andTable43–3).SomeformsofαistimulateK+Theβandγsubunitsarealwaysassociated(βγ)andap-2+channelsandinhibitCachannels,andsomeαsmole-peartofunctionasaheterodimer.Thebindingofahor-culeshavetheoppositeeffects.MembersoftheGqfam-monetoRsorRiresultsinareceptor-mediatedactiva-ilyactivatethephospholipaseCgroupofenzymes.ThetionofG,whichentailstheexchangeofGDPbyGTPβγcomplexeshavebeenassociatedwithK+channelonαandtheconcomitantdissociationofβγfromα.stimulationandphospholipaseCactivation.GproteinsTheαsproteinhasintrinsicGTPaseactivity.Theareinvolvedinmanyimportantbiologicprocessesinactiveform,αs•GTP,isinactivateduponhydrolysisofadditiontohormoneaction.NotableexamplesincludetheGTPtoGDP;thetrimericGscomplex(αβγ)isolfaction(αOLF)andvision(αt).Someexamplesarethenre-formedandisreadyforanothercycleofactiva-listedinTable43–3.GPCRsareimplicatedinanum-tion.CholeraandpertussistoxinscatalyzetheADP-berofdiseasesandaremajortargetsforpharmaceuticalribosylationofαsandαi-2(seeTable43–3),respec-agents.
458460/CHAPTER43NNHEEγγββαsαsGTPCGDPCNohormone:inactiveeffectorBoundhormone(H):activeeffectorFigure43–4.Componentsofthehormonereceptor–Gproteineffectorsystem.ReceptorsthatcoupletoeffectorsthroughGproteins(GPCR)typicallyhavesevenmembrane-spanningdomains.Intheabsenceofhormone(left),theheterotrimericG-proteincomplex(α,β,γ)isinaninactiveguano-sinediphosphate(GDP)-boundformandisprobablynotassociatedwiththereceptor.Thiscomplexisanchoredtotheplasmamembranethroughprenylatedgroupsontheβγsubunits(wavylines)andperhapsbymyristoylatedgroupsonαsubunits(notshown).Onbindingofhormone(H)tothere-ceptor,thereisapresumedconformationalchangeofthereceptor—asindicatedbythetiltedmem-branespanningdomains—andactivationoftheG-proteincomplex.ThisresultsfromtheexchangeofGDPwithguanosinetriphosphate(GTP)ontheαsubunit,afterwhichαandβγdissociate.Theαsub-unitbindstoandactivatestheeffector(E).Ecanbeadenylylcyclase,Ca2+,Na+,orCl−channels(α),orsitcouldbeaK+channel(αi),phospholipaseCβ(αq),orcGMPphosphodiesterase(αt).TheβγsubunitcanalsohavedirectactionsonE.(Modifiedandreproduced,withpermission,fromGrannerDKin:Princi-plesandPracticeofEndocrinologyandMetabolism,3rded.BeckerKL[editor].Lippincott,2000.)B.PROTEINKINASEInprokaryoticcells,cAMPbindstoaspecificproteincalledcataboliteregulatoryprotein(CRP)thatbindsTable43–2.SubclassificationofgroupII.AdirectlytoDNAandinfluencesgeneexpression.Ineu-hormones.karyoticcells,cAMPbindstoaproteinkinasecalledproteinkinaseA(PKA)thatisaheterotetramericmol-eculeconsistingoftworegulatorysubunits(R)andtwoHormonesThatStimulateHormonesThatInhibitcatalyticsubunits(C).cAMPbindingresultsinthefol-AdenylylCyclaseAdenylylCyclaselowingreaction:(Hs)(Hl)ACTHAcetylcholineADHα-Adrenergics44cAMP+R22C2Ra⋅(cAMP)+2C2β-AdrenergicsAngiotensinIICalcitoninSomatostatinTheR2C2complexhasnoenzymaticactivity,butCRHthebindingofcAMPbyRdissociatesRfromC,FSHtherebyactivatingthelatter(Figure43–5).TheactiveGlucagonCsubunitcatalyzesthetransferoftheγphosphateofhCGATPtoaserineorthreonineresidueinavarietyofpro-LHteins.Theconsensusphosphorylationsitesare-Arg-LPHArg/Lys-X-Ser/Thr-and-Arg-Lys-X-X-Ser-,whereXMSHcanbeanyaminoacid.PTHProteinkinaseactivitieswereoriginallydescribedasTSHbeing“cAMP-dependent”or“cAMP-independent.”This
459HORMONEACTION&SIGNALTRANSDUCTION/4611,2Table43–3.ClassesandfunctionsofselectedGproteins.ClassorTypeStimulusEffectorEffectGsαsGlucagon,β-adrenergics↑AdenylylcyclaseGluconeogenesis,lipolysis,↑CardiacCa2+,Cl−,andNa+channelsglycogenolysisαolfOdorant↑AdenylylcyclaseOlfactionGiαi-1,2,3Acetylcholine,↓AdenylylcyclaseSlowedheartrateα2-adrenergics↑PotassiumchannelsM2cholinergics↓Calciumchannelsα0Opioids,endorphins↑PotassiumchannelsNeuronalelectricalactivityαtLight↑cGMPphosphodiesteraseVisionGqαqM1cholinergicsα1-Adrenergics↑PhospholipaseC-β1↑Musclecontractionandα11α1-Adrenergics↑Phospholipasec-β2↑BloodpressureG12−α12?Clchannel?1Modifiedandreproduced,withpermission,fromGrannerDKin:PrinciplesandPracticeofEndocrinologyandMetabolism,3rded.BeckerKL(editor).Lippincott,2000.2ThefourmajorclassesorfamiliesofmammalianGproteins(Gs,Gi,Gq,andG12)arebasedonproteinsequencehomology.Representativemembersofeachareshown,alongwithknownstimuli,effectors,andwell-definedbiologiceffects.Nineiso-formsofadenylylcyclasehavebeenidentified(isoformsI–IX).Allisoformsarestimulatedbyαs;αiisoformsinhibittypesVandVI,andα0inhibitstypesIandV.Atleast16differentαsubunitshavebeenidentified.classificationhaschanged,asproteinphosphorylationismentbindingprotein(CREB).CREBbindstoanowrecognizedasbeingamajorregulatorymecha-cAMPresponsiveelement(CRE)(seeTable43–1)innism.Severalhundredproteinkinaseshavenowbeenitsnonphosphorylatedstateandisaweakactivatorofdescribed.Thekinasesarerelatedinsequenceandtranscription.WhenphosphorylatedbyPKA,CREBstructurewithinthecatalyticdomain,buteachisabindsthecoactivatorCREB-bindingproteinCBP/uniquemoleculewithconsiderablevariabilitywithre-p300(seebelow)andasaresultisamuchmorepotentspecttosubunitcomposition,molecularweight,au-transcriptionactivator.tophosphorylation,KmforATP,andsubstratespeci-D.PHOSPHODIESTERASESficity.ActionscausedbyhormonesthatincreasecAMPcon-C.PHOSPHOPROTEINScentrationcanbeterminatedinanumberofways,in-TheeffectsofcAMPineukaryoticcellsareallthoughtcludingthehydrolysisofcAMPto5′-AMPbyphos-tobemediatedbyproteinphosphorylation-dephosphor-phodiesterases(seeFigure43–5).Thepresenceoftheseylation,principallyonserineandthreonineresidues.hydrolyticenzymesensuresarapidturnoverofthesig-ThecontrolofanyoftheeffectsofcAMP,includingnal(cAMP)andhencearapidterminationofthebio-suchdiverseprocessesassteroidogenesis,secretion,ionlogicprocessoncethehormonalstimulusisremoved.transport,carbohydrateandfatmetabolism,enzymein-Thereareatleast11knownmembersofthephospho-duction,generegulation,synaptictransmission,anddiesterasefamilyofenzymes.Thesearesubjecttoregu-cellgrowthandreplication,couldbeconferredbyalationbytheirsubstrates,cAMPandcGMP;byhor-specificproteinkinase,byaspecificphosphatase,orbymones;andbyintracellularmessengerssuchascalcium,specificsubstratesforphosphorylation.Thesesubstratesprobablyactingthroughcalmodulin.Inhibitorsofhelpdefineatargettissueandareinvolvedindefiningphosphodiesterase,mostnotablymethylatedxanthinetheextentofaparticularresponsewithinagivencell.derivativessuchascaffeine,increaseintracellularcAMPForexample,theeffectsofcAMPongenetranscriptionandmimicorprolongtheactionsofhormonesthrougharemediatedbytheproteincyclicAMPresponseele-thissignal.
460462/CHAPTER43ATP•Mg2+R2C2InactivePKAActiveadenylylcyclasecAMPPhosphodiesteraseC2ActivePKA+R2Cell5′-AMPmembraneMg2+•ATPProteinPhosphoproteinPhosphatasePhysiologiceffectsFigure43–5.HormonalregulationofcellularprocessesthroughcAMP-dependentproteinkinase(PKA).PKAexistsinaninactiveformasanR2C2heterotetramerconsistingoftworegulatoryandtwocatalyticsubunits.ThecAMPgeneratedbytheactionofadenylylcyclase(acti-vatedasshowninFigure43–4)bindstotheregulatory(R)subunitofPKA.Thisresultsindissociationoftheregulatoryandcatalyticsubunitsandactivationofthelatter.Theactivecatalyticsubunitsphosphorylateanumberoftargetproteinsonserineandthreonineresidues.Phos-phatasesremovephosphatefromtheseresiduesandthusterminatethephysiologicresponse.AphosphodiesterasecanalsoterminatetheresponsebyconvertingcAMPto5′-AMP.E.PHOSPHOPROTEINPHOSPHATASESheat-stableproteininhibitorsregulatetypeIphos-Giventheimportanceofproteinphosphorylation,itisphataseactivity.Inhibitor-1isphosphorylatedandacti-notsurprisingthatregulationoftheproteindephos-vatedbycAMP-dependentproteinkinases;andin-phorylationreactionisanotherimportantcontrolhibitor-2,whichmaybeasubunitoftheinactivemechanism(seeFigure43–5).Thephosphoproteinphosphatase,isalsophosphorylated,possiblybyglyco-phosphatasesarethemselvessubjecttoregulationbygensynthasekinase-3.phosphorylation-dephosphorylationreactionsandbyavarietyofothermechanisms,suchasprotein-proteincGMPIsAlsoanIntracellularSignalinteractions.Infact,thesubstratespecificityofthephosphoserine-phosphothreoninephosphatasesmaybeCyclicGMPismadefromGTPbytheenzymegua-dictatedbydistinctregulatorysubunitswhosebindingnylylcyclase,whichexistsinsolubleandmembrane-isregulatedhormonally.Thebest-studiedroleofregu-boundforms.Eachoftheseisozymeshasuniquephysi-lationbythedephosphorylationofproteinsisthatofologicproperties.Theatriopeptins,afamilyofpeptidesglycogenmetabolisminmuscle.Twomajortypesofproducedincardiacatrialtissues,causenatriuresis,di-phosphoserine-phosphothreoninephosphataseshaveuresis,vasodilation,andinhibitionofaldosteronesecre-beendescribed.TypeIpreferentiallydephosphorylatestion.Thesepeptides(eg,atrialnatriureticfactor)bindtheβsubunitofphosphorylasekinase,whereastypeIItoandactivatethemembrane-boundformofguanylyldephosphorylatestheαsubunit.TypeIphosphataseiscyclase.ThisresultsinanincreaseofcGMPbyasmuchimplicatedintheregulationofglycogensynthase,phos-as50-foldinsomecases,andthisisthoughttomediatephorylase,andphosphorylasekinase.Thisphosphatasetheeffectsmentionedabove.OtherevidencelinksisitselfregulatedbyphosphorylationofcertainofitscGMPtovasodilation.Aseriesofcompounds,includ-subunits,andthesereactionsarereversedbytheactioningnitroprusside,nitroglycerin,nitricoxide,sodiumofoneofthetypeIIphosphatases.Inaddition,twonitrite,andsodiumazide,allcausesmoothmusclere-
461HORMONEACTION&SIGNALTRANSDUCTION/463laxationandarepotentvasodilators.Theseagentsin-B.CALMODULINcreasecGMPbyactivatingthesolubleformofguanylylThecalcium-dependentregulatoryproteiniscalmod-cyclase,andinhibitorsofcGMPphosphodiesterase(theulin,a17-kDaproteinthatishomologoustothemus-drugsildenafil[Viagra],forexample)enhanceandpro-cleproteintroponinCinstructureandfunction.longtheseresponses.TheincreasedcGMPactivatesCalmodulinhasfourCa2+bindingsites,andfulloccu-cGMP-dependentproteinkinase(PKG),whichinturnpancyofthesesitesleadstoamarkedconformationalphosphorylatesanumberofsmoothmuscleproteins.change,whichallowscalmodulintoactivateenzymesPresumably,thisisinvolvedinrelaxationofsmoothandionchannels.TheinteractionofCa2+withcalmod-muscleandvasodilation.ulin(withtheresultantchangeofactivityofthelatter)isconceptuallysimilartothebindingofcAMPtoPKASeveralHormonesActThroughandthesubsequentactivationofthismolecule.CalciumorPhosphatidylinositolsCalmodulincanbeoneofnumeroussubunitsofcom-plexproteinsandisparticularlyinvolvedinregulatingIonizedcalciumisanimportantregulatorofavarietyofvariouskinasesandenzymesofcyclicnucleotidegener-cellularprocesses,includingmusclecontraction,stimu-ationanddegradation.Apartiallistoftheenzymesreg-lus-secretioncoupling,thebloodclottingcascade,en-ulateddirectlyorindirectlybyCa2+,probablythroughzymeactivity,andmembraneexcitability.Itisalsoancalmodulin,ispresentedinTable43–4.intracellularmessengerofhormoneaction.Inadditiontoitseffectsonenzymesandiontrans-2+A.CALCIUMMETABOLISMport,Ca/calmodulinregulatestheactivityofmanystructuralelementsincells.Theseincludetheactin-2+Theextracellularcalcium(Ca)concentrationisaboutmyosincomplexofsmoothmuscle,whichisunderβ-5mmol/Landisveryrigidlycontrolled.Althoughsub-adrenergiccontrol,andvariousmicrofilament-medi-stantialamountsofcalciumareassociatedwithintracel-atedprocessesinnoncontractilecells,includingcelllularorganellessuchasmitochondriaandtheendoplas-motility,cellconformationchanges,mitosis,granulere-micreticulum,theintracellularconcentrationoffreeorlease,andendocytosis.2+ionizedcalcium(Ca)isverylow:0.05–10μmol/L.Inspiteofthislargeconcentrationgradientandafavor-C.CALCIUMISAMEDIATOROFHORMONEACTION2+abletransmembraneelectricalgradient,Caisre-2+AroleforCainhormoneactionissuggestedbythestrainedfromenteringthecell.Aconsiderableamountobservationsthattheeffectofmanyhormonesis(1)ofenergyisexpendedtoensurethattheintracellular2+2+2+bluntedbyCa-freemediaorwhenintracellularcal-Caiscontrolled,asaprolongedelevationofCain+2+ciumisdepleted;(2)canbemimickedbyagentsthatthecellisverytoxic.ANa/Caexchangemechanism2+2+2+increasecytosolicCa,suchastheCaionophorethathasahighcapacitybutlowaffinitypumpsCa2+A23187;and(3)influencescellularcalciumflux.Theoutofcells.TherealsoisaCa/protonATPase-depen-2++regulationofglycogenmetabolisminliverbyvaso-dentpumpthatextrudesCainexchangeforH.This2+pressinandα-adrenergiccatecholaminesprovidesahasahighaffinityforCabutalowcapacityandis2+goodexample.ThisisshownschematicallyinFiguresprobablyresponsibleforfine-tuningcytosolicCa.2+2+18–6and18–7.Furthermore,CaATPasespumpCafromthecy-tosoltothelumenoftheendoplasmicreticulum.There2+arethreewaysofchangingcytosolicCa:(1)Certainhormones(classII.C,Table42–3)bybindingtorecep-torsthatarethemselvesCa2+channels,enhancemem-Table43–4.EnzymesandproteinsregulatedbybranepermeabilitytoCa2+andtherebyincreaseCa2+calciumorcalmodulin.2+influx.(2)HormonesalsoindirectlypromoteCain-fluxbymodulatingthemembranepotentialattheAdenylylcyclaseplasmamembrane.Membranedepolarizationopens2+Ca-dependentproteinkinasesvoltage-gatedCa2+channelsandallowsforCa2+influx.Ca2+-Mg2+ATPase2+Ca2+-phospholipid-dependentproteinkinase(3)Cacanbemobilizedfromtheendoplasmicreticu-lum,andpossiblyfrommitochondrialpools.CyclicnucleotidephosphodiesteraseAnimportantobservationlinkingCa2+tohormoneSomecytoskeletalproteinsactioninvolvedthedefinitionoftheintracellulartargetsSomeionchannels(eg,L-typecalciumchannels)ofCa2+action.ThediscoveryofaCa2+-dependentreg-NitricoxidesynthaseulatorofphosphodiesteraseactivityprovidedthebasisPhosphorylasekinaseforabroadunderstandingofhowCa2+andcAMPin-Phosphoproteinphosphatase2BSomereceptors(eg,NMDA-typeglutamatereceptor)teractwithincells.
462464/CHAPTER43Anumberofcriticalmetabolicenzymesareregu-facereceptorssuchasthoseforacetylcholine,antidi-2+latedbyCa,phosphorylation,orboth,includinguretichormone,andα1-typecatecholaminesare,whenglycogensynthase,pyruvatekinase,pyruvatecarboxy-occupiedbytheirrespectiveligands,potentactivatorslase,glycerol-3-phosphatedehydrogenase,andpyruvateofphospholipaseC.Receptorbindingandactivationofdehydrogenase.phospholipaseCarecoupledbytheGqisoforms(Table43–3andFigure43–6).PhospholipaseCcatalyzestheD.PHOSPHATIDYLINOSITIDEMETABOLISMAFFECTShydrolysisofphosphatidylinositol4,5-bisphosphateto2+CA-DEPENDENTHORMONEACTIONinositoltrisphosphate(IP)and1,2-diacylglycerol(Fig-3Somesignalmustprovidecommunicationbetweentheure43–7).Diacylglycerolisitselfcapableofactivatinghormonereceptorontheplasmamembraneandthein-proteinkinaseC(PKC),theactivityofwhichalsode-2+2+tracellularCareservoirs.ThisisaccomplishedbypendsuponCa.IP3,byinteractingwithaspecificin-2+productsofphosphatidylinositolmetabolism.Cellsur-tracellularreceptor,isaneffectivereleaserofCafrom2+CaReceptorGproteinPhospholipaseCPIP2DiacylglycerolEndoplasmicreticulum+ProteinkinaseCInositol–P(PKC)3(IP3)Mitochondrion2+CaCalmodulin2+Ca-Calmodulin++SpecificMultifunctionalcalmodulinkinasecalmodulinkinaseProteinsPhosphoproteinsOtherproteinsPhysiologicresponsesFigure43–6.Certainhormone-receptorinteractionsresultintheactivationofphospholipaseC.Thisap-pearstoinvolveaspecificGprotein,whichalsomayactivateacalciumchannel.PhospholipaseCresultsin2+generationofinositoltrisphosphate(IP3),whichliberatesstoredintracellularCa,anddiacylglycerol(DAG),apotentactivatorofproteinkinaseC(PKC).Inthisscheme,theactivatedPKCphosphorylatesspecificsub-2+strates,whichthenalterphysiologicprocesses.Likewise,theCa-calmodulincomplexcanactivatespecifickinases,twoofwhichareshownhere.Theseactionsresultinphosphorylationofsubstrates,andthisleadsto2+alteredphysiologicresponses.ThisfigurealsoshowsthatCacanentercellsthroughvoltage-orligand-2+2+gatedCachannels.TheintracellularCaisalsoregulatedthroughstorageandreleasebythemitochon-driaandendoplasmicreticulum.(CourtesyofJHExton.)
463HORMONEACTION&SIGNALTRANSDUCTION/465R1R2POHOHPR1R2OHPhospholipaseC1,2-DiacylglycerolOH(DAG)PPPhosphatidylinositol4,5-bisphosphateOHOHPFigure43–7.(PIP2)PhospholipaseCcleavesPIP2intodiacylglycerolandinositoltrisphosphate.R1OHgenerallyisstearate,andR2isusuallyarachido-nate.IP3canbedephosphorylated(totheinac-PtiveI-1,4-P2)orphosphorylated(tothepotentiallyInositol1,4,5-trisphosphate(IP3)activeI-1,3,4,5-P4).intracellularstoragesitesintheendoplasmicreticulum.ligand-activatedtyrosinekinaseactivity.Severalrecep-Thus,thehydrolysisofphosphatidylinositol4,5-bis-tors—generallythoseinvolvedinbindingligandsin-phosphateleadstoactivationofPKCandpromotesanvolvedingrowthcontrol,differentiation,andthein-2+increaseofcytoplasmicCa.AsshowninFigure43–4,flammatoryresponse—eitherhaveintrinsictyrosinetheactivationofGproteinscanalsohaveadirectac-kinaseactivityorareassociatedwithproteinsthatare2+tiononCachannels.Theresultingelevationsofcy-tyrosinekinases.Anotherdistinguishingfeatureofthis2+2+tosolicCaactivateCa–calmodulin-dependentkinasesclassofhormoneactionisthatthesekinasespreferen-2+andmanyotherCa–calmodulin-dependentenzymes.tiallyphosphorylatetyrosineresidues,andtyrosineSteroidogenicagents—includingACTHandcAMPphosphorylationisinfrequent(<0.03%oftotalaminointheadrenalcortex;angiotensinII,K+,serotonin,acidphosphorylation)inmammaliancells.Athirddis-ACTH,andcAMPinthezonaglomerulosaofthetinguishingfeatureisthattheligand-receptorinterac-adrenal;LHintheovary;andLHandcAMPinthetionthatresultsinatyrosinephosphorylationeventini-Leydigcellsofthetestes—havebeenassociatedwithin-tiatesacascadethatmayinvolveseveralproteinkinases,creasedamountsofphosphatidicacid,phosphatidyl-phosphatases,andotherregulatoryproteins.inositol,andpolyphosphoinositides(seeChapter14)inA.INSULINTRANSMITSSIGNALStherespectivetargettissues.Severalotherexamplescouldbecited.BYSEVERALKINASECASCADESTherolesthatCa2+andpolyphosphoinositidebreak-Theinsulin,epidermalgrowthfactor(EGF),andIGF-Idownproductsmightplayinhormoneactionarepre-receptorshaveintrinsicproteintyrosinekinaseactivitiessentedinFigure43–6.Inthisschemetheactivatedpro-locatedintheircytoplasmicdomains.TheseactivitiesteinkinaseCcanphosphorylatespecificsubstrates,arestimulatedwhenthereceptorbindsligand.There-whichthenalterphysiologicprocesses.Likewise,theceptorsarethenautophosphorylatedontyrosineCa2+-calmodulincomplexcanactivatespecifickinases.residues,andthisinitiatesacomplexseriesofeventsThesethenmodifysubstratesandtherebyalterphysio-(summarizedinsimplifiedfashioninFigure43–8).Thelogicresponses.phosphorylatedinsulinreceptornextphosphorylatesinsulinreceptorsubstrates(thereareatleastfourofSomeHormonesActThroughthesemolecules,calledIRS1–4)ontyrosineresidues.aProteinKinaseCascadePhosphorylatedIRSbindstotheSrchomology2(SH2)domainsofavarietyofproteinsthataredirectly2+SingleproteinkinasessuchasPKA,PKC,andCa-involvedinmediatingdifferenteffectsofinsulin.Onecalmodulin(CaM)-kinases,whichresultinthephos-oftheseproteins,PI-3kinase,linksinsulinreceptorac-phorylationofserineandthreonineresiduesintargettivationtoinsulinactionthroughactivationofanum-proteins,playaveryimportantroleinhormoneaction.berofmolecules,includingthekinasePDK1(phospho-ThediscoverythattheEGFreceptorcontainsanintrin-inositide-dependentkinase-1).Thisenzymepropagatessictyrosinekinaseactivitythatisactivatedbythebind-thesignalthroughseveralotherkinases,includingPKBingoftheligandEGFwasanimportantbreakthrough.(akt),SKG,andaPKC(seelegendtoFigure43–8forTheinsulinandIGF-Ireceptorsalsocontainintrinsicdefinitionsandexpandedabbreviations).Analternative
464466/CHAPTER43RECOGNITION(HYPERGLYCEMIA)INSULINP-YY-PYYIRS1-4IRS1-4YYSIGNALP-YY-PGRB2/mSOSGENERATIONPI3-+PTEN+kinasemTORp21Ras+Raf-1PKBMEKSGK?p70S6KMAPaPKCkinaseProteintranslocationEnzymeactivityGenetranscriptionCellgrowthDNAsynthesisEFFECTSGlucosetransporterInsulinreceptorPEPCKHKIIEarlyresponseInsulinreceptorProteinphosphatasesGlucagonGlucokinasegenesIGF-IIreceptorPhosphodiesterases*IGFBP1>100othersOthersFigure43–8.Insulinsignalingpathways.Theinsulinsignalingpathwaysprovideanexcellentexampleofthe“recognition→hormonerelease→signalgeneration→effects”paradigmoutlinedinFigure43–1.Insulinisre-leasedinresponsetohyperglycemia.Bindingofinsulintoatargetcell-specificplasmamembranereceptorresultsinacascadeofintracellularevents.Stimulationoftheintrinsictyrosinekinaseactivityoftheinsulinreceptormarkstheinitialevent,resultinginincreasedtyrosine(Y)phosphorylation(Y→Y-P)ofthereceptorandthenoneormoreoftheinsulinreceptorsubstratemolecules(IRS1–4).ThisincreaseinphosphotyrosinestimulatestheactivityofmanyintracellularmoleculessuchasGTPases,proteinkinases,andlipidkinases,allofwhichplayaroleincertainmeta-bolicactionsofinsulin.Thetwobest-describedpathwaysareshown.First,phosphorylationofanIRSmolecule(probablyIRS-2)resultsindockingandactivationofthelipidkinase,PI-3kinase,whichgeneratesnovelinositollipidsthatmayactas“secondmessenger”molecules.These,inturn,activatePDK1andthenavarietyofdownstreamsig-nalingmolecules,includingproteinkinaseB(PKBorakt),SGK,andaPKC.Analternativepathwayinvolvestheactiva-tionofp70S6Kandperhapsotherasyetunidentifiedkinases.Second,phosphorylationofIRS(probablyIRS-1)re-sultsindockingofGRB2/mSOSandactivationofthesmallGTPase,p21RAS,whichinitiatesaproteinkinasecascadethatactivatesRaf-1,MEK,andthep42/p44MAPkinaseisoforms.Theseproteinkinasesareimportantintheregula-tionofproliferationanddifferentiationofseveralcelltypes.ThemTORpathwayprovidesanalternativewayofacti-vatingp70S6Kandappearstobeinvolvedinnutrientsignalingaswellasinsulinaction.Eachofthesecascadesmayinfluencedifferentphysiologicprocesses,asshown.Eachofthephosphorylationeventsisreversiblethroughtheactionofspecificphosphatases.Forexample,thelipidphosphatasePTENdephosphorylatestheproductofthePI-3kinasereaction,therebyantagonizingthepathwayandterminatingthesignal.Representativeeffectsofmajorac-tionsofinsulinareshownineachoftheboxes.TheasteriskafterphosphodiesteraseindicatesthatinsulinindirectlyaffectstheactivityofmanyenzymesbyactivatingphosphodiesterasesandreducingintracellularcAMPlevels.(IGFBP,insulin-likegrowthfactorbindingprotein;IRS1–4,insulinreceptorsubstrateisoforms1–4);PI-3kinase,phos-phatidylinositol3-kinase;PTEN,phosphataseandtensinhomologdeletedonchromosome10;PKD1,phosphoinosi-tide-dependentkinase;PKB,proteinkinaseB;SGK,serumandglucocorticoid-regulatedkinase;aPKC,atypicalpro-teinkinaseC;p70S6K,p70ribosomalproteinS6kinase;mTOR,mammaliantargetofrapamycin;GRB2,growthfactorreceptorbindingprotein2;mSOS,mammaliansonofsevenless;MEK,MAPkinasekinaseandERKkinase;MAPkinase,mitogen-activatedproteinkinase.)
465HORMONEACTION&SIGNALTRANSDUCTION/467pathwaydownstreamfromPKD1involvesp70S6Kandbalancingactionsofphosphatases.Twomechanismsperhapsotherasyetunidentifiedkinases.Asecondmajorareemployedtoinitiatethiscascade.Somehormones,pathwayinvolvesmTOR.Thisenzymeisdirectlyregu-suchasgrowthhormone,prolactin,erythropoietin,andlatedbyaminoacidsandinsulinandisessentialforthecytokines,initiatetheiractionbyactivatingatyro-p70S6Kactivity.Thispathwayprovidesadistinctionsinekinase,butthisactivityisnotanintegralpartofbetweenthePKBandp70S6Kbranchesdownstreamthehormonereceptor.Thehormone-receptorinterac-fromPKD1.Thesepathwaysareinvolvedinproteintionpromotesbindingandactivationofcytoplasmictranslocation,enzymeactivity,andtheregulation,byproteintyrosinekinases,suchasTyk-2,Jak1,orinsulin,ofgenesinvolvedinmetabolism(Figure43–8).Jak2.Thesekinasesphosphorylateoneormorecyto-AnotherSH2domain-containingproteinisGRB2,plasmicproteins,whichthenassociatewithotherdock-whichbindstoIRS-1andlinkstyrosinephosphoryla-ingproteinsthroughbindingtoSH2domains.Onetiontoseveralproteins,theresultofwhichisactivationsuchinteractionresultsintheactivationofafamilyofofacascadeofthreonineandserinekinases.Apathwaycytosolicproteinscalledsignaltransducersandactiva-showinghowthisinsulin-receptorinteractionactivatestorsoftranscription(STATs).Thephosphorylatedthemitogen-activatedprotein(MAP)kinasepathwaySTATproteindimerizesandtranslocatesintothenu-andtheanaboliceffectsofinsulinisillustratedinFig-cleus,bindstoaspecificDNAelementsuchasthein-ure43–8.Theexactrolesofmanyofthesedockingterferonresponseelement,andactivatestranscription.proteins,kinases,andphosphatasesremaintobeestab-ThisisillustratedinFigure43–9.OtherSH2dockinglished.eventsmayresultintheactivationofPI3-kinase,theMAPkinasepathway(throughSHCorGRB2),orGB.THEJAK/STATPATHWAYISUSEDprotein-mediatedactivationofphospholipaseC(PLCγ)BYHORMONESANDCYTOKINESwiththeattendantproductionofdiacylglycerolandac-Tyrosinekinaseactivationcanalsoinitiateaphosphor-tivationofproteinkinaseC.Itisapparentthatthereisylationanddephosphorylationcascadethatinvolvestheapotentialfor“cross-talk”whendifferenthormonesac-actionofseveralotherproteinkinasesandthecounter-tivatethesevarioussignaltransductionpathways.LigandRRRRRRJAKJAKPJAKJAKPPJAKJAKPPPPPSTATPPPPSH2PPXx=SHCDimerizationGRB2andPLCγnuclearPI-3KtranslocationGAPFigure43–9.InitiationofsignaltransductionbyreceptorslinkedtoJakki-nases.Thereceptors(R)thatbindprolactin,growthhormone,interferons,andcy-tokineslackendogenoustyrosinekinase.Uponligandbinding,thesereceptorsdimerizeandanassociatedprotein(Jak1,Jak2,orTYK)isphosphorylated.Jak-P,anactivekinase,phosphorylatesthereceptorontyrosineresidues.TheSTATpro-teinsassociatewiththephosphorylatedreceptorandthenarethemselvesphos-phorylatedbyJak-P.STATPdimerizes,translocatestothenucleus,bindstospe-cificDNAelements,andregulatestranscription.ThephosphotyrosineresiduesofthereceptoralsobindtoseveralSH2domain-containingproteins.ThisresultsinactivationoftheMAPkinasepathway(throughSHCorGRB2),PLCγ,orPI-3kinase.
466468/CHAPTER43C.THENF-BPATHWAYISGlucocorticoidhormonesaretherapeuticallyusefulREGULATEDBYGLUCOCORTICOIDSagentsforthetreatmentofavarietyofinflammatoryandimmunediseases.Theiranti-inflammatoryandim-ThetranscriptionfactorNF-κBisaheterodimericmunomodulatoryactionsareexplainedinpartbythein-complextypicallycomposedoftwosubunitstermedhibitionofNF-κBanditssubsequentactions.Evidencep50andp65(Figure43–10).Normally,NF-κBiskeptforthreemechanismsfortheinhibitionofNF-κBbysequesteredinthecytoplasminatranscriptionallyinac-glucocorticoidshasbeenpresented:(1)GlucocorticoidstiveformbymembersoftheinhibitorofNF-κB(IκB)increaseIκBmRNA,whichleadstoanincreaseofIκBfamily.ExtracellularstimulisuchasproinflammatoryproteinandmoreefficientsequestrationofNF-κBinthecytokines,reactiveoxygenspecies,andmitogensleadtocytoplasm.(2)TheglucocorticoidreceptorcompetesactivationoftheIκBkinasecomplex,IKK,whichisawithNF-κBforbindingtocoactivators.(3)Thegluco-heterohexamericstructureconsistingofα,β,andγsub-corticoidreceptordirectlybindstothep65subunitofunits.IKKphosphorylatesIκBontwoserineresidues,NF-κBandinhibitsitsactivation(Figure43–10).andthistargetsIκBforubiquitinationandsubsequentdegradationbytheproteasome.FollowingIκBdegra-dation,freeNF-κBcannowtranslocatetothenucleus,HORMONESCANINFLUENCEwhereitbindstoanumberofgenepromotersandacti-SPECIFICBIOLOGICEFFECTSBYvatestranscription,particularlyofgenesinvolvedintheMODULATINGTRANSCRIPTIONinflammatoryresponse.TranscriptionalregulationbyNF-κBismediatedbyavarietyofcoactivatorssuchasThesignalsgeneratedasdescribedabovehavetobeCREBbindingprotein(CBP),asdescribedbelow(Fig-translatedintoanactionthatallowsthecelltoeffec-ure43–13).tivelyadapttoachallenge(Figure43–1).MuchofthisNF-κBActivatorsProinflammatorycytokinesBacterialandviralinfectionReactiveoxygenspeciesMitogensIKKcomplexγγPPProteasomeααββIκBUbiquitin1IκBp50p65Cytoplasmp50p65Nucleus2Coactivators3p50p65TargetgeneFigure43–10.RegulationoftheNF-κBpathway.NF-κBconsistsoftwosub-units,p50andp65,whichregulatetranscriptionofmanygeneswheninthenucleus.NF-κBisrestrictedfromenteringthenucleusbyIκB,aninhibitorofNF-κB.IκBbindsto—andmasks—thenuclearlocalizationsignalofNF-κB.ThiscytoplasmicproteinisphosphorylatedbyanIKKcomplexwhichisacti-vatedbycytokines,reactiveoxygenspecies,andmitogens.PhosphorylatedIκBcanbeubiquitinylatedanddegraded,thusreleasingitsholdonNF-κB.Glu-cocorticoidsaffectmanystepsinthisprocess,asdescribedinthetext.
467HORMONEACTION&SIGNALTRANSDUCTION/469Figure43–11.Thehormoneresponsetranscrip-tionunit.ThehormoneresponsetranscriptionunitisanassemblyofDNAelementsandboundpro-teinsthatinteract,throughprotein-proteininterac-tions,withanumberofcoactivatororcorepressormolecules.AnessentialcomponentisthehormoneAFEresponseelementwhichbindstheligand()-HREboundreceptor(R).Alsoimportantaretheacces-soryfactorelements(AFEs)withboundtranscrip-AFEtionfactors.MorethantwodozenoftheseAFRaccessoryfactors(AFs),whichareoftenmembersofRthenuclearreceptorsuperfamily,havebeenlinkedAFp160tohormoneeffectsontranscription.TheAFscanin-teractwitheachother,withtheligandednuclearp300receptors,orwithcoregulators.ThesecomponentscommunicatewiththebasaltranscriptioncomplexthroughacoregulatorcomplexthatcanconsistofBTCGeneoneormoremembersofthep160,corepressor,codingregionmediator-related,orCBP/p300families(seeTable43–6).adaptationisaccomplishedthroughalterationsinthetioninitiationsite,buttheymaybelocatedwithintheratesoftranscriptionofspecificgenes.Manydifferentcodingregionofthegene,inintrons.HREswerede-observationshaveledtothecurrentviewofhowhor-finedbythestrategyillustratedinFigure39–11.Themonesaffecttranscription.Someoftheseareasfollows:consensussequencesillustratedinTable43–1werear-(1)Activelytranscribedgenesareinregionsof“open”rivedatthroughanalysisofseveralgenesregulatedbyachromatin(definedbyasusceptibilitytotheenzymegivenhormoneusingsimple,heterologousreportersys-DNaseI),whichallowsfortheaccessoftranscriptiontems(seeFigure39–10).AlthoughthesesimpleHREsfactorstoDNA.(2)Geneshaveregulatoryregions,andbindthehormone-receptorcomplexmoreavidlythantranscriptionfactorsbindtothesetomodulatethefre-surroundingDNA—orDNAfromanunrelatedquencyoftranscriptioninitiation.(3)Thehormone-source—andconferhormoneresponsivenesstoare-receptorcomplexcanbeoneofthesetranscriptionfac-portergene,itsoonbecameapparentthattheregula-tors.TheDNAsequencetowhichthisbindsiscalledatorycircuitryofnaturalgenesmustbemuchmorehormoneresponseelement(HRE;seeTable43–1forcomplicated.Glucocorticoids,progestins,mineralocor-examples).(4)Alternatively,otherhormone-generatedticoids,andandrogenshavevastlydifferentphysiologicsignalscanmodifythelocation,amount,oractivityofactions.Howcouldthespecificityrequiredfortheseef-transcriptionfactorsandtherebyinfluencebindingtofectsbeachievedthroughregulationofgeneexpressiontheregulatoryorresponseelement.(5)MembersofabythesameHRE(Table43–1)?Questionslikethislargesuperfamilyofnuclearreceptorsactwith—orinahaveledtoexperimentswhichhaveallowedforelabora-manneranalogousto—hormonereceptors.(6)Thesetionofaverycomplexmodeloftranscriptionregula-nuclearreceptorsinteractwithanotherlargegroupoftion.Forexample,theHREmustassociatewithothercoregulatorymoleculestoeffectchangesinthetran-DNAelements(andassociatedbindingproteins)toscriptionofspecificgenes.functionoptimally.Theextensivesequencesimilaritynotedbetweensteroidhormonereceptors,particularlySeveralHormoneResponseElementsintheirDNA-bindingdomains,ledtodiscoveryofthenuclearreceptorsuperfamilyofproteins.These—and(HREs)HaveBeenDefinedalargenumberofcoregulatorproteins—allowforaHormoneresponseelementsresembleenhancerele-widevarietyofDNA-proteinandprotein-proteininter-mentsinthattheyarenotstrictlydependentonposi-actionsandthespecificitynecessaryforhighlyregulatedtionandlocation.Theygenerallyarefoundwithinaphysiologiccontrol.Aschematicofsuchanassemblyisfewhundrednucleotidesupstream(5′)ofthetranscrip-illustratedinFigure43–11.
468470/CHAPTER43A/BCDEFNCAF-1DBDHingeLBDAF-2××GR,MR,PRTR,RAR,VDRCOUP-TF,TR2,GEN8AR,ERPPARα,β,γHNF-4,TLXFXR,CAR,LXR,PXR/SXRReceptors:SteroidclassRXRpartneredOrphansBinding:HomodimersHeterodimersHomodimersLigand:Steroids9-CisRA+(x)?DNAelement:InvertedDirectrepeatsDirectrepeatsrepeatFigure43–12.Thenuclearreceptorsuperfamily.Membersofthisfamilyaredividedintosixstructuraldomains(A–F).DomainA/BisalsocalledAF-1,orthemodulatorregion,becauseitisinvolvedinactivatingtranscription.TheCdo-mainconsistsoftheDNA-bindingdomain(DBD).TheDregioncontainsthehinge,whichprovidesflexibilitybetweentheDBDandtheligand-bindingdo-main(LBD,regionE).Theamino(N)terminalpartofregionEcontainsAF-2,an-otherdomainimportantfortransactivation.TheFregionispoorlydefined.Thefunctionsofthesedomainsarediscussedinmoredetailinthetext.Receptorswithknownligands,suchasthesteroidhormones,bindashomodimersonin-vertedrepeathalf-sites.OtherreceptorsformheterodimerswiththepartnerRXRondirectrepeatelements.Therecanbenucleotidespacersofonetofivebasesbetweenthesedirectrepeats(DR1–5).Anotherclassofreceptorsforwhichligandshavenotbeendetermined(orphanreceptors)bindashomo-dimerstodirectrepeatsandoccasionallyasmonomerstoasinglehalf-site.ThereIsaLargeFamilyofNuclearreceptor[RXR]partner),orasmonomers.Thetargetresponseelementconsistsofoneortwohalf-sitecon-ReceptorProteinssensussequencesarrangedasaninvertedordirectre-Thenuclearreceptorsuperfamilyconsistsofadiversesetpeat.Thespacingbetweenthelatterhelpsdetermineoftranscriptionfactorsthatwerediscoveredbecauseofabindingspecificity.Thus,adirectrepeatwiththree,sequencesimilarityintheirDNA-bindingdomains.Thisfour,orfivenucleotidespacerregionsspecifiesthefamily,nowwithmorethan50members,includesthebindingofthevitaminD,thyroid,andretinoicacidre-nuclearhormonereceptorsdiscussedabove,anumberofceptors,respectively,tothesameconsensusresponseotherreceptorswhoseligandswerediscoveredaftertheelement(Table43–1).Amultifunctionalligand-receptorswereidentified,andmanyputativeororphanbindingdomain(LBD)islocatedinthecarboxylter-receptorsforwhichaligandhasyettobediscovered.minalhalfofthereceptor.TheLBDbindshormonesThesenuclearreceptorshaveseveralcommonstruc-ormetaboliteswithselectivityandthusspecifiesapar-turalfeatures(Figure43–12).Allhaveacentrallylo-ticularbiologicresponse.TheLBDalsocontainsdo-catedDNA-bindingdomain(DBD)thatallowsthemainsthatmediatethebindingofheatshockproteins,receptortobindwithhighaffinitytoaresponseele-dimerization,nuclearlocalization,andtransactivation.ment.TheDBDcontainstwozincfingerbindingmo-Thelatterfunctionisfacilitatedbythecarboxyltermi-tifs(seeFigure39–14)thatdirectbindingeitherasho-naltranscriptionactivationfunction(AF-2domain),modimers,asheterodimers(usuallywitharetinoidXwhichformsasurfacerequiredfortheinteractionwith
469HORMONEACTION&SIGNALTRANSDUCTION/471GroupIGH,Prl,Insulin,GPCRHormonesCytokines,etcEGF,etcTNF,etcJakcAMPRetinoicacid,RASIRSPlasmaestrogen,membranevitaminD,NFκB•IκBMEKglucocorticoids,etcSTATsPKANFκBMAPKNuclearCREBreceptorsNuclearSTATsmembraneAP-1NFκBCBPp300Figure43–13.SeveralsignaltransductionpathwaysconvergeonCBP/p300.Ligandsthatassociatewithmembraneornuclearreceptorseven-tuallyconvergeonCBP/p300.Severaldifferentsignaltransductionpathwaysareemployed.EGF,epidermalgrowthfactor;GH,growthhormone;Prl,pro-lactin;TNF,tumornecrosisfactor;otherabbreviationsareexpandedinthetext.coactivators.AhighlyvariablehingeregionseparatesAnothergroupoforphanreceptorsthatasyethavenotheDBDfromtheLBD.Thisregionprovidesflexibil-knownligandbindashomodimersormonomerstodi-itytothereceptor,soitcanassumedifferentDNA-rectrepeatsequences.bindingconformations.Finally,thereisahighlyvari-AsillustratedinTable43–5,thediscoveryofthenu-ableaminoterminalregionthatcontainsanothertrans-clearreceptorsuperfamilyhasledtoanimportantun-activationdomainreferredtoasAF-1.Lesswellde-derstandingofhowavarietyofmetabolitesandxenobi-fined,theAF-1domainmayprovidefordistinctoticsregulategeneexpressionandthusthemetabolism,physiologicfunctionsthroughthebindingofdifferentdetoxification,andeliminationofnormalbodyprod-coregulatorproteins.Thisregionofthereceptor,uctsandexogenousagentssuchaspharmaceuticals.throughtheuseofdifferentpromoters,alternativeNotsurprisingly,thisareaisafertilefieldforinvestiga-splicesites,andmultipletranslationinitiationsites,tionofnewtherapeuticinterventions.providesforreceptorisoformsthatshareDBDandLBDidentitybutexertdifferentphysiologicresponsesALargeNumberofNuclearReceptorbecauseoftheassociationofvariouscoregulatorswithCoregulatorsAlsoParticipatethisvariableaminoterminalAF-1domain.inRegulatingTranscriptionItispossibletosortthislargenumberofreceptorsintogroupsinavarietyofways.HeretheyarediscussedChromatinremodeling,transcriptionfactormodificationaccordingtothewaytheybindtotheirrespectiveDNAbyvariousenzymeactivities,andthecommunicationelements(Figure43–12).Classichormonereceptorsforbetweenthenuclearreceptorsandthebasaltranscrip-glucocorticoids(GR),mineralocorticoids(MR),estro-tionapparatusareaccomplishedbyprotein-proteinin-gens(ER),androgens(AR),andprogestins(PR)bindteractionswithoneormoreofaclassofcoregulatorashomodimerstoinvertedrepeatsequences.Othermolecules.Thenumberofthesecoregulatormoleculeshormonereceptorssuchasthyroid(TR),retinoicacidnowexceeds100,notcountingspeciesvariationsand(RAR),andvitaminD(VDR)andreceptorsthatbindsplicevariants.ThefirstofthesetobedescribedwasthevariousmetaboliteligandssuchasPPARαβ,andγ,CREB-bindingprotein,CBP.CBP,throughanFXR,LXR,PXR/SXR,andCARbindasheterodimers,aminoterminaldomain,bindstophosphorylatedserinewithretinoidXreceptor(RXR)asapartner,todirect137ofCREBandmediatestransactivationinresponserepeatsequences(seeFigure43–12andTable43–5).tocAMP.Itthusisdescribedasacoactivator.CBPand
470472/CHAPTER431Table43–5.Nuclearreceptorswithspecialligands.ReceptorPartnerLigandProcessAffectedPeroxisomePPARαRXR(DR1)FattyacidsPeroxisomeproliferationProliferator-PPARβFattyacidsactivatedPPARγFattyacidsLipidandcarbohydratemetabolismEicosanoids,thiazolidinedionesFarnesoidXFXRRXR(DR4)Farnesol,bileacidsBileacidmetabolismLiverXLXRRXR(DR4)OxysterolsCholesterolmetabolismXenobioticXCARRXR(DR5)AndrostanesPhenobarbitalProtectionagainstcertaindrugs,toxicXenobioticsmetabolites,andxenobioticsPXRRXR(DR3)PregnanesXenobiotics1Manymembersofthenuclearreceptorsuperfamilywerediscoveredbycloning,andthecorrespondingligandswerethenidentified.Theseligandsarenothormonesintheclassicsense,buttheydohaveasimilarfunctioninthattheyactivatespecificmembersofthenuclearreceptorsuperfamily.Therecep-torsdescribedhereformheterodimerswithRXRandhavevariablenucleotidesequencesseparatingthedirectrepeatbindingelements(DR1–5).Thesereceptorsregulateavarietyofgenesencodingcy-tochromep450s(CYP),cytosolicbindingproteins,andATP-bindingcassette(ABC)transporterstoinflu-encemetabolismandprotectcellsagainstdrugsandnoxiousagents.itscloserelative,p300,interactdirectlyorindirectlywithanumberofsignalingmolecules,includingactiva-torprotein-1(AP-1),signaltransducersandactivatorsoftranscription(STATs),nuclearreceptors,andCREB(Figure39–11).CBP/p300alsobindstothep160familyofcoactivatorsdescribedbelowandtoanumberTable43–6.Somemammaliancoregulatorofotherproteins,includingviraltranscriptionfactorproteins.rskEla,thep90proteinkinase,andRNAhelicaseA.ItisimportanttonotethatCBP/p300alsohasintrinsicI.300-kDafamilyofcoactivatorshistoneacetyltransferase(HAT)activity.Theimpor-CBPCREB-bindingproteintanceofthisisdescribedbelow.Someofthemanyac-p300Proteinof300kDationsofCBP/p300,whichappeartodependonintrin-II.160-kDafamilyofcoactivatorssicenzymeactivitiesanditsabilitytoserveasascaffoldA.SRC-1Steroidreceptorcoactivator1forthebindingofotherproteins,areillustratedinFig-NCoA-1Nuclearreceptorcoactivator1ure43–11.Othercoregulatorsmayservesimilarfunc-B.TIF2Transcriptionalintermediaryfactor2tions.GRIP1Glucocorticoidreceptor-interactingproteinSeveralotherfamiliesofcoactivatormoleculeshaveNCoA-2Nuclearreceptorcoactivator2beendescribed.Membersofthep160familyofcoac-C.p/CIPp300/CBPcointegrator-associatedprotein1tivators,allofabout160kDa,include(1)SRC-1andACTRActivatorofthethyroidandretinoicacidNCoA-1;(2)GRIP1,TIF2,andNCoA-2;and(3)receptorsp/CIP,ACTR,AIB1,RAC3,andTRAM-1(TableAIBAmplifiedinbreastcancer43–6).Thedifferentnamesformemberswithinasub-RAC3Receptor-associatedcoactivator3familyoftenrepresentspeciesvariationsorminorspliceTRAM-1TRactivatormolecule1variants.Thereisabout35%aminoacididentitybe-III.Corepressorstweenmembersofthedifferentsubfamilies.Thep160NCoRNuclearreceptorcorepressorcoactivatorsshareseveralproperties.They(1)bindSMRTSilencingmediatorforRXRandTRnuclearreceptorsinanagonistandAF-2transactiva-IV.Mediator-relatedproteinstiondomain-dependentmanner;(2)haveaconservedTRAPsThyroidhormonereceptor-associatedaminoterminalbasichelix-loop-helix(bHLH)motifproteinsDRIPsVitaminDreceptor-interactingproteins(seeChapter39);(3)haveaweakcarboxylterminalARCActivator-recruitedcofactortransactivationdomainandastrongeraminoterminal
471HORMONEACTION&SIGNALTRANSDUCTION/473transactivationdomaininaregionthatisrequiredfor•ManyhormoneresponsesareaccomplishedthroughtheCBP/p16Ointeraction;(4)containatleastthreeofalterationsintherateoftranscriptionofspecifictheLXXLLmotifsrequiredforprotein-proteininter-genes.actionwithothercoactivators;and(5)oftenhaveHAT•Thenuclearreceptorsuperfamilyofproteinsplaysaactivity.TheroleofHATisparticularlyinteresting,ascentralroleintheregulationofgenetranscription.mutationsoftheHATdomaindisablemanyofthese•Thesereceptors,whichmayhavehormones,metabo-transcriptionfactors.Currentthinkingholdsthattheselites,ordrugsasligands,bindtospecificDNAele-HATactivitiesacetylatehistonesandresultinremodel-mentsashomodimersorasheterodimerswithRXR.ingofchromatinintoatranscription-efficientenviron-Some—orphanreceptors—havenoknownligandment;however,otherproteinsubstratesforHAT-butbindDNAandinfluencetranscription.mediatedacetylationhavebeenreported.Histone•Anotherlargefamilyofcoregulatorproteinsremodelacetylation/deacetylationisproposedtoplayacriticalchromatin,modifyothertranscriptionfactors,androleingeneexpression.bridgethenuclearreceptorstothebasaltranscriptionAsmallnumberofproteins,includingNCoRandapparatus.SMRT,comprisethecorepressorfamily.Theyfunc-tion,atleastinpart,asdescribedinFigure43–2.An-otherfamilyincludestheTRAPs,DRIPs,andARCREFERENCES(Table43–6).Theseso-calledmediator-relatedpro-teinsrangeinsizefrom80kDato240kDaandareArvanitakisLetal:ConstitutivelysignalingG-protein-coupledre-thoughttobeinvolvedinlinkingthenuclearreceptor-ceptorsandhumandisease.TrendsEndocrinolMetab1998;9:27.coactivatorcomplextoRNApolymeraseIIandtheothercomponentsofthebasaltranscriptionapparatus.BerridgeM:Inositoltriphosphateandcalciumsignalling.Nature1993;361:315.TheexactroleofthesecoactivatorsispresentlyChawlaAetal:Nuclearreceptorsandlipidphysiology:openingtheunderintensiveinvestigation.ManyoftheseproteinsXfiles.Science2001;294:1866.haveintrinsicenzymaticactivities.ThisisparticularlyDarnellJEJr,KerrIM,StarkGR:Jak-STATpathwaysandtrans-interestinginviewofthefactthatacetylation,phos-criptionalactivationinresponsetoIFNsandotherextracellu-phorylation,methylation,andubiquitination—aswelllarsignalingproteins.Science1994;264:1415.asproteolysisandcellulartranslocation—havebeenFantlWJ,JohnsonDE,WilliamsLT:Signallingbyreceptortyro-proposedtoaltertheactivityofsomeofthesecoregula-sinekinases.AnnuRevBiochem1993;62:453.torsandtheirtargets.GiguèreV:Orphannuclearreceptors:fromgenetofunction.En-Itappearsthatcertaincombinationsofcoregula-docrRev1999;20:689.tors—andthusdifferentcombinationsofactivatorsandGrunsteinM:Histoneacetylationinchromatinstructureandtrans-inhibitors—areresponsibleforspecificligand-inducedcription.Nature1997;389:349.actionsthroughvariousreceptors.HanouneJ,DeferN:Regulationandroleofadenylylcyclaseiso-forms.AnnuRevPharmacolToxicol2001;41:145.SUMMARYHermansonO,GlassCK,RosenfeldMG:Nuclearreceptorcoregu-lators:multiplemodesofreceptormodification.TrendsEn-•Hormones,cytokines,interleukins,andgrowthfac-docrinolMetab2002;13:55.torsuseavarietyofsignalingmechanismstofacilitateJakenS:ProteinkinaseCisozymesandsubstrates.CurrOpinCellcellularadaptiveresponses.Biol1996;8:168.•Theligand-receptorcomplexservesastheinitialsig-LucasP,GrannerD:Hormoneresponsedomainsingenetranscrip-tion.AnnuRevBiochem1992;61:1131.nalformembersofthenuclearreceptorfamily.MontminyM:TranscriptionalregulationbycyclicAMP.Annu•ClassIIhormones,whichbindtocellsurfacerecep-RevBiochem1997;66:807tors,generateavarietyofintracellularsignals.TheseMorrisAJ,MalbonCC:PhysiologicalregulationofGprotein-2+includecAMP,cGMP,Ca,phosphatidylinositides,linkedsignaling.PhysiolRev1999;79:1373.andproteinkinasecascades.WaltonKM,DixonJE:Proteintyrosinephosphatases.AnnuRevBiochem1993;62:101.
472SECTIONVISpecialTopicsNutrition,Digestion,&Absorption44DavidA.Bender,PhD,&PeterA.Mayes,PhD,DScBIOMEDICALIMPORTANCEandsteatorrhea.Lactoseintoleranceisduetolactasedeficiencyleadingtodiarrheaandintestinaldiscomfort.Besideswater,thedietmustprovidemetabolicfuelsAbsorptionofintactpeptidesthatstimulateantibody(mainlycarbohydratesandlipids),protein(forgrowthresponsescausesallergicreactions,andceliacdiseaseandturnoveroftissueproteins),fiber(forroughage),isanallergicreactiontowheatgluten.minerals(elementswithspecificmetabolicfunctions),andvitaminsandessentialfattyacids(organiccom-poundsneededinsmallamountsforessentialmetabolicDIGESTION&ABSORPTIONandphysiologicfunctions).Thepolysaccharides,tri-acylglycerols,andproteinsthatmakeupthebulkoftheOFCARBOHYDRATESdietmustbehydrolyzedtotheirconstituentmonosac-Thedigestionofcomplexcarbohydratesisbyhydroly-charides,fattyacids,andaminoacids,respectively,be-sistoliberateoligosaccharides,thenfreemono-anddi-foreabsorptionandutilization.Mineralsandvitaminssaccharides.Theincreaseinbloodglucoseafteratestmustbereleasedfromthecomplexmatrixoffoodbe-doseofacarbohydratecomparedwiththatafteranforetheycanbeabsorbedandutilized.equivalentamountofglucoseisknownastheglycemicGlobally,undernutritioniswidespread,leadingtoindex.Glucoseandgalactosehaveanindexof1,asdoimpairedgrowth,defectiveimmunesystems,andre-lactose,maltose,isomaltose,andtrehalose,whichgiveducedworkcapacity.Bycontrast,indevelopedcoun-risetothesemonosaccharidesonhydrolysis.Fructosetries,thereisoftenexcessivefoodconsumption(espe-andthesugaralcoholsareabsorbedlessrapidlyandciallyoffat),leadingtoobesityandtothedevelopmenthavealowerglycemicindex,asdoessucrose.Theofcardiovasculardiseaseandsomeformsofcancer.De-glycemicindexofstarchvariesbetweennear1tonearficienciesofvitaminA,iron,andiodineposemajorzeroduetovariableratesofhydrolysis,andthatofnon-healthconcernsinmanycountries,anddeficienciesofstarchpolysaccharidesiszero.Foodsthathavealowothervitaminsandmineralsareamajorcauseofillglycemicindexareconsideredtobemorebeneficialhealth.Indevelopedcountries,nutrientdeficiencyissincetheycauselessfluctuationininsulinsecretion.rare,thoughtherearevulnerablesectionsofthepopula-tionatrisk.IntakesofmineralsandvitaminsthatareadequatetopreventdeficiencymaybeinadequatetoAmylasesCatalyzepromoteoptimumhealthandlongevity.theHydrolysisofStarchExcessivesecretionofgastricacid,associatedwithHelicobacterpyloriinfection,canresultinthedevelop-Thehydrolysisofstarchbysalivaryandpancreaticmentofgastricandduodenalulcers;smallchangesinamylasescatalyzerandomhydrolysisofα(1→4)glyco-thecompositionofbilecanresultincrystallizationofsidebonds,yieldingdextrins,thenamixtureofglucose,cholesterolasgallstones;failureofexocrinepancreaticmaltose,andisomaltose(fromthebranchpointsinsecretion(asincysticfibrosis)leadstoundernutritionamylopectin).474
473NUTRITION,DIGESTION,&ABSORPTION/475DisaccharidasesAreBrushSGLT1transporterBorderEnzymesGlucoseproteinGlucoseNa+Thedisaccharidases—maltase,sucrase-isomaltase(aGalactoseGlucosebifunctionalenzymecatalyzinghydrolysisofsucroseandFructoseGalactoseisomaltose),lactase,andtrehalase—arelocatedonthebrushborderoftheintestinalmucosalcellswherethere-GLUT5sultantmonosaccharidesandothersarisingfromthedietareabsorbed.Inmostpeople,apartfromthoseofnorth-ernEuropeangeneticorigin,lactaseisgraduallylostthroughadolescence,leadingtolactoseintolerance.BrushborderLactoseremainsintheintestinallumen,whereitisasubstrateforbacterialfermentationtolactate,resultingindiscomfortanddiarrhea.Na+-K+IntestinalpumpThereAreTwoSeparateMechanismsepitheliumfortheAbsorptionofMonosaccharidesNa+ATPintheSmallIntestine3Na+GlucoseGlucoseandgalactoseareabsorbedbyasodium-depen-Fructose2K+dentprocess.TheyarecarriedbythesametransportGalactose2K+protein(SGLT1)andcompetewitheachotherforin-ADP+Pitestinalabsorption(Figure44–1).Othermonosaccha-ridesareabsorbedbycarrier-mediateddiffusion.Be-causetheyarenotactivelytransported,fructoseandTocapillariessugaralcoholsareonlyabsorbeddowntheirconcentra-GLUT2tiongradient,andafteramoderatelyhighintakesomeFigure44–1.Transportofglucose,fructose,andmayremainintheintestinallumen,actingasasub-strateforbacterialfermentation.galactoseacrosstheintestinalepithelium.TheSGLT1transporteriscoupledtotheNa+-K+pump,allowingglucoseandgalactosetobetransportedagainsttheirDIGESTION&ABSORPTIONOFLIPIDS+concentrationgradients.TheGLUT5Na-independentThemajorlipidsinthedietaretriacylglycerolsand,toafacilitativetransporterallowsfructoseaswellasglu-lesserextent,phospholipids.Thesearehydrophobiccoseandgalactosetobetransportedwiththeircon-moleculesandmustbehydrolyzedandemulsifiedtocentrationgradients.Exitfromthecellforallthesugarsverysmalldroplets(micelles)beforetheycanbeab-isviatheGLUT2facilitativetransporter.sorbed.Thefat-solublevitamins—A,D,E,andK—andavarietyofotherlipids(includingcholesterol)areabsorbeddissolvedinthelipidmicelles.Absorptionofthefat-solublevitaminsisimpairedonaverylowfatoftheproductsoflipiddigestionintomicellesandlipo-diet.somestogetherwithphospholipidsandcholesterolHydrolysisoftriacylglycerolsisinitiatedbylingualfromthebile.Becausethemicellesaresoluble,theyandgastriclipasesthatattackthesn-3esterbond,form-allowtheproductsofdigestion,includingthefat-ing1,2-diacylglycerolsandfreefattyacids,aidingemul-solublevitamins,tobetransportedthroughtheaqueoussification.Pancreaticlipaseissecretedintothesmallenvironmentoftheintestinallumenandpermitcloseintestineandrequiresafurtherpancreaticprotein,coli-contactwiththebrushborderofthemucosalcells,al-pase,foractivity.Itisspecificfortheprimaryesterlowinguptakeintotheepithelium,mainlyoftheje-links—ie,positions1and3intriacylglycerols—result-junum.Thebilesaltspassontotheileum,whereingin2-monoacylglycerolsandfreefattyacidsasthemostareabsorbedintotheenterohepaticcircula-majorend-productsofluminaltriacylglyceroldigestion.tion(Chapter26).Withintheintestinalepithelium,Monoacylglycerolsarehydrolyzedwithdifficultyto1-monoacylglycerolsarehydrolyzedtofattyacidsandglycerolandfreefattyacids,sothatlessthan25%ofin-glyceroland2-monoacylglycerolsarere-acylatedtotri-gestedtriacylglyceroliscompletelyhydrolyzedtoglyc-acylglycerolsviathemonoacylglycerolpathway.Glyc-erolandfattyacids(Figure44–2).Bilesalts,formedinerolreleasedintheintestinallumenisnotreutilizedbuttheliverandsecretedinthebile,enableemulsificationpassesintotheportalvein;glycerolreleasedwithinthe
474AcylAcylAcylPORTALVEINVESSELSGlycerolLYMPHATIC(LACTEALS)ChylomicronsAcylAcylAcylAcylAcylAcylTriacylglycerolPOHOHGlycerol3-phosphateGlycolysisATPKINASEGLYCEROLphatidicacidpathwayOHOHOHsPhoGlycerolINTESTINALEPITHELIUMMonoacylglycerolpathwayATPATPCoALIPASEFAAcyl-CoAINTESTINALOHAcylOHAcylOHOHACYL-CoASYNTHETASE72%6%ACYL-CoAATP,CoA22%SYNTHETASEAcylAcylOHOHAcylOHAcylOHOHOHOHOH2-Mono-1-Mono-GlycerolDigestionandabsorptionoftriacylglycerols.ThevaluesgivenforpercentageuptakemayvaryacylglycerolacylglycerolFAFALIPASELIPASE1,2-DiacylglycerolISOMERASEPANCREATICPANCREATICFAFALUMENLIPASEfromFigure44–2.widelybutindicatetherelativeimportanceofthethreeroutesshown.INTESTINALPANCREATICbilesaltmicelleAbsorptionAcylAcylAcyl123Triacylglycerol,100%476
475NUTRITION,DIGESTION,&ABSORPTION/477epitheliumisreutilizedfortriacylglycerolsynthesisviaseveraldifferentaminoacidtransporters,withspecificitythenormalphosphatidicacidpathway(Chapter24).forthenatureoftheaminoacidsidechain(largeorAlllong-chainfattyacidsabsorbedareconvertedtotri-small;neutral,acidic,orbasic).Thevariousaminoacidsacylglycerolinthemucosalcellsand,togetherwiththecarriedbyanyonetransportercompetewitheachotherotherproductsoflipiddigestion,secretedaschylomi-forabsorptionandtissueuptake.Dipeptidesandtripep-cronsintothelymphatics,enteringthebloodstreamviatidesenterthebrushborderoftheintestinalmucosalthethoracicduct(Chapter25).cells,wheretheyarehydrolyzedtofreeaminoacids,whicharethentransportedintothehepaticportalvein.DIGESTION&ABSORPTIONOFPROTEINSRelativelylargepeptidesmaybeabsorbedintact,eitherbyuptakeintomucosalepithelialcells(transcellular)orFewpeptidebondsthatarehydrolyzedbyproteolyticbypassingbetweenepithelialcells(paracellular).Manyenzymesareaccessiblewithoutpriordenaturationofdi-suchpeptidesarelargeenoughtostimulateantibodyfor-etaryproteins(byheatincookingandbytheactionofmation—thisisthebasisofallergicreactionstofoods.gastricacid).DIGESTION&ABSORPTIONSeveralGroupsofEnzymesCatalyzeOFVITAMINS&MINERALStheDigestionofProteinsVitaminsandmineralsarereleasedfromfoodduringTherearetwomainclassesofproteolyticdigestiveen-digestion—thoughthisisnotcomplete—andtheavail-zymes(proteases),withdifferentspecificitiesfortheabilityofvitaminsandmineralsdependsonthetypeofaminoacidsformingthepeptidebondtobehydrolyzed.foodand,especiallyforminerals,thepresenceofchelat-Endopeptidaseshydrolyzepeptidebondsbetweenspe-ingcompounds.Thefat-solublevitaminsareabsorbedcificaminoacidsthroughoutthemolecule.Theyaretheinthelipidmicellesthatresultfromfatdigestion;firstenzymestoact,yieldingalargernumberofsmallerwater-solublevitaminsandmostmineralsaltsarefragments,eg,pepsininthegastricjuiceandtrypsin,absorbedfromthesmallintestineeitherbyactivetrans-chymotrypsin,andelastasesecretedintothesmallin-portorbycarrier-mediateddiffusionfollowedbybind-testinebythepancreas.Exopeptidasescatalyzethehy-ingtointracellularbindingproteinstoachieveconcen-drolysisofpeptidebonds,oneatatime,fromtheendstrationuponuptake.VitaminB12absorptionrequiresaofpolypeptides.Carboxypeptidases,secretedinthespecifictransportprotein,intrinsicfactor;calciumab-pancreaticjuice,releaseaminoacidsfromthefreecar-sorptionisdependentonvitaminD;zincabsorptionboxylterminal,andaminopeptidases,secretedbytheprobablyrequiresazinc-bindingligandsecretedbytheintestinalmucosalcells,releaseaminoacidsfromtheexocrinepancreas;andtheabsorptionofironislimited.aminoterminal.Dipeptides,whicharenotsubstratesforexopeptidases,arehydrolyzedinthebrushborderofin-CalciumAbsorptionIsDependenttestinalmucosalcellsbydipeptidases.onVitaminDTheproteasesaresecretedasinactivezymogens;theactivesiteoftheenzymeismaskedbyasmallregionofInadditiontoitsroleinregulatingcalciumhomeosta-itspeptidechain,whichisremovedbyhydrolysisofasis,vitaminDisrequiredfortheintestinalabsorptionspecificpeptidebond.Pepsinogenisactivatedtopepsinofcalcium.Synthesisoftheintracellularcalcium-bygastricacidandbyactivatedpepsin(autocatalysis).Inbindingprotein,calbindin,requiredforcalciumab-thesmallintestine,trypsinogen,theprecursorofsorption,isinducedbyvitaminD,whichalsoaffectstrypsin,isactivatedbyenteropeptidase,whichisse-thepermeabilityofthemucosalcellstocalcium,anef-cretedbytheduodenalepithelialcells;trypsincanthenfectthatisrapidandindependentofproteinsynthesis.activatechymotrypsinogentochymotrypsin,proelas-Phyticacid(inositolhexaphosphate)incerealsbindstasetoelastase,procarboxypeptidasetocarboxypepti-calciumintheintestinallumen,preventingitsabsorp-dase,andproaminopeptidasetoaminopeptidase.tion.Otherminerals,includingzinc,arealsochelatedbyphytate.ThisismainlyaproblemamongpeopleFreeAminoAcids&SmallPeptidesArewhoconsumelargeamountsofunleavenedwholeAbsorbedbyDifferentMechanismswheatproducts;yeastcontainsanenzyme,phytase,whichdephosphorylatesphytate,sorenderingitinac-Theendproductoftheactionofendopeptidasesandtive.Highconcentrationsoffattyacidsintheintestinalexopeptidasesisamixtureoffreeaminoacids,di-andlumen—asaresultofimpairedfatabsorption—cantripeptides,andoligopeptides,allofwhichareabsorbed.alsoreducecalciumabsorptionbyforminginsolubleFreeaminoacidsareabsorbedacrosstheintestinalmu-calciumsalts;ahighintakeofoxalatecansometimescosabysodium-dependentactivetransport.Therearecausedeficiency,sincecalciumoxalateisinsoluble.
476478/CHAPTER44IronAbsorptionIsLimitedlizediscarbohydrate,fat,orprotein.Measurementof&StrictlyControlledbutIstheratioofthevolumeofcarbondioxideproducedtoEnhancedbyVitaminC&Ethanolvolumeofoxygenconsumed(respiratoryquotient;RQ)isanindicationofthemixtureofmetabolicfuelsbeingAlthoughirondeficiencyisacommonproblem,aboutoxidized(Table27–1).Amorerecenttechniqueper-10%ofthepopulationaregeneticallyatriskofironmitsestimationoftotalenergyexpenditureoverape-overload(hemochromatosis),andelementalironcanriodof1–2weeksusingdualisotopicallylabeledwater,leadtononenzymicgenerationoffreeradicals.Absorp-2H18O.2Hislostfromthebodyonlyinwater,while2tionofironisstrictlyregulated.Inorganicironisaccu-18Oislostinbothwaterandcarbondioxide;thediffer-mulatedinintestinalmucosalcellsboundtoanintra-enceintherateoflossofthetwolabelspermitsestima-cellularprotein,ferritin.Oncetheferritininthecellistionoftotalcarbondioxideproductionandthusoxy-saturatedwithiron,nomorecanenter.Ironcanonlygenconsumptionandenergyexpenditure.leavethemucosalcellifthereistransferrininplasmaBasalmetabolicrate(BMR)istheenergyexpendi-tobindto.Oncetransferrinissaturatedwithiron,anyturebythebodywhenatrest—butnotasleep—underthathasaccumulatedinthemucosalcellswillbelostcontrolledconditionsofthermalneutrality,measuredwhenthecellsareshed.Asaresultofthismucosalbar-atabout12hoursafterthelastmeal,anddependsonrier,onlyabout10%ofdietaryironisnormallyab-weight,age,andgender.Totalenergyexpenditurede-sorbedandonly1–5%frommanyplantfoods.pendsonthebasalmetabolicrate,theenergyrequired2+InorganicironisabsorbedonlyintheFe(reduced)forphysicalactivity,andtheenergycostofsynthesizingstate,andforthatreasonthepresenceofreducingagentsreservesinthefedstate.Itisthereforepossibletocalcu-willenhanceabsorption.Themosteffectivecompoundlateanindividual’senergyrequirementfrombodyisvitaminC,andwhileintakesof40–60mgofvitaminweight,age,gender,andlevelofphysicalactivity.BodyCperdayaremorethanadequatetomeetrequirements,weightaffectsBMRbecausethereisagreateramountanintakeof25–50mgpermealwillenhanceironab-ofactivetissueinalargerbody.ThedecreaseinBMRsorption,especiallywhenironsaltsareusedtotreatironwithincreasingage,evenwhenbodyweightremainsdeficiencyanemia.Ethanolandfructosealsoenhanceconstant,isduetomuscletissuereplacementbyadi-ironabsorption.Hemeironfrommeatisabsorbedsepa-posetissue,whichismetabolicallymuchlessactive.ratelyandisconsiderablymoreavailablethaninorganicSimilarly,womenhaveasignificantlylowerBMRthaniron.However,theabsorptionofbothinorganicanddomenofthesamebodyweightbecausewomen’sbod-hemeironisimpairedbycalcium—aglassofmilkwithieshaveproportionatelymoreadiposetissuethanmen.amealsignificantlyreducesavailability.EnergyRequirementsIncreaseENERGYBALANCE:WithActivityOVER-&UNDERNUTRITIONThemostusefulwayofexpressingtheenergycostofAftertheprovisionofwater,thebody’sfirstrequirementphysicalactivitiesisasamultipleofBMR.Sedentaryisformetabolicfuels—fats,carbohydrates,andaminoactivitiesuseonlyabout1.1–1.2×BMR.Bycontrast,acidsfromproteins(andethanol)(Table27–1).Foodin-vigorousexertion,suchasclimbingstairs,cross-countrytakeinexcessofenergyexpenditureleadstoobesity,skiing,walkinguphill,etc,mayuse6–8×BMR.whileintakelessthanexpenditureleadstoemaciationandwasting,asinmarasmusandkwashiorkor.BothTenPercentoftheEnergyYieldofaMealobesityandsevereundernutritionareassociatedwithin-MayBeExpendedinFormingReservescreasedmortality.Thebodymassindex,definedasweightinkilogramsdividedbyheightinmeterssquared,Thereisaconsiderableincreaseinmetabolicrateafteraiscommonlyusedasawayofexpressingrelativeobesitymeal,aphenomenonknownasdiet-inducedthermo-toheight.Adesirablerangeisbetween20and25.genesis.Asmallpartofthisistheenergycostofsecret-ingdigestiveenzymesandofactivetransportoftheEnergyRequirementsAreEstimatedbyproductsofdigestion;themajorpartisduetosynthe-MeasurementofEnergyExpendituresizingreservesofglycogen,triacylglycerol,andprotein.Energyexpenditurecanbedetermineddirectlybymea-ThereAreTwoExtremeFormssuringheatoutputfromthebodybutisnormallyesti-ofUndernutritionmatedindirectlyfromtheconsumptionofoxygen.Thereisanenergyexpenditureof20kJ/LofoxygenMarasmuscanoccurinbothadultsandchildrenandconsumedregardlessofwhetherthefuelbeingmetabo-occursinvulnerablegroupsofallpopulations.Kwash-
477NUTRITION,DIGESTION,&ABSORPTION/479iorkoronlyaffectschildrenandhasonlybeenreportedtheliverduetoaccumulationoffat.Itwasformerlybe-indevelopingcountries.Thedistinguishingfeatureoflievedthatthecauseofkwashiorkorwasalackofpro-kwashiorkoristhatthereisfluidretention,leadingtotein,withamoreorlessadequateenergyintake;how-edema.Marasmusisastateofextremeemaciation;itisever,analysisofthedietsofaffectedchildrenshowsthattheoutcomeofprolongednegativeenergybalance.Notthisisnotso.Childrenwithkwashiorkorarelessonlyhavethebody’sfatreservesbeenexhausted,butstuntedthanthosewithmarasmus,andtheedemabe-thereiswastageofmuscleaswell,andastheconditionginstoimproveearlyintreatment,whenthechildisprogressesthereislossofproteinfromtheheart,liver,stillreceivingalow-proteindiet.Verycommonly,anandkidneys.Theaminoacidsreleasedbythecatabo-infectionprecipitateskwashiorkor.Superimposedonlismoftissueproteinsareusedasasourceofmetabolicgeneralfooddeficiency,thereisprobablyadeficiencyfuelandassubstratesforgluconeogenesistomaintainaoftheantioxidantnutrientssuchaszinc,copper,supplyofglucoseforthebrainandredbloodcells.Asacarotene,andvitaminsCandE.Therespiratoryburstresultofthereducedsynthesisofproteins,thereisim-inresponsetoinfectionleadstotheproductionofoxy-pairedimmuneresponseandmoreriskfrominfections.genandhalogenfreeradicalsaspartofthecytotoxicImpairmentofcellproliferationintheintestinalmu-actionofstimulatedmacrophages.Thisaddedoxidantcosaoccurs,resultinginreductioninsurfaceareaofthestressmaywelltriggerthedevelopmentofkwashiorkor.intestinalmucosaandreductioninabsorptionofsuchnutrientsasareavailable.PROTEIN&AMINOACIDREQUIREMENTSPatientsWithAdvancedCancerProteinRequirementsCanBeDetermined&AIDSAreMalnourishedbyMeasuringNitrogenBalancePatientswithadvancedcancer,HIVinfectionandThestateofproteinnutritioncanbedeterminedbyAIDS,andanumberofotherchronicdiseasesarefre-measuringthedietaryintakeandoutputofnitrogenousquentlyundernourished—theconditioniscalledcompoundsfromthebody.Althoughnucleicacidsalsocachexia.Physically,theyshowallthesignsofmaras-containnitrogen,proteinisthemajordietarysourceofmus,butthereisconsiderablymorelossofbodypro-nitrogenandmeasurementoftotalnitrogenintaketeinthanoccursinstarvation.Thesecretionofcy-givesagoodestimateofproteinintake(mgN×6.25=tokinesinresponsetoinfectionandcancerincreasesthemgprotein,asnitrogenis16%ofmostproteins).Thecatabolismoftissueprotein.Thisdiffersfrommaras-outputofnitrogenfromthebodyismainlyinureaandmus,inwhichproteinsynthesisisreducedbutcatabo-smallerquantitiesofothercompoundsinurineandlisminunaffected.Patientsarehypermetabolic,ie,undigestedproteininfeces,andsignificantamountsthereisaconsiderableincreaseinbasalmetabolicrate.mayalsobelostinsweatandshedskin.Manytumorsmetabolizeglucoseanaerobicallytore-Thedifferencebetweenintakeandoutputofnitroge-leaselactate.Thisisusedforgluconeogenesisinthenouscompoundsisknownasnitrogenbalance.Threeliver,whichisenergy-consumingwithanetcostofsixstatescanbedefined:Inahealthyadult,nitrogenbal-ATPforeachmoleofglucosecycled(Chapter19).anceisinequilibriumwhenintakeequalsoutput,andThereisincreasedstimulationofuncouplingproteinsthereisnochangeinthetotalbodycontentofprotein.bycytokines,leadingtothermogenesisandincreasedInagrowingchild,apregnantwoman,orinrecoveryoxidationofmetabolicfuels.Futilecyclingoflipidsoc-fromproteinloss,theexcretionofnitrogenouscom-cursbecausehormone-sensitivelipaseisactivatedbyapoundsislessthanthedietaryintakeandthereisnetre-proteoglycansecretedbytumors,resultinginliberationtentionofnitrogeninthebodyasprotein,ie,positiveoffattyacidsfromadiposetissueandATP-expensivenitrogenbalance.Inresponsetotraumaorinfection—reesterificationinthelivertotriacylglycerols,whichareoriftheintakeofproteinisinadequatetomeetrequire-exportedinVLDL.ments—thereisnetlossofproteinnitrogenfromthebody,ie,negativenitrogenbalance.Thecontinualca-KwashiorkorAffectstabolismoftissueproteinscreatestherequirementfordietaryproteineveninanadultwhoisnotgrowing,UndernourishedChildrenthoughsomeoftheaminoacidsreleasedcanbereuti-Inadditiontothewastingofmuscletissue,lossofin-lized.Nitrogenbalancestudiesshowthattheaveragetestinalmucosa,andimpairedimmuneresponsesseendailyrequirementis0.6gofproteinperkilogramofinmarasmus,childrenwithkwashiorkorshowanum-bodyweight(thefactor0.75shouldbeusedtoallowforberofcharacteristicfeatures.Thedefiningcharacteristicindividualvariation),orapproximately50g/d.Averageisedema,associatedwithadecreasedconcentrationofintakesofproteinindevelopedcountriesareaboutplasmaproteins.Inaddition,thereisenlargementof80–100g/d,ie,14–15%ofenergyintake.Because
478480/CHAPTER44growingchildrenareincreasingtheproteininthebody,aloacetate,andα-ketoglutarate,respectively).There-theyhaveaproportionatelygreaterrequirementthanmainingaminoacidsareconsideredasnonessential,butadultsandshouldbeinpositivenitrogenbalance.Evenundersomecircumstancestherequirementforthemso,theneedisrelativelysmallcomparedwiththere-mayoutstriptheorganism’scapacityforsynthesis.quirementforproteinturnover.Insomecountries,pro-teinintakemaybeinadequatetomeettheserequire-ments,resultinginstuntingofgrowth.SUMMARY•DigestioninvolveshydrolyzingfoodmoleculesintoThereIsaLossofBodyProteininsmallermoleculesforabsorptionthroughtheResponsetoTrauma&Infectiongastrointestinalepithelium.Polysaccharidesareabsorbedasmonosaccharides;triacylglycerolsasOneofthemetabolicreactionstomajortrauma,such2-monoacylglycerols,fattyacids,andglycerol;andasaburn,abrokenlimb,orsurgery,isanincreaseinproteinsasaminoacids.thenetcatabolismoftissueproteins.Asmuchas6–7%•Digestivedisordersariseasaresultof(1)enzymede-ofthetotalbodyproteinmaybelostover10days.Pro-ficiency,eg,lactaseandsucrase;(2)malabsorption,longedbedrestresultsinconsiderablelossofproteineg,ofglucoseandgalactoseduetodefectsinthebecauseofatrophyofmuscles.ProteiniscatabolizedasNa+-glucosecotransporter(SGLT1);(3)absorptionnormal,butwithoutthestimulusofexerciseitisnotofunhydrolyzedpolypeptides,leadingtoimmuno-completelyreplaced.Lostproteinisreplacedduringlogicresponses,eg,asinceliacdisease;and(4)pre-convalescence,whenthereispositivenitrogenbalance.cipitationofcholesterolfrombileasgallstones.Anormaldietisadequatetopermitthisreplacement.•Besideswater,thedietmustprovidemetabolicfuelsTheRequirementIsNotforProteinItself(carbohydrateandfat)forbodilygrowthandactivity;proteinforsynthesisoftissueproteins;fiberforbutforSpecificAminoAcidsroughage;mineralsforspecificmetabolicfunctions;Notallproteinsarenutritionallyequivalent.Moreofcertainpolyunsaturatedfattyacidsofthen-3andn-6somethanofothersisneededtomaintainnitrogenfamiliesforeicosanoidsynthesisandotherfunctions;balancebecausedifferentproteinscontaindifferentandvitamins,organiccompoundsneededinsmallamountsofthevariousaminoacids.Thebody’sre-amountsformanyvariedessentialfunctions.quirementisforspecificaminoacidsinthecorrect•Twentydifferentaminoacidsarerequiredforpro-proportionstoreplacethebodyproteins.Theaminoteinsynthesis,ofwhichnineareessentialintheacidscanbedividedintotwogroups:essentialandhumandiet.Thequantityofproteinrequiredisaf-nonessential.Therearenineessentialorindispensablefectedbyproteinquality,energyintake,andphysicalaminoacids,whichcannotbesynthesizedinthebody:activity.histidine,isoleucine,leucine,lysine,methionine,phen-•Undernutritionoccursintwoextremeforms:maras-ylalanine,threonine,tryptophan,andvaline.Ifoneofmusinadultsandchildrenandkwashiorkorinchil-theseislackingorinadequate,then—regardlessofthedren.Overnutritionfromexcessenergyintakeisas-totalintakeofprotein—itwillnotbepossibletomain-sociatedwithdiseasessuchasobesity,type2diabetestainnitrogenbalancesincetherewillnotbeenoughofmellitus,atherosclerosis,cancer,andhypertension.thataminoacidforproteinsynthesis.Twoaminoacids—cysteineandtyrosine—canbesynthesizedinthebody,butonlyfromessentialaminoREFERENCESacidprecursors(cysteinefrommethionineandtyrosinefromphenylalanine).ThedietaryintakesofcysteineBenderDA,BenderAE:Nutrition:AReferenceHandbook.OxfordUnivPress,1997.andtyrosinethusaffecttherequirementsformethio-BüllerHA,GrandRJ:Lactoseintolerance.AnnuRevMednineandphenylalanine.Theremaining11aminoacids1990;41:141.inproteinsareconsideredtobenonessentialordispens-FullerMF,GarlickPJ:Humanaminoacidrequirements.Annuable,sincetheycanbesynthesizedaslongasthereisRevNutr1994;14:217.enoughtotalproteininthediet—ie,ifoneoftheseGarrowJS,JamesWPT,RalphA:HumanNutritionandDietetics,aminoacidsisomittedfromthediet,nitrogenbalance10thed.Churchill-Livingstone,2000.canstillbemaintained.However,onlythreeaminoNationalAcademyofSciencesreportondietandhealth.NutrRevacids—alanine,aspartate,andglutamate—canbecon-1989;47:142.sideredtobetrulydispensable;theyaresynthesizedNielsenFH:Nutritionalsignificanceoftheultratraceelements.fromcommonmetabolicintermediates(pyruvate,ox-NutrRev1988;46:337.
479Vitamins&Minerals45DavidA.Bender,PhD,&PeterA.Mayes,PhD,DScBIOMEDICALIMPORTANCEmia(iron),cretinismandgoiter(iodine).Ifpresentinexcessaswithselenium,toxicitysymptomsmayoccur.Vitaminsareagroupoforganicnutrientsrequiredinsmallquantitiesforavarietyofbiochemicalfunctionsandwhich,generally,cannotbesynthesizedbytheTHEDETERMINATIONOFNUTRIENTbodyandmustthereforebesuppliedinthediet.REQUIREMENTSDEPENDSONTHEThelipid-solublevitaminsareapolarhydrophobicCRITERIAOFADEQUACYCHOSENcompoundsthatcanonlybeabsorbedefficientlywhenForanynutrient,particularlymineralsandvitamins,thereisnormalfatabsorption.Theyaretransportedinthereisarangeofintakesbetweenthatwhichisclearlytheblood,likeanyotherapolarlipid,inlipoproteinsorinadequate,leadingtoclinicaldeficiencydisease,andattachedtospecificbindingproteins.Theyhavediversethatwhichissomuchinexcessofthebody’smetabolicfunctions,eg,vitaminA,vision;vitaminD,calciumcapacitythattheremaybesignsoftoxicity.Betweenandphosphatemetabolism;vitaminE,antioxidant;vi-thesetwoextremesisalevelofintakethatisadequatetaminK,bloodclotting.Aswellasdietaryinadequacy,fornormalhealthandthemaintenanceofmetabolicin-conditionsaffectingthedigestionandabsorptionofthetegrity.Individualsdonotallhavethesamerequire-lipid-solublevitamins—suchassteatorrheaanddisor-mentfornutrientsevenwhencalculatedonthebasisofdersofthebiliarysystem—canallleadtodeficiencybodysizeorenergyexpenditure.Thereisarangeofin-syndromes,including:nightblindnessandxeroph-dividualrequirementsofupto25%aroundthemean.thalmia(vitaminA);ricketsinyoungchildrenandos-Therefore,inordertoassesstheadequacyofdiets,itisteomalaciainadults(vitaminD);neurologicdisordersnecessarytosetareferencelevelofintakehighenoughandanemiaofthenewborn(vitaminE);andhemor-toensurethatnoonewilleithersufferfromdeficiencyrhageofthenewborn(vitaminK).Toxicitycanresultorbeatriskoftoxicity.IfitisassumedthatindividualfromexcessiveintakeofvitaminsAandD.VitaminArequirementsaredistributedinastatisticallynormalandβ-carotene(provitaminA),aswellasvitaminE,arefashionaroundtheobservedmeanrequirement,thenaantioxidantsandhavepossiblerolesinatherosclerosisrangeof+/−2×thestandarddeviation(SD)aroundandcancerprevention.themeanwillincludetherequirementsof95%oftheThewater-solublevitaminscomprisetheBcomplexpopulation.andvitaminCandfunctionasenzymecofactors.Folicacidactsasacarrierofone-carbonunits.DeficiencyofasinglevitaminoftheBcomplexisrare,sincepoorTHEVITAMINSAREADISPARATEGROUPdietsaremostoftenassociatedwithmultipledeficiencyOFCOMPOUNDSWITHAVARIETYstates.Nevertheless,specificsyndromesarecharacteris-OFMETABOLICFUNCTIONSticofdeficienciesofindividualvitamins,eg,beriberi(thiamin);cheilosis,glossitis,seborrhea(riboflavin);Avitaminisdefinedasanorganiccompoundthatisre-pellagra(niacin);peripheralneuritis(pyridoxine);quiredinthedietinsmallamountsforthemaintenancemegaloblasticanemia,methylmalonicaciduria,andofnormalmetabolicintegrity.Deficiencycausesaspe-perniciousanemia(vitaminB12);andmegaloblasticcificdisease,whichiscuredorpreventedonlybyrestor-anemia(folicacid).VitaminCdeficiencyleadstoingthevitamintothediet(Table45–1).However,vit-scurvy.aminD,whichcanbemadeintheskinafterexposureInorganicmineralelementsthathaveafunctionintosunlight,andniacin,whichcanbeformedfromthethebodymustbeprovidedinthediet.Whentheintakeessentialaminoacidtryptophan,donotstrictlycon-isinsufficient,deficiencysymptomsmayarise,eg,ane-formtothisdefinition.481
480482/CHAPTER45Table45–1.Thevitamins.VitaminFunctionsDeficiencyDiseaseARetinol,β-caroteneVisualpigmentsintheretina;regulationofNightblindness,xerophthalmia;geneexpressionandcelldifferentiation;keratinizationofskinβ-caroteneisanantioxidantDCalciferolMaintenanceofcalciumbalance;enhancesRickets=poormineralizationofbone;2+intestinalabsorptionofCaandmobilizesosteomalacia=bonedemineralizationbonemineralETocopherols,tocotrienolsAntioxidant,especiallyincellmembranesExtremelyrare—seriousneurologicdysfunctionKPhylloquinone,Coenzymeinformationofγ-carboxyglutamateImpairedbloodclotting,hemor-menaquinonesinenzymesofbloodclottingandbonematrixrhagicdiseaseB1ThiaminCoenzymeinpyruvateandα−ketoglutarate,Peripheralnervedamage(beriberi)ordehydrogenases,andtransketolase;poorlycentralnervoussystemlesionsdefinedfunctioninnerveconduction(Wernicke-Korsakoffsyndrome)B2RiboflavinCoenzymeinoxidationandreductionreactions;Lesionsofcornerofmouth,lips,andprostheticgroupofflavoproteinstongue;seborrheicdermatitisNiacinNicotinicacid,Coenzymeinoxidationandreductionreactions,Pellagra—photosensitivedermatitis,nicotinamidefunctionalpartofNADandNADPdepressivepsychosisB6Pyridoxine,pyridoxal,Coenzymeintransaminationanddecarboxy-Disordersofaminoacidmetabolism,pyridoxaminelationofaminoacidsandglycogenconvulsionsphosphorylase;roleinsteroidhormoneactionFolicacidCoenzymeintransferofone-carbonfragmentsMegaloblasticanemiaB12CobalaminCoenzymeintransferofone-carbonfragmentsPerniciousanemia=megaloblasticandmetabolismoffolicacidanemiawithdegenerationofthespinalcordPantothenicacidFunctionalpartofCoAandacylcarrierprotein:fattyacidsynthesisandmetabolismHBiotinCoenzymeincarboxylationreactionsingluco-Impairedfatandcarbohydratemetab-neogenesisandfattyacidsynthesisolism,dermatitisCAscorbicacidCoenzymeinhydroxylationofprolineandScurvy—impairedwoundhealing,lysineincollagensynthesis;antioxidant;lossofdentalcement,subcutaneousenhancesabsorptionofironhemorrhageprovitaminA,astheycanbecleavedtoyieldretinalde-LIPID-SOLUBLEVITAMINShydeandthenceretinolandretinoicacid.Theα-,β-,andγ-carotenesandcryptoxanthinarequantitativelythemostimportantprovitaminAcarotenoids.Al-RETINOIDS&CAROTENOIDSthoughitwouldappearthatonemoleculeofβ-HAVEVITAMINAACTIVITYcaroteneshouldyieldtwoofretinol,thisisnotsoinpractice;6μgofβ-caroteneisequivalentto1μgof(Figure45–1)preformedretinol.ThetotalamountofvitaminAinfoodsisthereforeexpressedasmicrogramsofretinolRetinoidscompriseretinol,retinaldehyde,andequivalents.Beta-caroteneandotherprovitaminAretinoicacid(preformedvitaminA,foundonlyincarotenoidsarecleavedintheintestinalmucosabyfoodsofanimalorigin);carotenoids,foundinplants,carotenedioxygenase,yieldingretinaldehyde,whichiscomprisecarotenesandrelatedcompounds,knownasreducedtoretinol,esterified,andsecretedinchylomi-
481VITAMINS&MINERALS/483H3CCHCHHCCH3333H3CCH3CHCH3CH33β-CaroteneHCCHCH3CH3HCCHCH3CH33333CH2OHCHOCHRetinolCHRetinaldehyde33CH3CH3CH3H3CCH3H3CCH3COOHCHCHFigure45–1.β-Caroteneandthemajorvita-33minAvitamers.*ShowsthesiteofcleavageofAll-trans-retinoicacidH3Cβ-caroteneintotwomoleculesofretinaldehydeCOOHbycarotenedioxygenase.9-cis-retinoicacidcronstogetherwithestersformedfromdietaryretinol.ciency,boththetimetakentoadapttodarknessandtheTheintestinalactivityofcarotenedioxygenaseislow,abilitytoseeinpoorlightareimpaired.sothatarelativelylargeproportionofingestedβ-carotenemayappearinthecirculationunchanged.RetinoicAcidHasaRoleWhiletheprincipalsiteofcarotenedioxygenaseattackintheRegulationofGeneisthecentralbondofβ-carotene,asymmetriccleavageExpression&TissueDifferentiationmayalsooccur,leadingtotheformationof8′-,10′-,and12′-apo-carotenals,whichareoxidizedtoretinoicAmostimportantfunctionofvitaminAisinthecon-acidbutcannotbeusedassourcesofretinolorretin-trolofcelldifferentiationandturnover.All-trans-aldehyde.retinoicacidand9-cis-retinoicacid(Figure45–1)regu-lategrowth,development,andtissuedifferentiation;VitaminAHasaFunctioninVisiontheyhavedifferentactionsindifferenttissues.LikethesteroidhormonesandvitaminD,retinoicacidbindstoIntheretina,retinaldehydefunctionsastheprostheticnuclearreceptorsthatbindtoresponseelementsofgroupofthelight-sensitiveopsinproteins,formingDNAandregulatethetranscriptionofspecificgenes.rhodopsin(inrods)andiodopsin(incones).AnyoneTherearetwofamiliesofnuclearretinoidreceptors:theconecellcontainsonlyonetypeofopsinandissensitiveretinoicacidreceptors(RARs)bindall-trans-retinoictoonlyonecolor.Inthepigmentepitheliumoftheacidor9-cis-retinoicacid,andtheretinoidXreceptorsretina,all-trans-retinolisisomerizedto11-cis-retinol(RXRs)bind9-cis-retinoicacid.andoxidizedto11-cis-retinaldehyde.Thisreactswithalysineresidueinopsin,formingtheholoproteinVitaminADeficiencyIsaMajorPublicrhodopsin.AsshowninFigure45–2,theabsorptionofHealthProblemWorldwidelightbyrhodopsincausesisomerizationoftheretinalde-hydefrom11-cistoall-trans,andaconformationalVitaminAdeficiencyisthemostimportantpreventablechangeinopsin.Thisresultsinthereleaseofretinalde-causeofblindness.Theearliestsignofdeficiencyisahydefromtheproteinandtheinitiationofanerveim-lossofsensitivitytogreenlight,followedbyimpair-pulse.Theformationoftheinitialexcitedformofmentofadaptationtodimlight,followedbynightrhodopsin,bathorhodopsin,occurswithinpicosecondsblindness.Moreprolongeddeficiencyleadstoxeroph-ofillumination.Thereisthenaseriesofconformationalthalmia:keratinizationofthecorneaandskinandchangesleadingtotheformationofmetarhodopsinII,blindness.VitaminAalsohasanimportantroleindif-whichinitiatesaguaninenucleotideamplificationcas-ferentiationofimmunesystemcells,andmilddefi-cadeandthenanerveimpulse.Thefinalstepishydroly-ciencyleadstoincreasedsusceptibilitytoinfectiousdis-sistoreleaseall-trans-retinaldehydeandopsin.Thekeyeases.Furthermore,thesynthesisofretinol-bindingtoinitiationofthevisualcycleistheavailabilityofproteininresponsetoinfectionisreduced(itisanega-11-cis-retinaldehyde,andhencevitaminA.Indefi-tiveacutephaseprotein),decreasingthecirculatingvi-
482484/CHAPTER45CHCHataxia,andanorexia,allassociatedwithincreasedcere-HCCH3333CHOH2brospinalfluidpressure),theliver(hepatomegalywithhistologicchangesandhyperlipidemia),calciumho-CHAll-trans-retinol3meostasis(thickeningofthelongbones,hypercalcemiaCHandcalcificationofsofttissues),andtheskin(excessiveHCCH333dryness,desquamation,andalopecia).11-cis-RetinolCH3H3CCH2OHVITAMINDISREALLYAHORMONECH3H3CCH3VitaminDisnotstrictlyavitaminsinceitcanbesyn-11-cis-Retinaldehydethesizedintheskin,andundermostconditionsthatisCH3H3Citsmajorsource.OnlywhensunlightisinadequateisaHC=OC=Odietarysourcerequired.ThemainfunctionofvitaminH2NDisintheregulationofcalciumabsorptionandho-CHLysineresidueHCCH3meostasis;mostofitsactionsaremediatedbyway33inopsinNHofnuclearreceptorsthatregulategeneexpression.CHDeficiency—leadingtoricketsinchildrenandosteo-3H3CC=OHC=Nmalaciainadults—continuestobeaprobleminnorth-Rhodopsin(visualpurple)ernlatitudes,wheresunlightexposureispoor.-15NHLIGHT10secCHCHC=OVitaminDIsSynthesizedintheSkinHCCH3333C=N7-Dehydrocholesterol(anintermediateinthesynthesisCH3NHofcholesterolthataccumulatesintheskin),undergoesPhotorhodopsinanonenzymicreactiononexposuretoultravioletlight,45psecyieldingprevitaminD(Figure45–3).Thisundergoesa5'GMPBathorhodopsinfurtherreactionoveraperiodofhourstoformthevita-+cGMPNachannelclosed30nsecminitself,cholecalciferol,whichisabsorbedintothe+NachannelopenLumirhodopsinbloodstream.Intemperateclimates,theplasmaconcen-75μsecInactiveActivetrationofvitaminDishighestattheendofsummerphosphodiesteraseandlowestattheendofwinter.Beyondabout40de-MetarhodopsinIGDPTransducin-GTPgreesnorthorsouthinwinter,thereisverylittleultra-10msecConformationalchangesinproteinMetarhodopsinIIvioletradiationofappropriatewavelength.minutesGTPTransducin-GDPPiMetarhodopsinIIIVitaminDIsMetabolizedtotheActiveMetabolite,Calcitriol,inLiver&KidneyHCCHCH3CH3H33C=OIntheliver,cholecalciferol,whichhasbeensynthesizedintheskinorderivedfromfood,ishydroxylatedtoCHAll-trans-retinaldehyde+opsin3formthe25-hydroxyderivativecalcidiol(Figure45–4).ThisisreleasedintothecirculationboundtoavitaminFigure45–2.Theroleofretinaldehydeinvision.D-bindingglobulinwhichisthemainstorageformofthevitamin.Inthekidney,calcidiolundergoeseither1-hydroxylationtoyieldtheactivemetabolite1,25-dihy-tamin,andthereforethereisfurtherimpairmentofim-droxyvitaminD(calcitriol)or24-hydroxylationtoyieldmuneresponses.aninactivemetabolite,24,25-dihydroxyvitaminD(24-hydroxycalcidiol).ErgocalciferolfromfortifiedfoodsVitaminAIsToxicinExcessundergoessimilarhydroxylationstoyieldercalcitriol.ThereisonlyalimitedcapacitytometabolizevitaminVitaminDMetabolismBothRegulatesA,andexcessiveintakesleadtoaccumulationbeyond&IsRegulatedbyCalciumHomeostasisthecapacityofbindingproteins,sothatunboundvita-minAcausestissuedamage.Symptomsoftoxicityaf-ThemainfunctionofvitaminDisinthecontrolofcal-fectthecentralnervoussystem(headache,nausea,ciumhomeostasis,andinturnvitaminDmetabolismis
483VITAMINS&MINERALS/485OHThermalisomerizationLIGHTCholecalciferol(calciol;vitaminD3)HOCH3CH27-DehydrocholesterolPrevitaminDHOFigure45–3.SynthesisofvitaminDintheskin.regulatedbyfactorsthatrespondtoplasmaconcentra-VitaminDDeficiencyAffectstionsofcalciumandphosphate.CalcitriolactstoreduceChildren&Adultsitsownsynthesisbyinducingthe24-hydroxylaseandrepressingthe1-hydroxylaseinthekidney.ItsprincipalInthevitaminDdeficiencydiseaserickets,thebonesfunctionistomaintaintheplasmacalciumconcentra-ofchildrenareundermineralizedasaresultofpoorab-tion.Calcitriolachievesthisinthreeways:itincreasessorptionofcalcium.Similarproblemsoccurinadoles-intestinalabsorptionofcalcium,reducesexcretionofcentswhoaredeficientduringtheirgrowthspurt.Os-calcium(bystimulatingresorptioninthedistalrenalteomalaciainadultsresultsfromdemineralizationoftubules),andmobilizesbonemineral.Inaddition,cal-boneinwomenwhohavelittleexposuretosunlight,citriolisinvolvedininsulinsecretion,synthesisandse-oftenafterseveralpregnancies.AlthoughvitaminDiscretionofparathyroidandthyroidhormones,inhibitionessentialforpreventionandtreatmentofosteomalaciaofproductionofinterleukinbyactivatedTlymphocytesintheelderly,thereislittleevidencethatitisbeneficialandofimmunoglobulinbyactivatedBlymphocytes,intreatingosteoporosis.differentiationofmonocyteprecursorcells,andmod-VitaminDIsToxicinExcessulationofcellproliferation.Initsactions,itbehaveslikeasteroidhormone,bindingtoanuclearreceptorSomeinfantsaresensitivetointakesofvitaminDasprotein.lowas50μg/d,resultinginanelevatedplasmaconcen-OHOHCalciol-25-hydroxylaseCalcidiol-1-hydroxylaseCH2CH2CH2CalcitriolCalcidiol(1,25-hydroxycholecalciferol)HOCholecalciferolHO(25-hydroxycholecalciferol)HOOH(calciol;vitaminD3)Calcidiol-24-hydroxylaseCalcidiol-24-hydroxylaseOHOHOHOHCalcidiol-1-hydroxylaseCHCH2224-hydroxycalcidiolCalcitetrolHOHOOHFigure45–4.MetabolismofvitaminD.
484486/CHAPTER45trationofcalcium.Thiscanleadtocontractionofreducedbacktotocopherolbyreactionwithvitaminbloodvessels,highbloodpressure,andcalcinosis—theCfromplasma(Figure45–6).Theresultantmonode-calcificationofsofttissues.Althoughexcessdietaryvita-hydroascorbatefreeradicalthenundergoesenzymicorminDistoxic,excessiveexposuretosunlightdoesnotnonenzymicreactiontoyieldascorbateanddehy-leadtovitaminDpoisoningbecausethereisalimiteddroascorbate,neitherofwhichisafreeradical.Thecapacitytoformtheprecursor7-dehydrocholesterolstabilityofthetocopheroxylfreeradicalmeansthatitandtotakeupcholecalciferolfromtheskin.canpenetratefartherintocellsand,potentially,propa-gateachainreaction.Therefore,vitaminEmay,likeVITAMINEDOESNOTHAVEAPRECISELYotherantioxidants,alsohavepro-oxidantactions,es-DEFINEDMETABOLICFUNCTIONpeciallyathighconcentrations.Thismayexplainwhy,althoughstudieshaveshownanassociationbetweenNounequivocaluniquefunctionforvitaminEhashighbloodconcentrationsofvitaminEandalowerbeendefined.However,itdoesactasalipid-solublean-incidenceofatherosclerosis,theeffectofhighdosesoftioxidantincellmembranes,wheremanyofitsfunc-vitaminEhavebeendisappointing.tionscanbeprovidedbysyntheticantioxidants.Vita-minEisthegenericdescriptorfortwofamiliesofDietaryVitaminEDeficiencycompounds,thetocopherolsandthetocotrienols(Fig-ure45–5).Thedifferentvitamers(compoundshavinginHumansIsUnknownsimilarvitaminactivity)havedifferentbiologicpoten-Inexperimentalanimals,vitaminEdeficiencyresultsincies;themostactiveisD-α-tocopherol,anditisusualresorptionoffetusesandtesticularatrophy.Dietaryde-toexpressvitaminEintakeinmilligramsofD-α-tocoph-ficiencyofvitaminEinhumansisunknown,thougherolequivalents.SyntheticDL-α-tocopheroldoesnotpatientswithseverefatmalabsorption,cysticfibrosis,havethesamebiologicpotencyasthenaturallyoccur-andsomeformsofchronicliverdiseasesufferdefi-ringcompound.ciencybecausetheyareunabletoabsorbthevitaminortransportit,exhibitingnerveandmusclemembraneVitaminEIstheMajorLipid-Solubledamage.Prematureinfantsarebornwithinadequatere-AntioxidantinCellMembranesservesofthevitamin.Theirerythrocytemembranesare&PlasmaLipoproteinsabnormallyfragileasaresultofperoxidation,whichleadstohemolyticanemia.ThemainfunctionofvitaminEisasachain-break-ing,freeradicaltrappingantioxidantincellmem-branesandplasmalipoproteins.ItreactswiththelipidVITAMINKISREQUIREDFORSYNTHESISperoxideradicalsformedbyperoxidationofpolyun-OFBLOOD-CLOTTINGPROTEINSsaturatedfattyacidsbeforetheycanestablishachainreaction.Thetocopheroxylfreeradicalproductisrela-VitaminKwasdiscoveredasaresultofinvestigationstivelyunreactiveandultimatelyformsnonradicalintothecauseofableedingdisorder—hemorrhagiccompounds.Commonly,thetocopheroxylradicalis(sweetclover)disease—ofcattle,andofchickensfedonafat-freediet.ThemissingfactorinthedietofthechickenswasvitaminK,whilethecattlefeedcontaineddicumarol,anantagonistofthevitamin.AntagonistsofvitaminKareusedtoreducebloodcoagulationinR1patientsatriskofthrombosis—themostwidelyusedHOagentiswarfarin.ROThreecompoundshavethebiologicactivityofvita-2CH3TocopherolR3minK(Figure45–7):phylloquinone,thenormaldi-etarysource,foundingreenvegetables;menaqui-R1nones,synthesizedbyintestinalbacteria,withdifferingHOlengthsofside-chain;menadione,menadiol,andROmenadioldiacetate,syntheticcompoundsthatcanbe2CH3Tocotrienolmetabolizedtophylloquinone.Menaquinonesareab-R3sorbedtosomeextentbutitisnotcleartowhatextentFigure45–5.ThevitaminEvitamers.Inα-tocoph-theyarebiologicallyactiveasitispossibletoinduceerolandtocotrienolR1,R2,andR3areall⎯CH3groups.signsofvitaminKdeficiencysimplybyfeedingaphyl-Intheβ-vitamersR2isH;intheγ-vitamersR1isH,andinloquinonedeficientdiet,withoutinhibitingintestinaltheδ-vitamersR1andR2arebothH.bacterialaction.
485VITAMINS&MINERALS/487FreeradicalchainreactionPUFAOOPUFAOOHRTocOHTocORPHOSPHOLIPASEO2A2PUFAH(inphospholipid)MEMBRANESCYTOSOLVitaminCox,VitaminCred,PUFAOOH,GSSGGSHH2O2GSHGLUTATHIONESUPEROXIDECATALASESePEROXIDASEDISMUTASE–O2H2O,GSSGSuperoxidePUFAOHFigure45–6.Interactionandsynergismbetweenantioxidantsystemsoperatinginthelipidphase(membranes)ofthecellandtheaqueousphase(cytosol).(R•,freeradical;PUFA-OO•,peroxylfreeradicalofpolyunsaturatedfattyacidinmembranephospholipid;PUFA-OOH,hydroperoxypolyunsaturatedfattyacidinmembranephospholipidreleasedashydroperoxyfreefattyacidintocytosolbytheactionofphospholipaseA2;PUFA-OH,hydroxypolyunsaturatedfattyacid;TocOH,vitaminE(α-tocopherol);TocO•,freeradicalofα-tocopherol;Se,selenium;GSH,reducedglu-tathione;GS-SG,oxidizedglutathione,whichisreturnedtothereducedstateafterreactionwithNADPHcatalyzedbyglutathionereductase;PUFA-H,polyunsaturatedfattyacid.)VitaminKIstheCoenzymequinonereductase.Inthepresenceofwarfarin,vitaminforCarboxylationofGlutamateKepoxidecannotbereducedbutaccumulates,andisinthePostsyntheticModificationexcreted.IfenoughvitaminK(aquinone)isprovidedofCalcium-BindingProteinsinthediet,itcanbereducedtotheactivehydro-quinonebythewarfarin-insensitiveenzyme,andcar-VitaminKisthecofactorforthecarboxylationofgluta-boxylationcancontinue,withstoichiometricutilizationmateresiduesinthepost-syntheticmodificationofpro-ofvitaminKandexcretionoftheepoxide.Ahighdoseteinstoformtheunusualaminoacidγ-carboxygluta-ofvitaminKistheantidotetoanoverdoseofwarfarin.mate(Gla),whichchelatesthecalciumion.Initially,ProthrombinandseveralotherproteinsofthebloodvitaminKhydroquinoneisoxidizedtotheepoxideclottingsystem(FactorsVII,IXandX,andproteinsC(Figure45–8),whichactivatesaglutamateresidueinandS)eachcontainbetweenfourandsixγ-carboxygluta-theproteinsubstratetoacarbanion,thatreactsnon-materesidueswhichchelatecalciumionsandsopermitenzymicallywithcarbondioxidetoformγ-carboxyglut-thebindingofthebloodclottingproteinstomembranes.amate.VitaminKepoxideisreducedtothequinonebyInvitaminKdeficiencyorinthepresenceofwarfarin,anawarfarin-sensitivereductase,andthequinoneisre-abnormalprecursorofprothrombin(preprothrombin)ducedtotheactivehydroquinonebyeitherthesamecontaininglittleornoγ-carboxyglutamate,andincapablewarfarin-sensitivereductaseorawarfarin-insensitiveofchelatingcalcium,isreleasedintothecirculation.
486488/CHAPTER45OVitaminKIsAlsoImportantCH3intheSynthesisofBoneCalcium-BindingProteinsHO3TreatmentofpregnantwomenwithwarfarincanleadtoPhylloquinonefetalboneabnormalities(fetalwarfarinsyndrome).Twoproteinsarepresentinbonethatcontainγ-carboxygluta-Omate,osteocalcinandbonematrixGlaprotein.Osteocal-CH3cinalsocontainshydroxyproline,soitssynthesisisdepen-dentonbothvitaminsKandC;inaddition,itssynthesisHOnisinducedbyvitaminD.ThereleaseintothecirculationMenaquinoneCH3ofosteocalcinprovidesanindexofvitaminDstatus.COOHOCH3CH3WATER-SOLUBLEVITAMINSOHOMenadiolCOVITAMINB1(THIAMIN)HASAKEYROLECHINCARBOHYDRATEMETABOLISM3MenadiolodiacetateThiaminhasacentralroleinenergy-yieldingmetabo-(acetomenaphthone)lism,andespeciallythemetabolismofcarbohydrateFigure45–7.ThevitaminKvitamers.Menadiol(or(Figure45–9).Thiamindiphosphateisthecoenzymemenadione)andmenadioldiacetatearesyntheticcom-forthreemulti-enzymecomplexesthatcatalyzeoxida-poundsthatareconvertedtomenaquinoneinthelivertivedecarboxylationreactions:pyruvatedehydrogenaseandhavevitaminKactivity.incarbohydratemetabolism;α-ketoglutaratedehydro-OOCCHCOOCH2HNCHCOCarboxyglutamateresiduenon-CO2enzymicCH2COOO2CHCOOCHCH22HNCHCOHNCHCOVITAMINKGlutamateresidueEPOXIDASEGlutamatecarbanionOHOCHCH33ORROHOVitaminKhydroquinoneVitaminKepoxideDisulfideSulfhydryl+NADPVITAMINKQUINONEVITAMINKEPOXIDEQUINONEREDUCTASEOREDUCTASEREDUCTASESulfhydrylNADPHCH3DisulfideROFigure45–8.TheroleofvitaminKintheVitaminKquinonebiosynthesisofγ-carboxyglutamate.
487VITAMINS&MINERALS/489OOH3CH3CNNH2H3CCH2CH2CH2OHCH2CH2OPOPONSNCH2NOO+SHThiaminThiamindiphosphateCarbanionFigure45–9.Thiamin,thiamindiphosphate,andthecarbanionform.genaseinthecitricacidcycle;andthebranched-chainpyruvatedehydrogenasemeansthatindeficiencythereisketo-aciddehydrogenaseinvolvedinthemetabolismofimpairedconversionofpyruvatetoacetylCoA.Insub-leucine,isoleucine,andvaline.Itisalsothecoenzymejectsonarelativelyhighcarbohydratediet,thisresultsinfortransketolase,inthepentosephosphatepathway.Inincreasedplasmaconcentrationsoflactateandpyruvate,eachcase,thethiamindiphosphateprovidesareactivewhichmaycauselife-threateninglacticacidosis.carbononthethiazolemoietythatformsacarbanion,whichthenaddstothecarbonylgroupof,forinstance,ThiaminNutritionalStatusCanpyruvate.Theadditioncompoundthendecarboxylates,eliminatingCO.ElectricalstimulationofnerveleadsBeAssessedbyErythrocyte2toafallinmembranethiamintriphosphateandreleaseTransketolaseActivationoffreethiamin.ItislikelythatthiamintriphosphateTheactivationofapo-transketolase(theenzymepro-actsasaphosphatedonorforphosphorylationofthetein)inerythrocytelysatebythiamindiphosphatenervemembranesodiumtransportchannel.addedinvitrohasbecometheacceptedindexofthi-aminnutritionalstatus.ThiaminDeficiencyAffectstheNervousSystem&HeartVITAMINB2(RIBOFLAVIN)HASThiamindeficiencycanresultinthreedistinctsyn-ACENTRALROLEINENERGY-dromes:achronicperipheralneuritis,beriberi,whichYIELDINGMETABOLISMmayormaynotbeassociatedwithheartfailureandedema;acutepernicious(fulminating)beriberi(shoshinRiboflavinfulfillsitsroleinmetabolismasthecoenzymesberiberi),inwhichheartfailureandmetabolicabnor-flavinmononucleotide(FMN)andflavinadeninemalitiespredominate,withoutperipheralneuritis;anddinucleotide(FAD)(Figure45–10).FMNisformedbyWernicke’sencephalopathywithKorsakoff’spsy-ATP-dependentphosphorylationofriboflavin,whereaschosis,whichisassociatedespeciallywithalcoholandFADissynthesizedbyfurtherreactionofFMNwithdrugabuse.ThecentralroleofthiamindiphosphateinATPinwhichitsAMPmoietyistransferredtotheOHOHOHOHOHOHOCH2CHCHCHCH2OHCH2CHCHCHCH2OPOH3CNNOH3CNNOONNHCNHCN33OORiboflavinFMNNH2OHOHOHOONNCH2CHCHCHCH2OPOPOCH2ONNH3CNNOOONHCN3OHOHOFADFigure45–10.Riboflavinandthecoenzymesflavinmononucleotide(FMN)andflavinadeninedinucleotide(FAD).
488490/CHAPTER45FMN.ThemaindietarysourcesofriboflavinaremilkCOOCONH2anddairyproducts.Inaddition,becauseofitsintenseyellowcolor,riboflaviniswidelyusedasafoodadditive.NNNicotinicacidNicotinamideCONHFlavinCoenzymesAreElectronCarriers2NHinOxidoreductionReactions+2NOHOHOONNTheseincludethemitochondrialrespiratorychain,keyCH2OPOPOCH2NNenzymesinfattyacidandaminoacidoxidation,andOOOthecitricacidcycle.ReoxidationofthereducedflavinOinoxygenasesandmixed-functionoxidasesproceedsbywayofformationoftheflavinradicalandflavinhy-OHOHdroperoxide,withtheintermediategenerationofsuper-NADoxideandperhydroxylradicalsandhydrogenperoxide.Figure45–11.Niacin(nicotinicacidandnicotin-Becauseofthis,flavinoxidasesmakeasignificantcon-amide)andnicotinamideadeninedinucleotide(NAD).tributiontothetotaloxidantstressofthebody.*ShowsthesiteofphosphorylationinNADP.RiboflavinDeficiencyIsWidespreadButNotFatalAlthoughriboflavinisfundamentallyinvolvedinme-diarrhea,and,ifuntreated,death.Althoughthenutri-tabolism,anddeficienciesarefoundinmostcountries,tionaletiologyofpellagraiswellestablishedandtrypto-itisnotfatalasthereisveryefficientconservationofphanorniacinwillpreventorcurethedisease,addi-tissueriboflavin.Riboflavindeficiencyischaracterizedtionalfactors,includingdeficiencyofriboflavinorbycheilosis,lingualdesquamationandaseborrheicder-vitaminB6,bothofwhicharerequiredforsynthesisofmatitis.Riboflavinnutritionalstatusisassessedbymea-nicotinamidefromtryptophan,maybeimportant.InsurementoftheactivationoferythrocyteglutathionemostoutbreaksofpellagratwiceasmanywomenasreductasebyFADaddedinvitro.menareaffected,probablytheresultofinhibitionoftryptophanmetabolismbyestrogenmetabolites.NIACINISNOTSTRICTLYAVITAMINPellagraCanOccurasaResultNiacinwasdiscoveredasanutrientduringstudiesofpel-ofDiseaseDespiteanAdequatelagra.Itisnotstrictlyavitaminsinceitcanbesyn-IntakeofTryptophan&Niacinthesizedinthebodyfromtheessentialaminoacidtryptophan.Twocompounds,nicotinicacidandnico-Anumberofgeneticdiseasesthatresultindefectsoftinamide,havethebiologicactivityofniacin;itsmeta-tryptophanmetabolismareassociatedwiththedevelop-bolicfunctionisasthenicotinamideringofthecoen-mentofpellagradespiteanapparentlyadequateintakezymesNADandNADPinoxidation-reductionreactionsofbothtryptophanandniacin.Hartnupdiseaseisa(Figure45–11).About60mgoftryptophanisequivalentraregeneticconditioninwhichthereisadefectoftheto1mgofdietaryniacin.Theniacincontentoffoodsismembranetransportmechanismfortryptophan,result-expressedasmgniacinequivalents=mgpreformedniacininginlargelossesduetointestinalmalabsorptionand+1/60×mgtryptophan.Becausemostoftheniacininfailureoftherenalresorptionmechanism.Incarcinoidcerealsisbiologicallyunavailable,thisisdiscounted.syndromethereismetastasisofaprimarylivertumorofenterochromaffincellswhichsynthesize5-hydroxy-NADIstheSourceofADP-Ribosetryptamine.Overproductionof5-hydroxytryptaminemayaccountforasmuchas60%ofthebody’strypto-Inadditiontoitscoenzymerole,NADisthesourceofphanmetabolism,causingpellagrabecauseofthediver-ADP-ribosefortheADP-ribosylationofproteinsandsionawayfromNADsynthesis.polyADP-ribosylationofnucleoproteinsinvolvedintheDNArepairmechanism.NiacinIsToxicinExcessPellagraIsCausedbyDeficiencyNicotinicacidhasbeenusedtotreathyperlipidemiawhenoftheorderof1–6g/darerequired,causingdila-ofTryptophan&Niacintionofbloodvesselsandflushing,withskinirritation.Pellagraischaracterizedbyaphotosensitivedermatitis.Intakesofbothnicotinicacidandnicotinamideinex-Astheconditionprogresses,thereisdementia,possiblycessof500mg/dcancauseliverdamage.
489VITAMINS&MINERALS/491VITAMINB6ISIMPORTANTINAMINOVitaminB6DeficiencyIsRareACID&GLYCOGENMETABOLISMAlthoughclinicaldeficiencydiseaseisrare,thereisevi-&INSTEROIDHORMONEACTIONdencethatasignificantproportionofthepopulationSixcompoundshavevitaminBactivity(Figure45–12):havemarginalvitaminB6status.Moderatedeficiency6resultsinabnormalitiesoftryptophanandmethioninepyridoxine,pyridoxal,pyridoxamine,andtheir5′-metabolism.Increasedsensitivitytosteroidhormoneac-phosphates.Theactivecoenzymeispyridoxal5′-phos-tionmaybeimportantinthedevelopmentofhormone-phate.Approximately80%ofthebody’stotalvitaminB6dependentcancerofthebreast,uterus,andprostate,ispresentaspyridoxalphosphateinmuscle,mostlyasso-ciatedwithglycogenphosphorylase.ThisisnotavailableandvitaminB6statusmayaffecttheprognosis.inB6deficiencybutisreleasedinstarvation,whenglyco-genreservesbecomedepleted,andisthenavailable,espe-VitaminB6StatusIsAssessedbyAssayingciallyinliverandkidney,tomeetincreasedrequirementErythrocyteAminotransferasesforgluconeogenesisfromaminoacids.ThemostwidelyusedmethodofassessingvitaminB6statusisbytheactivationoferythrocyteaminotrans-VitaminB6HasSeveralRolesferasesbypyridoxalphosphateaddedinvitro,expressedinMetabolismastheactivationcoefficient.Pyridoxalphosphateisacoenzymeformanyenzymesinvolvedinaminoacidmetabolism,especiallyinInExcess,VitaminB6Causestransaminationanddecarboxylation.Itisalsotheco-SensoryNeuropathyfactorofglycogenphosphorylase,wherethephosphategroupiscatalyticallyimportant.Inaddition,vitaminBThedevelopmentofsensoryneuropathyhasbeenre-6isimportantinsteroidhormoneactionwhereitre-portedinpatientstaking2–7gofpyridoxineperdayformovesthehormone-receptorcomplexfromDNAavarietyofreasons(thereissomeslightevidencethatitbinding,terminatingtheactionofthehormones.Invi-iseffectiveintreatingpremenstrualsyndrome).TheretaminB6deficiency,thisresultsinincreasedsensitivitywassomeresidualdamageafterwithdrawalofthesehightotheactionsoflowconcentrationsofestrogens,an-doses;otherreportssuggestthatintakesinexcessof200drogens,cortisol,andvitaminD.mg/dareassociatedwithneurologicdamage.VITAMINB12ISFOUNDONLYINFOODSOFANIMALORIGINOCHOHCHOH22Theterm“vitaminB”isusedasagenericdescriptorHOCHKINASEOPOCH122OH2OHforthecobalamins—thosecorrinoids(cobaltcon-PHOSPHATASEOtainingcompoundspossessingthecorrinring)havingNCHNCH33PyridoxinePyridoxinephosphatethebiologicactivityofthevitamin(Figure45–13).Somecorrinoidsthataregrowthfactorsformicroor-OXIDASEganismsnotonlyhavenovitaminB12activitybutmayalsobeantimetabolitesofthevitamin.AlthoughitisOHC=OHC=Osynthesizedexclusivelybymicroorganisms,forpracti-KINASEHOCH2OHOPOCH2OHcalpurposesvitaminB12isfoundonlyinfoodsofani-PHOSPHATASEOmalorigin,therebeingnoplantsourcesofthisvita-NCHNCH33min.Thismeansthatstrictvegetarians(vegans)areatPyridoxalPyridoxalphosphateriskofdevelopingB12deficiency.ThesmallamountsofthevitaminformedbybacteriaonthesurfaceofAMINOTRANSFERASESOXIDASEfruitsmaybeadequatetomeetrequirements,butOpreparationsofvitaminB12madebybacterialfermen-CHNHCHNH2222tationareavailable.KINASEHOCH2OHOPOCH2OHPHOSPHATASEONCH3NCH3VitaminB12AbsorptionRequiresTwoPyridoxaminePyridoxaminephosphateBindingProteinsFigure45–12.InterconversionofthevitaminB6VitaminB12isabsorbedboundtointrinsicfactor,avitamers.smallglycoproteinsecretedbytheparietalcellsofthe
490492/CHAPTER45CH2CONH2mentationinruminants.ItundergoesvitaminB12-H3CCHCHCONHdependentrearrangementtosuccinyl-CoA,catalyzed222HCbymethylmalonyl-CoAisomerase(Figure19–2).The3H2NCOCH2CH2NactivityofthisenzymeisgreatlyreducedinvitaminBRCH312HNCOCH22NCHdeficiency,leadingtoanaccumulationofmethyl-HCCoN33malonyl-CoAandurinaryexcretionofmethylmalonicH3CNCHCHCONHacid,whichprovidesameansofassessingvitaminB22212CH3nutritionalstatus.HNCOCH22CH3CH2VitaminBDeficiencyCauses12CH2PerniciousAnemiaCOPerniciousanemiaariseswhenvitaminBdeficiency12NHblocksthemetabolismoffolicacid,leadingtofunc-CHOtionalfolatedeficiency.Thisimpairserythropoiesis,2causingimmatureprecursorsoferythrocytestobere-H3CCOPONCH3Hleasedintothecirculation(megaloblasticanemia).TheOHONCHcommonestcauseofperniciousanemiaisfailureofthe3absorptionofvitaminB12ratherthandietarydefi-ciency.Thiscanbeduetofailureofintrinsicfactorse-HOCHOcretioncausedbyautoimmunediseaseofparietalcells2ortogenerationofanti-intrinsicfactorantibodies.Figure45–13.VitaminB12(cobalamin).Rmaybevariedtogivethevariousformsofthevitamin,eg,THEREAREMULTIPLEFORMS––R=CNincyanocobalamin;R=OHinhydroxocobal-OFFOLATEINTHEDIETamin;R=5′-deoxyadenosylin5′-deoxyadenosylcobal-amin;R=H2Oinaquocobalamin;andR=CH3inTheactiveformoffolicacid(pteroylglutamate)ismethylcobalamin.tetrahydrofolate(Figure45–15).Thefolatesinfoodsmayhaveuptosevenadditionalglutamateresidueslinkedbyγ-peptidebonds.Inaddition,alloftheone-carbonsubstitutedfolatesinFigure45–15mayalsobegastricmucosa.Gastricacidandpepsinreleasethevita-presentinfoods.minfromproteinbindinginfoodandmakeitavailableTheextenttowhichthedifferentformsoffolatecantobindtocobalophilin,abindingproteinsecretedinbeabsorbedvaries,andthismustbeallowedforincal-thesaliva.Intheduodenum,cobalophilinishy-culatingfolateintakes.drolyzed,releasingthevitaminforbindingtointrinsicfactor.Pancreaticinsufficiencycanthereforebeafac-torinthedevelopmentofvitaminB12deficiency,re-sultingintheexcretionofcobalophilin-boundvitaminB12.IntrinsicfactorbindsthevariousvitaminB12vita-SHH3CSmers,butnotothercorrinoids.VitaminB12isabsorbed(CH)(CH)fromthedistalthirdoftheileumviareceptorsthat2222bindtheintrinsicfactor-vitaminB12complexbutnotHCNH+HCNH+33freeintrinsicfactororfreevitamin.COO–COO–ThereAreThreeVitaminHomocysteineMethionineMETHIONINEB12-DependentEnzymesSYNTHASEMethylmalonylCoAmutase,leucineaminomutase,andmethioninesynthase(Figure45–14)arevitaminMethylcobalaminB12-dependentenzymes.MethylmalonylCoAisformedMethylH4folateB12H4folateasanintermediateinthecatabolismofvalineandbythecarboxylationofpropionylCoAarisingintheca-Figure45–14.Homocysteinuriaandthefolatetrap.tabolismofisoleucine,cholesterol,and,rarely,fattyVitaminB12deficiencyleadstoinhibitionofmethionineacidswithanoddnumberofcarbonatoms—ordirectlysynthaseactivitycausinghomocysteinuriaandthefrompropionate,amajorproductofmicrobialfer-trappingoffolateasmethyltetrahydrofolate.
491VITAMINS&MINERALS/493OCOOOHHHNCH2NCNCHN510HHNNNCH22HTetrahydrofolate(THF)CH2CO(Glu)nHCOHCOOHHOHHHNCH2NNCH2NNN5-FormylTHF10-FormylTHFHNNNHNNN22HHHCNHOHHOHCH2HNCH2NNCH2NNN5-FormiminoTHF5,10-MethyleneTHFHNNNHNNN22HHCH3OHHOHCHHNCH2NNCH2NNN+Figure45–15.Tetrahydrofolicacidandthe5-MethylTHF5,10-MethenylTHFHNNNHNNN22one-carbonsubstitutedfolates.HHTetrahydrofolateIsaCarrierceuticallyintheagentknownasfolinicacidandintheofOne-CarbonUnitssynthetic(racemic)compoundleucovorin.Themajorpointofentryforone-carbonfragmentsTetrahydrofolatecancarryone-carbonfragmentsat-intosubstitutedfolatesismethylenetetrahydrofolatetachedtoN-5(formyl,formimino,ormethylgroups),(Figure45–16),whichisformedbythereactionofN-10(formylgroup),orbridgingN-5toN-10(meth-glycine,serine,andcholinewithtetrahydrofolate.Serineyleneormethenylgroups).5-Formyl-tetrahydrofolateisisthemostimportantsourceofsubstitutedfolatesmorestablethanfolateandisthereforeusedpharma-forbiosyntheticreactions,andtheactivityofserinehy-Sourcesofone-carbonunitsSynthesisusingone-carbonunitsSerineSerineGlycineMethylene-THFMethyl-THFMethionineCholineTMP+dihydrofolateHistidineFormimino-THFMethenyl-THFDNAFormyl-methionineFormateFormyl-THFPurinesCO2Figure45–16.Sourcesandutilizationofone-carbonsubstitutedfolates.
492494/CHAPTER45droxymethyltransferaseisregulatedbythestateoffolateFolicAcidSupplementsReducesubstitutionandtheavailabilityoffolate.ThereactionistheRiskofNeuralTubeDefectsreversible,andinliveritcanformserinefromglycineas&Hyperhomocysteinemiaasubstrateforgluconeogenesis.Methylene,methenyl,and10-formyltetrahydrofolatesareinterconvertible.Supplementsof400μg/doffolatebegunbeforecon-Whenone-carbonfolatesarenotrequired,theoxidationceptionresultinasignificantreductionintheincidenceofformyltetrahydrofolatetoyieldcarbondioxidepro-ofneuraltubedefectsasfoundinspinabifida.Ele-videsameansofmaintainingapooloffreefolate.vatedbloodhomocysteineisanassociatedriskfactorforatherosclerosis,thrombosis,andhypertension.InhibitorsofFolateMetabolismTheconditionisduetoimpairedabilitytoformProvideCancerChemotherapy&methyl-tetrahydrofolatebymethylene-tetrahydrofolatereductase,causingfunctionalfolatedeficiencyandre-Antibacterial&AntimalarialDrugssultinginfailuretoremethylatehomocysteinetome-Themethylationofdeoxyuridinemonophosphatethionine.Peoplewiththecausativeabnormalvariantof(dUMP)tothymidinemonophosphate(TMP),cat-methylene-tetrahydrofolatereductasedonotdevelopalyzedbythymidylatesynthase,isessentialforthesyn-hyperhomocysteinemiaiftheyhavearelativelyhighin-thesisofDNA.Theone-carbonfragmentofmethy-takeoffolate,butitisnotyetknownwhetherthisaf-lene-tetrahydrofolateisreducedtoamethylgroupwithfectstheincidenceofcardiovasculardisease.releaseofdihydrofolate,whichisthenreducedbacktotetrahydrofolatebydihydrofolatereductase.Thymi-FolateEnrichmentofFoodsdylatesynthaseanddihydrofolatereductaseareespe-MayPutSomePeopleatRiskciallyactiveintissueswithahighrateofcelldivision.Methotrexate,ananalogof10-methyl-tetrahydrofo-Folatesupplementswillrectifythemegaloblasticanemialate,inhibitsdihydrofolatereductaseandhasbeenex-ofvitaminB12deficiencybutmayhastenthedevelop-ploitedasananticancerdrug.Thedihydrofolatereduc-mentofthe(irreversible)nervedamagefoundinB12de-tasesofsomebacteriaandparasitesdifferfromtheficiency.Thereisalsoantagonismbetweenfolicacidandhumanenzyme;inhibitorsoftheseenzymescanbeusedtheanticonvulsantsusedinthetreatmentofepilepsy.asantibacterialdrugs,eg,trimethoprim,andanti-malarialdrugs,eg,pyrimethamine.DIETARYBIOTINDEFICIENCYISUNKNOWNVitaminB12DeficiencyCausesFunctionalThestructuresofbiotin,biocytin,andcarboxy-biotinFolateDeficiency—theFolateTrap(theactivemetabolicintermediate)areshowninFigureWhenactingasamethyldonor,S-adenosylmethionine45–17.Biotiniswidelydistributedinmanyfoodsasformshomocysteine,whichmayberemethylatedbybiocytin(ε-amino-biotinyllysine),whichisreleasedonmethyltetrahydrofolatecatalyzedbymethioninesyn-proteolysis.Itissynthesizedbyintestinalflorainexcessthase,avitaminB12-dependentenzyme(Figure45–14).ofrequirements.DeficiencyisunknownexceptamongThereductionofmethylene-tetrahydrofolatetomethyl-peoplemaintainedformanymonthsonparenteralnu-tetrahydrofolateisirreversible,andsincethemajorsourcetritionandaverysmallnumberwhoeatabnormallyoftetrahydrofolatefortissuesismethyl-tetrahydrofolate,largeamountsofuncookedeggwhite,whichcontainstheroleofmethioninesynthaseisvitalandprovidesalinkavidin,aproteinthatbindsbiotinandrendersitun-betweenthefunctionsoffolateandvitaminB12.Impair-availableforabsorption.mentofmethioninesynthaseinB12deficiencyresultsintheaccumulationofmethyl-tetrahydrofolate—the“fo-BiotinIsaCoenzymelatetrap.”Thereisthereforefunctionaldeficiencyoffo-ofCarboxylaseEnzymeslatesecondarytothedeficiencyofvitaminB12.BiotinfunctionstotransfercarbondioxideinasmallFolateDeficiencyCausesnumberofcarboxylationreactions.AholocarboxylasesynthetaseactsonalysineresidueoftheapoenzymesofMegaloblasticAnemiaacetyl-CoAcarboxylase,pyruvatecarboxylase,propi-Deficiencyoffolicaciditself—ordeficiencyofvitaminonyl-CoAcarboxylase,ormethylcrotonyl-CoAcarboxy-B12,whichleadstofunctionalfolicaciddeficiency—af-lasetoreactwithfreebiotintoformthebiocytinresiduefectscellsthataredividingrapidlybecausetheyhaveaoftheholoenzyme.Thereactiveintermediateis1-N-largerequirementforthymidineforDNAsynthesis.carboxybiocytin,formedfrombicarbonateinanATP-Clinically,thisaffectsthebonemarrow,leadingtodependentreaction.Thecarboxylgroupisthentrans-megaloblasticanemia.ferredtothesubstrateforcarboxylation(Figure21–1).
493VITAMINS&MINERALS/495Othe⎯SHprostheticgroupofCoAandACP.CoAHNNHtakespartinreactionsofthecitricacidcycle,fattyacidBiotinsynthesisandoxidation,acetylations,andcholesterolsynthesis.ACPparticipatesinfattyacidsynthesis.TheCOOSvitaminiswidelydistributedinallfoodstuffs,anddefi-ciencyhasnotbeenunequivocallyreportedinhumanObeingsexceptinspecificdepletionstudies.HNNHBiotinyl-lysine(biocytin)COHASCORBICACIDISAVITAMINCNSCHFORONLYSOMESPECIESONHVitaminC(Figure45–19)isavitaminforhumanbeingsOandotherprimates,theguineapig,bats,passerinebirds,OOCNNHCarboxy-biocytinandmostfishesandinvertebrates;otheranimalssynthe-HCOsizeitasanintermediateintheuronicacidpathwayofCNSCHglucosemetabolism(Chapter20).InthosespeciesforOwhichitisavitamin,thereisablockinthatpathwaydueNHtoabsenceofgulonolactoneoxidase.Bothascorbicacidanddehydroascorbicacidhavevitaminactivity.Figure45–17.Biotin,biocytin,andcarboxy-biocytin.VitaminCIstheCoenzymeforTwoGroupsofHydroxylasesBiotinalsohasaroleinregulationofthecellcycle,Ascorbicacidhasspecificrolesinthecopper-containingactingtobiotinylatekeynuclearproteins.hydroxylasesandtheα-ketoglutarate-linkediron-con-taininghydroxylases.ItalsoincreasestheactivityofaASPARTOFCoAANDACP,numberofotherenzymesinvitro,thoughthisisanon-PANTOTHENICACIDACTSASspecificreducingaction.Inaddition,ithasanumberofnonenzymiceffectsduetoitsactionasareducingagentACARRIEROFACYLRADICALSandoxygenradicalquencher.Pantothenicacidhasacentralroleinacylgroupmetab-Dopamine-hydroxylaseisacopper-containingolismwhenactingasthepantetheinefunctionalmoietyenzymeinvolvedinthesynthesisofthecatecholaminesofcoenzymeAoracylcarrierprotein(ACP)(Figurenorepinephrineandepinephrinefromtyrosineinthe45–18).Thepantetheinemoietyisformedaftercombi-adrenalmedullaandcentralnervoussystem.Duringhy-droxylation,theCu+isoxidizedtoCu2+;reductionbacknationofpantothenatewithcysteine,whichprovidesOCOHOCNHCHCHSH22CHCH22CHCH22NHNHCOCOCHOHCHOHNH2HCCCHHCCCH3333OONNCH2OHCH2OPOPOCH2ONNHOOPantothenicacidCoenzymeA(CoASH)OOHFigure45–18.PantothenicacidandcoenzymeA.*ShowsthesiteofacylationOPObyfattyacids.O
494496/CHAPTER45CH2OHCH2OHCH2OHHOCH2HOCH2HOCH2OOOOOOOHOHO.OHOOAscorbateMonodehydroascorbateDehydroascorbate(semidehydroascorbate)Figure45–19.VitaminC.toCu+specificallyrequiresascorbate,whichisoxidizedvitaminCpreventthecommoncoldorreducethedu-tomonodehydroascorbate.Similaractionsofascorbaterationofitssymptoms.occurintyrosinedegradationatthep-hydroxy-phenylpyruvatehydroxylasestepandatthehomogenti-2+satedioxygenasestep,whichneedsFe(Figure30–12).Anumberofpeptidehormoneshaveacarboxylter-MINERALSAREREQUIREDminalamidewhichisderivedfromaglycineterminalFORBOTHPHYSIOLOGIC&residue.Thisglycineishydroxylatedontheα-carbonbyacopper-containingenzyme,peptidylglycinehy-BIOCHEMICALFUNCTIONSdroxylase,which,again,requiresascorbateforreduc-2+tionofCu.Manyoftheessentialminerals(Table45–2)arewidelyAnumberofiron-containing,ascorbate-requiringdistributedinfoods,andmostpeopleeatinganormalhydroxylasesshareacommonreactionmechanisminmixeddietarelikelytoreceiveadequateintakes.Thewhichhydroxylationofthesubstrateislinkedtodecar-boxylationofα-ketoglutarate(Figure28–11).Manyoftheseenzymesareinvolvedinthemodificationofpre-cursorproteins.ProlineandlysinehydroxylasesareTable45–2.Classificationofessentialmineralsrequiredforthepostsyntheticmodificationofprocol-accordingtotheirfunction.lagentocollagen,andprolinehydroxylaseisalsore-quiredinformationofosteocalcinandtheC1qcom-ponentofcomplement.Aspartateβ-hydroxylaseisFunctionMineralrequiredforthepostsyntheticmodificationofthepre-StructuralfunctionCalcium,magnesium,phosphatecursorofproteinC,thevitaminK-dependentproteaseInvolvedinmembraneSodium,potassiumwhichhydrolyzesactivatedfactorVinthebloodclot-function:principaltingcascade.Trimethyllysineandγ-butyrobetainehy-cationsofextracellular-droxylasesarerequiredforthesynthesisofcarnitine.andintracellularfluids,respectivelyVitaminCDeficiencyCausesScurvyFunctionasprostheticCobalt,copper,iron,molybde-SignsofvitaminCdeficiencyinscurvyincludeskingroupsinenzymesnum,selenium,zincchanges,fragilityofbloodcapillaries,gumdecay,toothRegulatoryroleorroleCalcium,chromium,iodine,loss,andbonefracture,manyofwhichcanbeattrib-inhormoneactionmagnesium,manganese,sodium,utedtodeficientcollagensynthesis.potassiumThereMayBeBenefitsFromHigherKnowntobeessential,Silicon,vanadium,nickel,tinbutfunctionunknownIntakesofVitaminCHaveeffectsintheFluoride,lithiumAtintakesaboveapproximately100mg/d,thebody’sbody,butessentialityiscapacitytometabolizevitaminCissaturated,andanynotestablishedfurtherintakeisexcretedintheurine.However,inad-ditiontoitsotherroles,vitaminCenhancestheabsorp-WithoutknownnutritionalAluminum,arsenic,antimony,functionbuttoxicinboron,bromine,cadmium,ce-tionofiron,andthisdependsonthepresenceofthevi-excesssium,germanium,lead,mercury,tamininthegut.Therefore,increasedintakesmaybesilver,strontiumbeneficial.Evidenceisunconvincingthathighdosesof
495VITAMINS&MINERALS/497amountsrequiredvaryfromtheorderofgramsperdaydecarboxylationofα-ketoacidsandoftransketolaseforsodiumandcalcium,throughmilligramsperdayinthepentosephosphatepathway.Riboflavinand(eg,iron)tomicrogramsperdayforthetraceelements.niacinareimportantcofactorsinoxidoreductionre-Ingeneral,mineraldeficienciesareencounteredwhenactions,respectivelypresentinflavoproteinenzymesfoodscomefromoneregion,wherethesoilmaybede-andinNADandNADP.ficientinsomeminerals,eg,iodinedeficiency.Where•PantothenicacidispresentincoenzymeAandacylthedietcomesfromavarietyofdifferentregions,min-carrierprotein,whichactascarriersforacylgroupseraldeficienciesareunlikely.However,irondeficiencyinmetabolicreactions.Pyridoxine,aspyridoxalisageneralproblembecauseifironlossesfromthephosphate,isthecoenzymeforseveralenzymesofbodyarerelativelyhigh(eg,fromheavymenstrualaminoacidmetabolism,includingtheaminotrans-bloodloss),itisdifficulttoachieveanadequateintakeferases,andofglycogenphosphorylase.Biotinisthetoreplacethelosses.Foodsfromsoilscontaininghighcoenzymeforseveralcarboxylaseenzymes.levelsofseleniumcausetoxicity,andincreasedintakes•Besidesotherfunctions,vitaminB12andfolicacidofcommonsalt(sodiumchloride)causehypertensiontakepartinprovidingone-carbonresiduesforDNAinsusceptibleindividuals.synthesis,deficiencyresultinginmegaloblasticane-mia.VitaminCisawater-solubleantioxidantthatSUMMARYmaintainsvitaminEandmanymetalcofactorsinthereducedstate.•Vitaminsareorganicnutrientswithessentialmeta-bolicfunctions,generallyrequiredinsmallamounts•Inorganicmineralelementsthathaveafunctionininthedietbecausetheycannotbesynthesizedbythethebodymustbeprovidedinthediet.Wheninsuffi-body.Thelipid-solublevitamins(A,D,E,andK)cient,deficiencysymptomsmayarise,andifpresentarehydrophobicmoleculesrequiringnormalfatab-inexcesstheymaybetoxic.sorptionfortheirefficientabsorptionandtheavoid-anceofdeficiencysymptoms.REFERENCES•VitaminA(retinol),presentincarnivorousdiets,andBenderDA,BenderAE:Nutrition:AReferenceHandbook.Oxfordtheprovitamin(β-carotene),foundinplants,formUnivPress,1997.retinaldehyde,utilizedinvision,andretinoicacid,BenderDA:NutritionalBiochemistryoftheVitamins.2nded.Cam-whichactsinthecontrolofgeneexpression.VitaminbridgeUnivPress,2003.Disasteroidprohormoneyieldingtheactivehor-GarrowJS,JamesWPT,RalphA:HumanNutritionandDietetics,monederivativecalcitriol,whichregulatescalcium10thed.Churchill-Livingstone,2000.andphosphatemetabolism.VitaminDdeficiencyHalliwellB,ChiricoS:Lipidperoxidation:itsmechanism,mea-leadstoricketsandosteomalacia.surement,andsignificance.AmJClinNutr1993;57(5•VitaminE(tocopherol)isthemostimportantan-Suppl):715S.tioxidantinthebody,actinginthelipidphaseofKrinskyNI:Actionsofcarotenoidsinbiologicalsystems.AnnuRevmembranesandprotectingagainsttheeffectsoffreeNutr1993;13:561.radicals.VitaminKfunctionsascofactortoacar-PadhH:VitaminC:newerinsightsintoitsbiochemicalfunctions.NutrRev1991;49:65.boxylasethatactsonglutamateresiduesofclottingfactorprecursorproteinstoenablethemtochelateShaneB:Folylpolyglutamatesynthesisandroleintheregulationofone-carbonmetabolism.VitamHorm1989;45:263.calcium.WisemanH,HalliwellB:DamagetoDNAbyreactiveoxygenand•Thewater-solublevitaminsoftheBcomplexactasnitrogenspecies:roleininflammatorydiseaseandprogressionenzymecofactors.Thiaminisacofactorinoxidativetocancer.BiochemJ1996;313:17.
496IntracellularTraffic&SortingofProteins46RobertK.Murray,MD,PhDBIOMEDICALIMPORTANCEthesignalpeptidearegivenbelow.Proteinssynthesizedonfreepolyribosomeslackthisparticularsignalpep-Proteinsmusttravelfrompolyribosomestomanydif-tideandaredeliveredintothecytosol.Theretheyareferentsitesinthecelltoperformtheirparticularfunc-directedtomitochondria,nuclei,andperoxisomesbytions.Somearedestinedtobecomponentsofspecificspecificsignals—orremaininthecytosoliftheylackaorganelles,othersforthecytosolorforexport,andyetsignal.Anyproteinthatcontainsatargetingsequenceotherswillbelocatedinthevariouscellularmem-thatissubsequentlyremovedisdesignatedasaprepro-branes.Thus,thereisconsiderableintracellulartraffictein.Insomecasesasecondpeptideisalsoremoved,ofproteins.ManystudieshaveshownthattheGolgiandinthateventtheoriginalproteinisknownasapre-apparatusplaysamajorroleinthesortingofproteinsproprotein(eg,preproalbumin;Chapter50).fortheircorrectdestinations.AmajorinsightwastheProteinssynthesizedandsortedintheroughERrecognitionthatforproteinstoattaintheirproperloca-branch(Figure46–2)includemanydestinedforvari-tions,theygenerallycontaininformation(asignalorousmembranes(eg,oftheER,Golgiapparatus,lyso-codingsequence)thattargetsthemappropriately.Oncesomes,andplasmamembrane)andforsecretion.Lyso-anumberofthesignalsweredefined,itbecameappar-somalenzymesarealsoincluded.Thus,suchproteinsentthatcertaindiseasesresultfrommutationsthataf-mayresideinthemembranesorlumensoftheERorfectthesesignals.Inthischapterwediscusstheintracel-followthemajortransportrouteofintracellularpro-lulartrafficofproteinsandtheirsortingandbrieflyteinstotheGolgiapparatus.Furthersignal-mediatedconsidersomeofthedisordersthatresultwhenabnor-sortingofcertainproteinsoccursintheGolgiappara-malitiesoccur.tus,resultingindeliverytolysosomes,membranesoftheGolgiapparatus,andothersites.ProteinsdestinedMANYPROTEINSARETARGETEDfortheplasmamembraneorforsecretionpassthroughBYSIGNALSEQUENCESTOTHEIRtheGolgiapparatusbutgenerallyarenotthoughttoCORRECTDESTINATIONScarryspecificsortingsignals;theyarebelievedtoreachtheirdestinationsbydefault.Theproteinbiosyntheticpathwaysincellscanbecon-TheentirepathwayofER→Golgiapparatus→sideredtobeonelargesortingsystem.Manyproteinsplasmamembraneisoftencalledthesecretoryorexo-carrysignals(usuallybutnotalwaysspecificsequencescytoticpathway.Eventsalongthisroutewillbegivenofaminoacids)thatdirectthemtotheirdestination,specialattention.MostoftheproteinsreachingthethusensuringthattheywillendupintheappropriateGolgiapparatusortheplasmamembranearecarriedinmembraneorcellcompartment;thesesignalsareafun-transportvesicles;abriefdescriptionoftheformationdamentalcomponentofthesortingsystem.Usuallytheoftheseimportantparticleswillbegivensubsequently.signalsequencesarerecognizedandinteractwithcom-Otherproteinsdestinedforsecretionarecarriedinse-plementaryareasofproteinsthatserveasreceptorsforcretoryvesicles(Figure46–2).Theseareprominentintheproteinsthatcontainthem.thepancreasandcertainotherglands.Theirmobiliza-Amajorsortingdecisionismadeearlyinproteintionanddischargeareregulatedandoftenreferredtoasbiosynthesis,whenspecificproteinsaresynthesizedei-“regulatedsecretion,”whereasthesecretorypathwaytheronfreeoronmembrane-boundpolyribosomes.involvingtransportvesiclesiscalled“constitutive.”ThisresultsintwosortingbranchescalledthecytosolicExperimentalapproachesthathaveaffordedmajorbranchandtheroughendoplasmicreticulum(RER)insightstotheprocessesdescribedinthischapterin-branch(Figure46–1).Thissortingoccursbecausepro-clude(1)useofyeastmutants;(2)applicationofre-teinssynthesizedonmembrane-boundpolyribosomescombinantDNAtechniques(eg,mutatingoreliminat-containasignalpeptidethatmediatestheirattach-ingparticularsequencesinproteins,orfusingnewmenttothemembraneoftheER.Furtherdetailsonsequencesontothem;and(3)developmentofinvitro498
497INTRACELLULARTRAFFIC&SORTINGOFPROTEINS/499Proteinsabout20–80aminoacidsinlength,whichisnothighlyMitochondrialconservedbutcontainsmanypositivelychargedaminoacids(eg,LysorArg).ThepresequenceisequivalenttoNuclear(1)CytosolicasignalpeptidemediatingattachmentofpolyribosomesPeroxisomaltomembranesoftheER(seebelow),butinthisin-Cytosolicstancetargetingproteinstothematrix;iftheleaderse-Polyribosomesquenceiscleavedoff,potentialmatrixproteinswillnotERmembranereachtheirdestination.GAmembraneTranslocationisbelievedtooccurposttranslation-(2)RoughERPlasmamembraneally,afterthematrixproteinsarereleasedfromthecy-tosolicpolyribosomes.InteractionswithanumberofSecretorycytosolicproteinsthatactaschaperones(seebelow)Lysosomalenzymesandastargetingfactorsoccurpriortotranslocation.Figure46–1.DiagrammaticrepresentationoftheTwodistincttranslocationcomplexesaresituatedtwobranchesofproteinsortingoccurringbysynthesisintheouterandinnermitochondrialmembranes,re-on(1)cytosolicand(2)membrane-boundpolyribo-ferredto(respectively)asTOM(translocase-of-the-somes.Themitochondrialproteinslistedareencodedoutermembrane)andTIM(translocase-of-the-innermembrane).Eachcomplexhasbeenanalyzedandbynucleargenes.Someofthesignalsusedinfurtherfoundtobecomposedofanumberofproteins,someofsortingoftheseproteinsarelistedinTable46–4.(ER,whichactasreceptorsfortheincomingproteinsandendoplasmicreticulum;GA,Golgiapparatus.)othersascomponentsofthetransmembraneporesthroughwhichtheseproteinsmustpass.Proteinsmustbeintheunfoldedstatetopassthroughthecom-systems(eg,tostudytranslocationintheERandmech-plexes,andthisismadepossiblebyATP-dependentanismsofvesicleformation).bindingtoseveralchaperoneproteins.TherolesofThesortingofproteinsbelongingtothecytosolicchaperoneproteinsinproteinfoldingarediscussedlaterbranchreferredtoaboveisdescribednext,startingwithinthischapter.Inmitochondria,theyareinvolvedinmitochondrialproteins.translocation,sorting,folding,assembly,anddegrada-tionofimportedproteins.Aproton-motiveforceacrosstheinnermembraneisrequiredforimport;itisTHEMITOCHONDRIONBOTHIMPORTSmadeupoftheelectricpotentialacrossthemembrane(insidenegative)andthepHgradient(seeChapter&SYNTHESIZESPROTEINS12).ThepositivelychargedleadersequencemaybeMitochondriacontainmanyproteins.Thirteenpro-helpedthroughthemembranebythenegativechargeteins(mostlymembranecomponentsoftheelectroninthematrix.Thepresequenceissplitoffinthematrixtransportchain)areencodedbythemitochondrialbyamatrix-processingpeptidase(MPP).Contactgenomeandsynthesizedinthatorganelleusingitsownwithotherchaperonespresentinthematrixisessentialprotein-synthesizingsystem.However,themajority(attocompletetheoverallprocessofimport.Interactionleastseveralhundred)areencodedbynucleargenes,withmt-Hsp70(Hsp=heatshockprotein)ensuresaresynthesizedoutsidethemitochondriaoncytosolicproperimportintothematrixandpreventsmisfoldingpolyribosomes,andmustbeimported.Yeastcellshaveoraggregation,whileinteractionwiththemt-Hsp60-provedtobeaparticularlyusefulsystemforanalyzingHsp10systemensuresproperfolding.Thelatterpro-themechanismsofimportofmitochondrialproteins,teinsresemblethebacterialGroELchaperonins,asub-partlybecauseithasprovedpossibletogenerateavari-classofchaperonesthatformcomplexcage-likeetyofmutantsthathaveilluminatedthefundamentalassembliesmadeupofheptamericringstructures.Theprocessesinvolved.Mostprogresshasbeenmadeintheinteractionsofimportedproteinswiththeabovechap-studyofproteinspresentinthemitochondrialmatrix,eronesrequirehydrolysisofATPtodrivethem.suchastheF1ATPasesubunits.OnlythepathwayofThedetailsofhowpreproteinsaretranslocatedhaveimportofmatrixproteinswillbediscussedinanydetailnotbeenfullyelucidated.Itispossiblethattheelectrichere.potentialassociatedwiththeinnermitochondrialmem-Matrixproteinsmustpassfromcytosolicpolyribo-branecausesaconformationalchangeintheunfoldedsomesthroughtheouterandinnermitochondrialpreproteinbeingtranslocatedandthatthishelpstopullmembranestoreachtheirdestination.Passagethroughitacross.Furthermore,thefactthatthematrixismorethetwomembranesiscalledtranslocation.Theyhavenegativethantheintermembranespacemay“attract”anaminoterminalleadersequence(presequence),thepositivelychargedaminoterminalofthepreprotein
498PlasmamembraneCytosolEarlySecretoryendosomeConstitutivestorage(excretory)granuletransportPrelysosomevesicle(orlateendosome)LysosomeTGNGolgitransapparatusmedialcisCGNEndoplasmicreticulumNuclearenvelopeFigure46–2.Diagrammaticrepresentationoftheroughendoplasmicreticu-lumbranchofproteinsorting.NewlysynthesizedproteinsareinsertedintotheERmembraneorlumenfrommembrane-boundpolyribosomes(smallblackcir-clesstuddingthecytosolicfaceoftheER).ThoseproteinsthataretransportedoutoftheER(indicatedbysolidblackarrows)dosofromribosome-freetransi-tionalelements.Suchproteinsmaythenpassthroughthevarioussubcompart-mentsoftheGolgiuntiltheyreachtheTGN,theexitsideoftheGolgi.IntheTGN,proteinsaresegregatedandsorted.Secretoryproteinsaccumulateinsecretorystoragegranulesfromwhichtheymaybeexpelledasshownintheupperright-handsideofthefigure.Proteinsdestinedfortheplasmamembraneorthosethataresecretedinaconstitutivemannerarecarriedouttothecellsurfaceintrans-portvesicles,asindicatedintheuppermiddleareaofthefigure.Someproteinsmayreachthecellsurfacevialateandearlyendosomes.Otherproteinsenterprelysosomes(lateendosomes)andareselectivelytransferredtolysosomes.Theendocyticpathwayillustratedintheupperleft-handareaofthefigureisconsid-eredelsewhereinthischapter.RetrievalfromtheGolgiapparatustotheERisnotconsideredinthisscheme.(CGN,cis-Golginetwork;TGN,trans-Golginetwork.)(CourtesyofEDegen.)500
499INTRACELLULARTRAFFIC&SORTINGOFPROTEINS/501toenterthematrix.Closecontactbetweenthemem-Thesemacromoleculesincludehistones,ribosomalpro-branesitesintheouterandinnermembranesinvolvedteinsandribosomalsubunits,transcriptionfactors,andintranslocationisnecessary.mRNAmolecules.ThetransportisbidirectionalandTheabovedescribesthemajorpathwayofproteinsoccursthroughthenuclearporecomplexes(NPCs).destinedforthemitochondrialmatrix.However,cer-Thesearecomplexstructureswithamassapproxi-tainproteinsinsertintotheoutermitochondrialmately30timesthatofaribosomeandarecomposedmembranefacilitatedbytheTOMcomplex.Othersofabout100differentproteins.Thediameterofanstopintheintermembranespace,andsomeinsertintoNPCisapproximately9nmbutcanincreaseuptoap-theinnermembrane.Yetothersproceedintothema-proximately28nm.Moleculessmallerthanabout40trixandthenreturntotheinnermembraneorinter-kDacanpassthroughthechanneloftheNPCbydiffu-membranespace.Anumberofproteinscontaintwosion,butspecialtranslocationmechanismsexistforsignalingsequences—onetoenterthemitochondriallargermolecules.Thesemechanismsareunderintensivematrixandtheothertomediatesubsequentrelocationinvestigation,butsomeimportantfeatureshavealready(eg,intotheinnermembrane).Certainmitochondrialemerged.proteinsdonotcontainpresequences(eg,cytochromeHereweshallmainlydescribenuclearimportofc,whichlocatesintheintermembranespace),andoth-certainmacromolecules.Thegeneralpicturethathaserscontaininternalpresequences.Overall,proteinsemergedisthatproteinstobeimported(cargomole-employavarietyofmechanismsandroutestoattaincules)carryanuclearlocalizationsignal(NLS).Onetheirfinaldestinationsinmitochondria.exampleofanNLSistheaminoacidsequence(Pro)2-Generalfeaturesthatapplytotheimportofproteins(Lys)4-Ala-Lys-Val,whichismarkedlyrichinbasicly-intoorganelles,includingmitochondriaandsomeofsineresidues.DependingonwhichNLSitcontains,atheotherorganellestobediscussedbelow,aresumma-cargomoleculeinteractswithoneofafamilyofsolublerizedinTable46–1.proteinscalledimportins,andthecomplexdocksattheNPC.AnotherfamilyofproteinscalledRanplaysaIMPORTINS&EXPORTINSAREcriticalregulatoryroleintheinteractionofthecomplexINVOLVEDINTRANSPORTwiththeNPCandinitstranslocationthroughtheNPC.RanproteinsaresmallmonomericnuclearGTP-OFMACROMOLECULESINasesand,likeotherGTPases,existineitherGTP-&OUTOFTHENUCLEUSboundorGDP-boundstates.Theyarethemselvesreg-Ithasbeenestimatedthatmorethanamillionmacro-ulatedbyguaninenucleotideexchangefactorsmoleculesperminutearetransportedbetweenthenu-(GEFs;eg,theproteinRCC1ineukaryotes),whicharecleusandthecytoplasminanactiveeukaryoticcell.locatedinthenucleus,andRanguanine-activatingproteins(GAPs),whicharepredominantlycytoplas-mic.TheGTP-boundstateofRanisfavoredinthenu-Table46–1.SomegeneralfeaturesofproteincleusandtheGDP-boundstateinthecytoplasm.Theimporttoorganelles.1conformationsandactivitiesofRanmoleculesvaryde-pendingonwhetherGTPorGDPisboundtothem(theGTP-boundstateisactive;seediscussionofGpro-•ImportofaproteinintoanorganelleusuallyoccursinthreeteinsinChapter43).Theasymmetrybetweennucleusstages:recognition,translocation,andmaturation.andcytoplasm—withrespecttowhichofthesetwonu-•TargetingsequencesontheproteinarerecognizedinthecleotidesisboundtoRanmolecules—isthoughttobecytoplasmoronthesurfaceoftheorganelle.crucialinunderstandingtherolesofRanintransferring•Theproteinisunfoldedfortranslocation,astatemain-tainedinthecytoplasmbychaperones.complexesunidirectionallyacrosstheNPC.When•Threadingoftheproteinthroughamembranerequiresen-cargomoleculesarereleasedinsidethenucleus,theim-ergyandorganellarchaperonesonthetranssideoftheportinsrecirculatetothecytoplasmtobeusedagain.membrane.Figure46–3summarizessomeoftheprincipalfeatures•Cyclesofbindingandreleaseoftheproteintothechaper-intheaboveprocess.oneresultinpullingofitspolypeptidechainthroughtheOthersmallmonomericGTPases(eg,ARF,Rab,membrane.Ras,andRho)areimportantinvariouscellularpro-•Otherproteinswithintheorganellecatalyzefoldingofthecessessuchasvesicleformationandtransport(ARFandprotein,oftenattachingcofactorsoroligosaccharidesandRab;seebelow),certaingrowthanddifferentiationassemblingthemintoactivemonomersoroligomers.processes(Ras),andformationoftheactincytoskele-1DatafromMcNewJA,GoodmanJM:Thetargetingandassemblyton.AprocessinvolvingGTPandGDPisalsocrucialofperoxisomalproteins:someoldrulesdonotapply.TrendsinthetransportofproteinsacrossthemembraneoftheBiochemSci1998;21:54.ER(seebelow).
500502/CHAPTER46Targeting1NucleusαβNucleusRanGTPCytoplasmRanGTPCytoplasmGDPOFFGTPONRanGAPRanDocking+PiRanexchangeGEF+2RanGDPRanGDPαβRanGTPGDP3RanGEF?RanBP1RanBP1GDPαβRan4GTPαβTerminationTranslocationRan6GGTPTP5RanPiα+RanβGAPGGTPTP7?RanRan+GGTPTPGDPα+β8RecyclefactorsFigure46–3.SchematicrepresentationoftheproposedroleofRanintheimportofcargocarryinganNLSsignal.(1)ThetargetingcomplexformswhentheNLSreceptor(α,animportin)bindsNLScargoandthedockingfactor(β).(2)Dockingoccursatfilamentoussitesthatpro-trudefromtheNPC.Ran-GDPdocksindependently.(3)TransfertothetranslocationchannelistriggeredwhenaRanGEFconvertsRan-GDPtoRan-GTP.(4)TheNPCcatalyzestranslocationofthetargetingcomplex.(5)Ran-GTPisrecycledtoRan-GDPbydockedRanGAP.(6)Ran-GTPdis-ruptsthetargetingcomplexbybindingtoasiteonβthatoverlapswithabindingsite.(7)NLScargodissociatesfromα,andRan-GTPmaydissociatefromβ.(8)αandβfactorsarerecycledtothecytoplasm.Inset:TheRantranslocationswitchisoffinthecytoplasmandoninthenucleus.Ran-GTPpromotesNLS-andNES-directedtranslocation.However,cytoplasmicRanisenrichedinRan-GDP(OFF)byanactiveRanGAP,andnuclearpoolsareenrichedinRan-GTP(ON)byanactiveGEF.RanBP1promotesthecontraryactivitiesofthesetwofactors.Directlinkageofnu-clearandcytoplasmicpoolsofRanoccursthroughtheNPCbyanunknownshuttlingmecha-nism.Pi,inorganicphosphate;NLS,nuclearlocalizationsignal;NPC,nuclearporecomplex;GEF,guaninenucleotideexchangefactor;GAP,guanine-activatingprotein;NES,nuclearexportsig-nal;BP,bindingprotein.(Reprinted,withpermission,fromGoldfarbDS:Whosefingerisontheswitch?Science1997;276:1814.)
501INTRACELLULARTRAFFIC&SORTINGOFPROTEINS/503Proteinssimilartoimportins,referredtoasex-thesynthesisofbileacids,andamarkedreductionofportins,areinvolvedinexportofmanymacromole-plasmalogens.Theconditionisbelievedtobeduetoculesfromthenucleus.Cargomoleculesforexportmutationsingenesencodingcertainproteins—socarrynuclearexportsignals(NESs).Ranproteinsarecalledperoxins—involvedinvariousstepsofperoxi-involvedinthisprocessalso,anditisnowestablishedsomebiogenesis(suchastheimportofproteinsde-thattheprocessesofimportandexportshareanumberscribedabove),oringenesencodingcertainperoxiso-ofcommonfeatures.malenzymesthemselves.TwocloselyrelatedconditionsareneonataladrenoleukodystrophyandinfantileRefsumdisease.ZellwegersyndromeandthesetwoMOSTCASESOFZELLWEGERSYNDROMEconditionsrepresentaspectrumofoverlappingfea-AREDUETOMUTATIONSINGENEStures,withZellwegersyndromebeingthemostsevereINVOLVEDINTHEBIOGENESIS(manyproteinsaffected)andinfantileRefsumdiseaseOFPEROXISOMEStheleastsevere(onlyoneorafewproteinsaffected).Table46–2listssomefeaturesoftheseandrelatedcon-Theperoxisomeisanimportantorganelleinvolvedinditions.aspectsofthemetabolismofmanymolecules,includingfattyacidsandotherlipids(eg,plasmalogens,choles-THESIGNALHYPOTHESISEXPLAINSterol,bileacids),purines,aminoacids,andhydrogenHOWPOLYRIBOSOMESBINDTOTHEperoxide.Theperoxisomeisboundedbyasinglemem-ENDOPLASMICRETICULUMbraneandcontainsmorethan50enzymes;catalaseandurateoxidasearemarkerenzymesforthisorganelle.ItsAsindicatedabove,theroughERbranchisthesecondproteinsaresynthesizedoncytosolicpolyribosomesandofthetwobranchesinvolvedinthesynthesisandsort-foldpriortoimport.Thepathwaysofimportofanum-ingofproteins.Inthisbranch,proteinsaresynthesizedberofitsproteinsandenzymeshavebeenstudied,someonmembrane-boundpolyribosomesandtranslocatedbeingmatrixcomponentsandothersmembranecom-intothelumenoftheroughERpriortofurthersortingponents.Atleasttwoperoxisomal-matrixtargeting(Figure46–2).sequences(PTSs)havebeendiscovered.One,PTS1,isThesignalhypothesiswasproposedbyBlobelandatripeptide(ie,Ser-Lys-Leu[SKL],butvariationsofSabatinipartlytoexplainthedistinctionbetweenfreethissequencehavebeendetected)locatedatthecar-andmembrane-boundpolyribosomes.Theyfoundthatboxylterminalofanumberofmatrixproteins,includ-proteinssynthesizedonmembrane-boundpolyribo-ingcatalase.Another,PTS2,consistingofabout26–36somescontainedapeptideextension(signalpeptide)aminoacids,hasbeenfoundinatleastfourmatrixpro-teins(eg,thiolase)and,unlikePTS1,iscleavedafterentryintothematrix.ProteinscontainingPTS1se-Table46–2.Disordersduetoperoxisomal1quencesformcomplexeswithasolublereceptorproteinabnormalities.(PTS1R)andproteinscontainingPTS2sequencescomplexwithanother,PTS2R.Theresultingcom-MIMNumber2plexestheninteractwithamembranereceptor,Pex14p.Zellwegersyndrome214100ProteinsinvolvedinfurthertransportofproteinsintoNeonataladrenoleukodystrophy202370thematrixarealsopresent.Mostperoxisomalmem-InfantileRefsumdisease266510braneproteinshavebeenfoundtocontainneitherofHyperpipecolicacidemia239400theabovetwotargetingsequences,butapparentlycon-Rhizomelicchondrodysplasiapunctata215100tainothers.TheimportsystemcanhandleintactAdrenoleukodystrophy300100oligomers(eg,tetramericcatalase).ImportofmatrixPseudo-neonataladrenoleukodystrophy264470proteinsrequiresATP,whereasimportofmembranePseudo-Zellwegersyndrome261510proteinsdoesnot.Hyperoxaluriatype1259900InterestinimportofproteinsintoperoxisomeshasAcatalasemia115500beenstimulatedbystudiesonZellwegersyndrome.Glutaryl-CoAoxidasedeficiency231690Thisconditionisapparentatbirthandischaracterized1Reproduced,withpermission,fromSeashoreMR,WappnerRS:byprofoundneurologicimpairment,victimsoftenGeneticsinPrimaryCare&ClinicalMedicine.Appleton&Lange,dyingwithinayear.Thenumberofperoxisomescan1996.varyfrombeingalmostnormaltobeingvirtuallyabsent2MIM=MendelianInheritanceinMan.Eachnumberspecifiesaref-insomepatients.Biochemicalfindingsincludeanaccu-erenceinwhichinformationregardingeachoftheabovecondi-mulationofverylongchainfattyacids,abnormalitiesoftionscanbefound.
502504/CHAPTER46attheiraminoterminalswhichmediatedtheirattach-andtheβsubunitspansthemembrane.WhentheSRP-menttothemembranesoftheER.Asnotedabove,signalpeptidecomplexinteractswiththereceptor,theproteinswhoseentiresynthesisoccursonfreepolyribo-exchangeofGDPforGTPisstimulated.Thisformofsomeslackthissignalpeptide.Animportantaspectofthereceptor(withGTPbound)hasahighaffinityforthesignalhypothesiswasthatitsuggested—asturnstheSRPandthusreleasesthesignalpeptide,whichbindsouttobethecase—thatallribosomeshavethesametothetranslocationmachinery(translocon)alsopresentstructureandthatthedistinctionbetweenmembrane-intheERmembrane.Theαsubunitthenhydrolyzesitsboundandfreeribosomesdependssolelyonthefor-boundGTP,restoringGDPandcompletingaGTP-mer’scarryingproteinsthathavesignalpeptides.MuchGDPcycle.Theunidirectionalityofthiscyclehelpsdriveevidencehasconfirmedtheoriginalhypothesis.Becausetheinteractionofthepolyribosomeanditssignalpeptidemanymembraneproteinsaresynthesizedonmem-withtheERmembraneintheforwarddirection.brane-boundpolyribosomes,thesignalhypothesisplaysThetransloconconsistsofanumberofmembraneanimportantroleinconceptsofmembraneassembly.proteinsthatformaprotein-conductingchannelintheSomecharacteristicsofsignalpeptidesaresummarizedERmembranethroughwhichthenewlysynthesizedinTable46–3.proteinmaypass.ThechannelappearstobeopenonlyFigure46–4illustratestheprincipalfeaturesinrela-whenasignalpeptideispresent,preservingconductancetiontothepassageofasecretedproteinthroughtheacrosstheERmembranewhenitcloses.Theconduc-membraneoftheER.Itincorporatesfeaturesfromthetanceofthechannelhasbeenmeasuredexperimentally.originalsignalhypothesisandfromsubsequentwork.SpecificfunctionsofanumberofcomponentsoftheThemRNAforsuchaproteinencodesanaminotermi-transloconhavebeenidentifiedorsuggested.TRAMnalsignalpeptide(alsovariouslycalledaleaderse-(translocatingchain-associatedmembrane)proteinmayquence,atransientinsertionsignal,asignalsequence,bindthesignalsequenceasitinitiallyinteractswiththeorapresequence).ThesignalhypothesisproposedthattransloconandtheSec61pcomplex(consistingofthreetheproteinisinsertedintotheERmembraneattheproteins)bindstheheavysubunitoftheribosome.sametimeasitsmRNAisbeingtranslatedonpolyribo-Theinsertionofthesignalpeptideintotheconduct-somes,so-calledcotranslationalinsertion.Asthesig-ingchannel,whiletheotherendoftheparentproteinisnalpeptideemergesfromthelargesubunitoftheribo-stillattachedtoribosomes,istermed“cotranslationalsome,itisrecognizedbyasignalrecognitionparticleinsertion.”Theprocessofelongationoftheremaining(SRP)thatblocksfurthertranslationafterabout70portionoftheproteinprobablyfacilitatespassageoftheaminoacidshavebeenpolymerized(40buriedinthenascentproteinacrossthelipidbilayerastheribosomeslargeribosomalsubunitand30exposed).TheblockisremainattachedtothemembraneoftheER.Thus,thereferredtoaselongationarrest.TheSRPcontainssixrough(orribosome-studded)ERisformed.Itisimpor-proteinsandhasa7SRNAassociatedwithitthatistantthattheproteinbekeptinanunfoldedstatepriorcloselyrelatedtotheAlufamilyofhighlyrepeatedtoenteringtheconductingchannel—otherwise,itmayDNAsequences(Chapter36).TheSRP-imposedblocknotbeabletogainaccesstothechannel.isnotreleaseduntiltheSRP-signalpeptide-polyribo-RibosomesremainattachedtotheERduringsyn-somecomplexhasboundtotheso-calleddockingpro-thesisofsignalpeptide-containingproteinsbutarere-tein(SRP-R,areceptorfortheSRP)ontheERmem-leasedanddissociatedintotheirtwotypesofsubunitsbrane;theSRPthusguidesthesignalpeptidetothewhentheprocessiscompleted.ThesignalpeptideisSRP-Randpreventsprematurefoldingandexpulsionhydrolyzedbysignalpeptidase,locatedontheluminaloftheproteinbeingsynthesizedintothecytosol.sideoftheERmembrane(Figure46–4),andthenisTheSRP-Risanintegralmembraneproteincom-apparentlyrapidlydegradedbyproteases.posedofαandβsubunits.TheαsubunitbindsGDPCytochromeP450(Chapter53),anintegralERmembraneprotein,doesnotcompletelycrossthemem-brane.Instead,itresidesinthemembranewithitssig-Table46–3.Somepropertiesofsignalpeptides.nalpeptideintact.Itspassagethroughthemembraneispreventedbyasequenceofaminoacidscalledahalt-or•Usually,butnotalways,locatedattheaminoterminalstop-transfersignal.•Containapproximately12–35aminoacidsSecretoryproteinsandproteinsdestinedformem-•MethionineisusuallytheaminoterminalaminoacidbranesdistaltotheERcompletelytraversethemem-•Containacentralclusterofhydrophobicaminoacidsbranebilayerandaredischargedintothelumenofthe•ContainatleastonepositivelychargedaminoacidnearER.N-Glycanchains,ifpresent,areadded(Chaptertheiraminoterminal47)astheseproteinstraversetheinnerpartoftheER•UsuallycleavedoffatthecarboxylterminalendofanAlamembrane—aprocesscalled“cotranslationalglycosyla-residuebysignalpeptidasetion.”Subsequently,theproteinsarefoundinthe
503INTRACELLULARTRAFFIC&SORTINGOFPROTEINS/5055′3′SignalcodonsAUGSignalpeptideSRPSignalpeptidaseRibosomereceptorSignalreceptorFigure46–4.DiagramofthesignalhypothesisforthetransportofsecretedproteinsacrosstheERmembrane.TheribosomessynthesizingaproteinmovealongthemessengerRNAspecifyingtheaminoacidsequenceoftheprotein.(Themessengerisrepresentedbythelinebetween5′and3′.)ThecodonAUGmarksthestartofthemessagefortheprotein;thehatchedlinesthatfollowAUGrepresentthecodonsforthesignalsequence.Astheproteingrowsoutfromthelargerribosomalsubunit,thesignalsequenceisexposedandboundbythesignalrecognitionparticle(SRP).Transla-tionisblockeduntilthecomplexbindstothe“dockingprotein,”alsodesignatedSRP-R(repre-sentedbythesolidbar)ontheERmembrane.Thereisalsoareceptor(openbar)fortheribosomeitself.TheinteractionoftheribosomeandgrowingpeptidechainwiththeERmembraneresultsintheopeningofachannelthroughwhichtheproteinistransportedtotheinteriorspaceoftheER.Duringtranslocation,thesignalsequenceofmostproteinsisremovedbyanenzymecalledthe“signalpeptidase,”locatedattheluminalsurfaceoftheERmembrane.Thecompletedproteiniseventuallyreleasedbytheribosome,whichthenseparatesintoitstwocomponents,thelargeandsmallribosomalsubunits.TheproteinendsupinsidetheER.Seetextforfurtherdetails.(Slightlymodifiedandreproduced,withpermission,fromMarxJL:Newlymadeproteinszipthroughthecell.Sci-ence1980;207:164.Copyright©1980bytheAmericanAssociationfortheAdvancementofScience.)lumenoftheGolgiapparatus,wherefurtherchangesinPROTEINSFOLLOWSEVERALROUTESglycanchainsoccur(Figure47–9)priortointracellularTOBEINSERTEDINTOORATTACHEDdistributionorsecretion.ThereisstrongevidencethatTOTHEMEMBRANESOFTHEthesignalpeptideisinvolvedintheprocessofproteininsertionintoERmembranes.Mutantproteins,con-ENDOPLASMICRETICULUMtainingalteredsignalpeptidesinwhichahydrophobicTheroutesthatproteinsfollowtobeinsertedintotheaminoacidisreplacedbyahydrophilicone,arenotin-membranesoftheERincludethefollowing.sertedintoERmembranes.Nonmembraneproteins(eg,α-globin)towhichsignalpeptideshavebeenat-A.COTRANSLATIONALINSERTIONtachedbygeneticengineeringcanbeinsertedintotheFigure46–5showsavarietyofwaysinwhichproteinslumenoftheERorevensecreted.aredistributedintheplasmamembrane.Inparticular,Thereisconsiderableevidencethatasecondtrans-theaminoterminalsofcertainproteins(eg,theLDLre-posonintheERmembraneisinvolvedinretrogradeceptor)canbeseentobeontheextracytoplasmicface,transportofvariousmoleculesfromtheERlumentowhereasforotherproteins(eg,theasialoglycoproteinre-thecytosol.Thesemoleculesincludeunfoldedormis-ceptor)thecarboxylterminalsareonthisface.Toex-foldedglycoproteins,glycopeptides,andoligosaccha-plainthesedispositions,onemustconsidertheinitialrides.SomeatleastofthesemoleculesaredegradedinbiosyntheticeventsattheERmembrane.TheLDLre-proteasomes.Thus,thereistwo-waytrafficacrosstheceptorenterstheERmembraneinamanneranalogousERmembrane.toasecretoryprotein(Figure46–4);itpartlytraverses
504506/CHAPTER46NNNNNEXTRACYTOPLASMICCFACENCCPHOSPHOLIPIDBILAYERNCCYTOPLASMICVarioustransporters(eg,glucose)CFACECCCNNInsulinandInfluenzaneuraminidaseGprotein–coupledreceptorsIGF-IreceptorsAsialoglycoproteinreceptorTransferrinreceptorHLA-DRinvariantchainLDLreceptorHLA-AheavychainInfluenzahemagglutininFigure46–5.Variationsinthewayinwhichproteinsareinsertedintomembranes.Thisschematicrepresentation,whichillustratesanumberofpossibleorientations,showstheseg-mentsoftheproteinswithinthemembraneasα-helicesandtheothersegmentsaslines.TheLDLreceptor,whichcrossesthemembraneonceandhasitsaminoterminalontheexterior,iscalledatypeItransmembraneprotein.Theasialoglycoproteinreceptor,whichalsocrossesthemembraneoncebuthasitscarboxylterminalontheexterior,iscalledatypeIItransmembraneprotein.Thevarioustransportersindicated(eg,glucose)crossthemembraneanumberoftimesandarecalledtypeIIItransmembraneproteins;theyarealsoreferredtoaspolytopicmembraneproteins.(N,aminoterminal;C,carboxylterminal.)(Adapted,withpermission,fromWicknerWT,LodishHF:Multiplemechanismsofproteininsertionintoandacrossmembranes.Science1985;230:400.Copyright©1985bytheAmericanAssociationfortheAdvancementofScience.)theERmembrane,itssignalpeptideiscleaved,anditscleavedinsertionsequencesandashalt-transfersignals,aminoterminalprotrudesintothelumen.However,itisrespectively.Eachpairofhelicalsegmentsisinsertedasaretainedinthemembranebecauseitcontainsahighlyhairpin.Sequencesthatdeterminethestructureofahydrophobicsegment,thehalt-orstop-transfersignal.proteininamembranearecalledtopogenicsequences.ThissequenceformsthesingletransmembranesegmentAsexplainedinthelegendtoFigure46–5,theaboveoftheproteinandisitsmembrane-anchoringdomain.threeproteinsareexamplesoftypeI,typeII,andtypeThesmallpatchofERmembraneinwhichthenewlyIIItransmembraneproteins.synthesizedLDLreceptorislocatedsubsequentlybudsoffasacomponentofatransportvesicle,probablyfromB.SYNTHESISONFREEPOLYRIBOSOMESthetransitionalelementsoftheER(Figure46–2).As&SUBSEQUENTATTACHMENTTOTHEdescribedbelowinthediscussionofasymmetryofpro-teinsandlipidsinmembraneassembly,thedispositionENDOPLASMICRETICULUMMEMBRANEofthereceptorintheERmembraneispreservedintheAnexampleiscytochromeb5,whichenterstheERvesicle,whicheventuallyfuseswiththeplasmamem-membranespontaneously.brane.Incontrast,theasialoglycoproteinreceptorpos-sessesaninternalinsertionsequence,whichinsertsintoC.RETENTIONATTHELUMINALASPECTthemembranebutisnotcleaved.Thisactsasananchor,anditscarboxylterminalisextrudedthroughthemem-OFTHEENDOPLASMICRETICULUMbrane.Themorecomplexdispositionofthetrans-BYSPECIFICAMINOACIDSEQUENCESporters(eg,forglucose)canbeexplainedbythefactAnumberofproteinspossesstheaminoacidsequencethatalternatingtransmembraneα-helicesactasun-KDEL(Lys-Asp-Glu-Leu)attheircarboxylterminal.
505INTRACELLULARTRAFFIC&SORTINGOFPROTEINS/507Thissequencespecifiesthatsuchproteinswillbeat-denotetransportstepsthatmaybeindependentoftar-tachedtotheinneraspectoftheERinarelativelyloosegetingsignals,whereastheverticalopenarrowsrepre-manner.ThechaperoneBiP(seebelow)isonesuchsentstepsthatdependonspecificsignals.Thus,flowofprotein.Actually,KDEL-containingproteinsfirsttravelcertainproteins(includingmembraneproteins)fromtotheGolgi,interacttherewithaspecificKDELrecep-theERtotheplasmamembrane(designated“bulktorprotein,andthenreturnintransportvesiclestotheflow,”asitisnonselective)probablyoccurswithoutanyER,wheretheydissociatefromthereceptor.targetingsequencesbeinginvolved,ie,bydefault.Ontheotherhand,insertionofresidentproteinsintotheD.RETROGRADETRANSPORTFROMERandGolgimembranesisdependentuponspecificTHEGOLGIAPPARATUSsignals(eg,KDELorhalt-transfersequencesfortheCertainothernon-KDEL-containingproteinsdestinedER).Similarly,transportofmanyenzymestolysosomesforthemembranesoftheERalsopasstotheGolgiandisdependentupontheMan6-Psignal(Chapter47),thenreturn,byretrogradevesiculartransport,totheERandasignalmaybeinvolvedforentryofproteinsintotobeinsertedtherein(seebelow).secretorygranules.Table46–4summarizesinforma-Theforegoingparagraphsdemonstratethatavari-tiononsequencesthatareknowntobeinvolvedintar-etyofroutesareinvolvedinassemblyoftheproteinsofgetingvariousproteinstotheircorrectintracellularsites.theERmembranes;asimilarsituationprobablyholdsforothermembranes(eg,themitochondrialmem-CHAPERONESAREPROTEINSbranesandtheplasmamembrane).Precisetargetingse-THATPREVENTFAULTYFOLDINGquenceshavebeenidentifiedinsomeinstances(eg,&UNPRODUCTIVEINTERACTIONSKDELsequences).Thetopicofmembranebiogenesisisdiscussedfur-OFOTHERPROTEINStherlaterinthischapter.ExitfromtheERmaybetherate-limitingstepinthesecretorypathway.Inthiscontext,ithasbeenfoundPROTEINSMOVETHROUGHCELLULARthatcertainproteinsplayaroleintheassemblyorCOMPARTMENTSTOSPECIFICproperfoldingofotherproteinswithoutthemselvesDESTINATIONSbeingcomponentsofthelatter.Suchproteinsarecalledmolecularchaperones;anumberofimportantproper-AschemerepresentingthepossibleflowofproteinstiesoftheseproteinsarelistedinTable46–5,andthealongtheER→Golgiapparatus→plasmamembranenamesofsomeofparticularimportanceintheERarerouteisshowninFigure46–6.ThehorizontalarrowslistedinTable46–6.Basically,theystabilizeunfoldedLysosomescismedialtransCellERGolgiGolgiGolgisurfaceSecretorystoragevesiclesFigure46–6.Flowofmembraneproteinsfromtheendoplas-micreticulum(ER)tothecellsurface.Horizontalarrowsdenotestepsthathavebeenproposedtobesignalindependentandthusrepresentbulkflow.Theopenverticalarrowsintheboxesdenoteretentionofproteinsthatareresidentinthemembranesoftheorganelleindicated.Theopenverticalarrowsoutsidetheboxesindicatesignal-mediatedtransporttolysosomesandsecre-torystoragegranules.(Reproduced,withpermission,fromPfefferSR,RothmanJE:Biosyntheticproteintransportandsortingbytheen-doplasmicreticulumandGolgi.AnnuRevBiochem1987;56:829.)
506508/CHAPTER46Table46–4.SomesequencesorcompoundsthatTable46–6.Somechaperonesandenzymesdirectproteinstospecificorganelles.involvedinfoldingthatarelocatedintheroughendoplasmicreticulum.TargetingSequenceorCompoundOrganelleTargeted•BiP(immunoglobulinheavychainbindingprotein)SignalpeptidesequenceMembraneofER•GRP94(glucose-regulatedprotein)•CalnexinAminoterminalLuminalsurfaceofER•CalreticulinKDELsequence•PDI(proteindisulfideisomerase)(Lys-Asp-Glu-Leu)•PPI(peptidylprolylcis-transisomerase)AminoterminalsequenceMitochondrialmatrix(20–80residues)1NLS(eg,Pro2-Lys2-Ala-NucleusSeveralexamplesofchaperoneswereintroducedLys-Val)abovewhenthesortingofmitochondrialproteinswasPTS1(eg,Ser-Lys-Leu)Peroxisomediscussed.Theimmunoglobulinheavychainbindingprotein(BiP)islocatedinthelumenoftheER.ThisMannose6-phosphateLysosomeproteinwillbindabnormallyfoldedimmunoglobulin1NLS,nuclearlocalizationsignal;PTS,peroxisomal-matrixtarget-heavychainsandcertainotherproteinsandpreventingsequence.themfromleavingtheER,inwhichtheyaredegraded.Anotherimportantchaperoneiscalnexin,acalcium-bindingproteinlocatedintheERmembrane.Thispro-teinbindsawidevarietyofproteins,includingmixedhistocompatibility(MHC)antigensandavarietyoforpartiallyfoldedintermediates,allowingthemtimetoserumproteins.AsmentionedinChapter47,calnexinfoldproperly,andpreventinappropriateinteractions,bindsthemonoglycosylatedspeciesofglycoproteinsthuscombatingtheformationofnonfunctionalstruc-thatoccurduringprocessingofglycoproteins,retainingtures.MostchaperonesexhibitATPaseactivityandthemintheERuntiltheglycoproteinhasfoldedprop-bindADPandATP.Thisactivityisimportantfortheirerly.Calreticulin,whichisalsoacalcium-bindingpro-effectonfolding.TheADP-chaperonecomplexoftentein,haspropertiessimilartothoseofcalnexin;itisnothasahighaffinityfortheunfoldedprotein,which,membrane-bound.Chaperonesarenottheonlypro-whenbound,stimulatesreleaseofADPwithreplace-teinsintheERlumenthatareconcernedwithpropermentbyATP.TheATP-chaperonecomplex,inturn,foldingofproteins.Twoenzymesarepresentthatplayreleasessegmentsoftheproteinthathavefoldedprop-anactiveroleinfolding.Proteindisulfideisomeraseerly,andthecycleinvolvingADPandATPbindingis(PDI)promotesrapidreshufflingofdisulfidebondsrepeateduntilthefoldedproteinisreleased.untilthecorrectsetisachieved.Peptidylprolylisom-erase(PPI)acceleratesfoldingofproline-containingproteinsbycatalyzingthecis-transisomerizationofX-Probonds,whereXisanyaminoacidresidue.Table46–5.Somepropertiesofchaperoneproteins.TRANSPORTVESICLESAREKEYPLAYERSININTRACELLULARPROTEINTRAFFIC•Presentinawiderangeofspeciesfrombacteriatohumans•Manyareso-calledheatshockproteins(Hsp)Mostproteinsthataresynthesizedonmembrane-•SomeareinduciblebyconditionsthatcauseunfoldingofboundpolyribosomesandaredestinedfortheGolginewlysynthesizedproteins(eg,elevatedtemperatureandapparatusorplasmamembranereachthesesitesinsidevariouschemicals)transportvesicles.Theprecisemechanismsbywhich•Theybindtopredominantlyhydrophobicregionsofun-proteinssynthesizedintheroughERareinsertedintofoldedandaggregatedproteinsthesevesiclesarenotknown.Thoseinvolvedintrans-•TheyactinpartasaqualitycontroloreditingmechanismportfromtheERtotheGolgiapparatusandvicefordetectingmisfoldedorotherwisedefectiveproteinsversa—andfromtheGolgitotheplasmamembrane—•MostchaperonesshowassociatedATPaseactivity,withATParemainlyclathrin-free,unlikethecoatedvesiclesin-orADPbeinginvolvedintheprotein-chaperoneinteractionvolvedinendocytosis(seediscussionsoftheLDLre-•Foundinvariouscellularcompartmentssuchascytosol,ceptorinChapters25and26).Forthesakeofclarity,mitochondria,andthelumenoftheendoplasmicreticulumthenon-clathrin-coatedvesicleswillbereferredtoin
507INTRACELLULARTRAFFIC&SORTINGOFPROTEINS/509thistextastransportvesicles.ThereisevidencethatTable46–7.FactorsinvolvedintheformationofproteinsdestinedforthemembranesoftheGolgiappa-non-clathrin-coatedvesiclesandtheirtransport.ratuscontainspecificsignalsequences.Ontheotherhand,mostproteinsdestinedfortheplasmamembrane•ARF:ADP-ribosylationfactor,aGTPaseorforsecretiondonotappeartocontainspecificsig-•Coatomer:Afamilyofatleastsevencoatproteins(α,β,γ,δ,nals,reachingthesedestinationsbydefault.ε,β′,andζ).Differenttransportvesicleshavedifferentcom-plementsofcoatproteins.TheGolgiApparatusIsInvolvedin•SNAP:SolubleNSFattachmentfactorGlycosylation&SortingofProteins•SNARE:SNAPreceptor•v-SNARE:VesicleSNARETheGolgiapparatusplaystwoimportantrolesinmem-•t-SNARE:TargetSNAREbranesynthesis.First,itisinvolvedintheprocessing•GTP-γ-S:AnonhydrolyzableanalogofGTP,usedtotesttheoftheoligosaccharidechainsofmembraneandotherinvolvementofGTPN-linkedglycoproteinsandalsocontainsenzymesin-•NEM:N-Ethylmaleimide,achemicalthatalkylatessulfhy-volvedinO-glycosylation(seeChapter47).Second,itdrylgroupsisinvolvedinthesortingofvariousproteinspriorto•NSF:NEM-sensitivefactor,anATPasetheirdeliverytotheirappropriateintracellulardestina-•Rabproteins:Afamilyofras-relatedproteinsfirstobservedtions.AllpartsoftheGolgiapparatusparticipateintheinratbrain;theyareGTPasesandareactivewhenGTPisfirstrole,whereasthetrans-Golgiisparticularlyin-found•Sec1:Amemberofafamilyofproteinsthatattachtovolvedinthesecondandisveryrichinvesicles.Becauset-SNAREsandaredisplacedfromthembyRabproteins,oftheircentralroleinproteintransport,considerabletherebyallowingv-SNARE–t-SNAREinteractionstooccur.researchhasbeenconductedinrecentyearsconcerningtheformationandfateoftransportvesicles.AModelofNon-Clathrin-CoatedVesiclesStep2:Membrane-associatedARFrecruitsthecoatInvolvesSNAREs&OtherFactorsproteinsthatcomprisethecoatomershellfromthecytosol,formingacoatedbud.VesicleslieattheheartofintracellulartransportofStep3:Thebudpinchesoffinaprocessinvolvingmanyproteins.Recently,significantprogresshasbeenacyl-CoA—andprobablyATP—tocompletethemadeinunderstandingtheeventsinvolvedinvesicleformationofthecoatedvesicle.formationandtransport.ThishastranspiredbecauseofStep4:Coatdisassembly(involvingdissociationoftheuseofanumberofapproaches.Theseincludees-ARFandcoatomershell)followshydrolysisoftablishmentofcell-freesystemswithwhichtostudyboundGTP;uncoatingisnecessaryforfusiontovesicleformation.Forinstance,itispossibletoobserve,occur.byelectronmicroscopy,buddingofvesiclesfromGolgipreparationsincubatedwithcytosolandATP.Thede-Step5:Vesicletargetingisachievedviamembersofvelopmentofgeneticapproachesforstudyingvesiclesafamilyofintegralproteins,termedv-SNAREs,inyeasthasalsobeencrucial.Thepictureiscomplex,thattagthevesicleduringitsbudding.v-SNAREswithitsownnomenclature(Table46–7),andinvolvespairwithcognatet-SNAREsinthetargetmembraneavarietyofcytosolicandmembraneproteins,GTP,todockthevesicle.ATP,andaccessoryfactors.Itispresumedthatsteps4and5arecloselycoupledBasedlargelyonaproposalbyRothmanandcol-andthatstep4mayfollowstep5,withARFandtheleagues,anterogradevesiculartransportcanbeconsid-coatomershellrapidlydissociatingafterdocking.eredtooccurineightsteps(Figure46–7).Thebasicconceptisthateachtransportvesiclebearsauniquead-Step6:Thegeneralfusionmachinerythenassem-dressmarkerconsistingofoneormorev-SNAREpro-blesonthepairedSNAREcomplex;itincludesanteins,whileeachtargetmembranebearsoneormoreATPase(NSF;NEM-sensitivefactor)andtheSNAPcomplementaryt-SNAREproteinswithwhichthe(solubleNSFattachmentfactor)proteins.SNAPsformerinteractspecifically.bindtotheSNARE(SNAPreceptor)complex,en-Step1:CoatassemblyisinitiatedwhenARFisac-ablingNSFtobind.tivatedbybindingGTP,whichisexchangedforStep7:HydrolysisofATPbyNSFisessentialforGDP.ThisleadstotheassociationofGTP-boundfusion,aprocessthatcanbeinhibitedbyNEM(N-ARFwithitsputativereceptor(hatchedinFigureethylmaleimide).Certainotherproteinsandcalcium46–7)inthedonormembrane.arealsorequired.
508510/CHAPTER46Coated3vesicle4t-SNARECoatedGTPGTPGTPGTP-γ-SbudGTP5GTPGTPGTPSNAPsNSFAcyl-CoAGTPGTPATPGTPPiGDPBuddingGTPGTP6GTPSNAPsNSF2+Ca220SfusionCoatomerparticlev-SNAREATP1GTPNEM7FusionGTPBFAGDPGDPGDPARFNocodazoleDonorTargetmembranemembrane8(eg,ER)(eg,CGN)Figure46–7.Modelofthestepsinaroundofanterogradevesiculartransport.Thecyclestartsinthebottomleft-handsideofthefigure,wheretwomoleculesofARFarerepresentedassmallovalscontainingGDP.Thestepsinthecyclearedescribedinthetext.MostoftheabbreviationsusedareexplainedinTable46–7.TherolesofRabandSec1proteins(seetext)intheoverallprocessarenotdealtwithinthisfigure.(CGN,cis-Golginetwork;BFA,BrefeldinA.)(AdaptedfromRothmanJE:Mechanismsofintracellularproteintransport.Nature1994;372:55.)(CourtesyofEDegen.)Step8:Retrogradetransportoccurstorestartthedoubtremaintobediscovered.COPIvesiclesarein-cycle.ThislaststepmayretrievecertainproteinsvolvedinbidirectionaltransportfromtheERtotheorrecyclev-SNAREs.Nocodazole,amicrotubule-Golgiandinthereversedirection,whereasCOPIIvesi-disruptingagent,inhibitsthisstep.clesareinvolvedmainlyintransportintheformerdi-rection.Clathrin-containingvesiclesareinvolvedintransportfromthetrans-GolginetworktoprelysosomesBrefeldinAInhibitstheCoatingProcessandfromtheplasmamembranetoendosomes,respec-tively.Regardingselectionofcargomoleculesbyvesi-Thefollowingpointsexpandandclarifytheabove.cles,thisappearstobeprimarilyafunctionofthecoat(a)Toparticipateinstep1,ARFmustfirstbemodi-proteinsofvesicles.Cargomoleculesmayinteractwithfiedbyadditionofmyristicacid(C14:0),employingcoatproteinseitherdirectlyorviaintermediaryproteinsmyristoyl-CoAastheacyldonor.Myristoylationisonethatattachtocoatproteins,andtheythenbecomeen-ofanumberofenzyme-catalyzedposttranslationalmod-closedintheirappropriatevesicles.ifications,involvingadditionofcertainlipidstospecific(c)ThefungalmetabolitebrefeldinApreventsresiduesofproteins,thatfacilitatethebindingofpro-GTPfrombindingtoARFinstep1andthusinhibitsteinstothecytosolicsurfacesofmembranesorvesicles.theentirecoatingprocess.Initspresence,theGolgiap-Othersareadditionofpalmitate,farnesyl,andgeranyl-paratusappearstodisintegrate,andfragmentsarelost.geranyl;thetwolattermoleculesarepolyisoprenoidsItmaydothisbyinhibitingtheguaninenucleotideex-containing15and20carbonatoms,respectively.changerinvolvedinstep1.(b)Atleastthreedifferenttypesofcoatedvesicles(d)GTP--S(anonhydrolyzableanalogofGTPhavebeendistinguished:COPI,COPII,andclathrin-oftenusedininvestigationsoftheroleofGTPinbio-coatedvesicles;thefirsttwoarereferredtohereaschemicalprocesses)blocksdisassemblyofthecoatfromtransportvesicles.Manyothertypesofvesiclesnocoatedvesicles,leadingtoabuild-upofcoatedvesicles.
509INTRACELLULARTRAFFIC&SORTINGOFPROTEINS/511(e)AfamilyofRas-likeproteins,calledtheRabpro-AsymmetryofBothProteins&LipidsIsteinfamily,arerequiredinseveralstepsofintracellularMaintainedDuringMembraneAssemblyproteintransport,regulatedsecretion,andendocytosis.TheyaresmallmonomericGTPasesthatattachtotheVesiclesformedfrommembranesoftheERandGolgicytosolicfacesofmembranesviageranylgeranylchains.apparatus,eithernaturallyorpinchedoffbyhomoge-TheyattachintheGTP-boundstate(notshowninnization,exhibittransverseasymmetriesofbothlipidFigure46–7)tothebuddingvesicle.Anotherfamilyofandprotein.Theseasymmetriesaremaintainedduringproteins(Sec1)bindstot-SNAREsandpreventsinter-fusionoftransportvesicleswiththeplasmamembrane.actionwiththemandtheircomplementaryv-SNAREs.Theinsideofthevesiclesafterfusionbecomestheout-Whenavesicleinteractswithitstargetmembrane,Rabsideoftheplasmamembrane,andthecytoplasmicsideproteinsdisplaceSec1proteinsandthev-SNARE-ofthevesiclesremainsthecytoplasmicsideofthemem-t-SNAREinteractionisfreetooccur.Itappearsthatbrane(Figure46–8).SincethetransverseasymmetryoftheRabandSec1familiesofproteinsregulatethespeedthemembranesalreadyexistsinthevesiclesoftheERofvesicleformation,opposingeachother.Rabproteinswellbeforetheyarefusedtotheplasmamembrane,ahavebeenlikenedtothrottlesandSec1proteinstomajorproblemofmembraneassemblybecomesunder-dampersontheoverallprocessofvesicleformation.standinghowtheintegralproteinsareinsertedintothe(f)Studiesusingv-andt-SNAREproteinsreconsti-lipidbilayeroftheER.Thisproblemwasaddressedtutedintoseparatelipidbilayervesicleshaveindicatedearlierinthischapter.thattheyformSNAREpins,ie,SNAREcomplexesthatPhospholipidsarethemajorclassoflipidinmem-linktwomembranes(vesicles).SNAPsandNSFarere-branes.TheenzymesresponsibleforthesynthesisofquiredforformationofSNAREpins,butoncetheyphospholipidsresideinthecytoplasmicsurfaceofthehaveformedtheycanapparentlyleadtospontaneouscisternaeoftheER.Asphospholipidsaresynthesizedatfusionofmembranesatphysiologictemperature,sug-thatsite,theyprobablyself-assembleintothermody-gestingthattheyaretheminimalmachineryrequirednamicallystablebimolecularlayers,therebyexpandingformembranefusion.themembraneandperhapspromotingthedetachment(g)Thefusionofsynapticvesicleswiththeplasmaofso-calledlipidvesiclesfromit.Ithasbeenproposedmembraneofneuronsinvolvesaseriesofeventssimilarthatthesevesiclestraveltoothersites,donatingtheirtothatdescribedabove.Forexample,onev-SNAREislipidstoothermembranes;however,littleisknowndesignatedsynaptobrevinandtwot-SNAREsaredes-aboutthismatter.Asindicatedabove,cytosolicpro-ignatedsyntaxinandSNAP25(synaptosome-associ-teinsthattakeupphospholipidsfromonemembraneatedproteinof25kDa).BotulinumBtoxinisoneofandreleasethemtoanother(ie,phospholipidexchangethemostlethaltoxinsknownandthemostseriousproteins)havebeendemonstrated;theyprobablyplayacauseoffoodpoisoning.Onecomponentofthistoxinroleincontributingtothespecificlipidcompositionofisaproteasethatappearstocleaveonlysynaptobrevin,variousmembranes.thusinhibitingreleaseofacetylcholineattheneuro-muscularjunctionandpossiblyprovingfatal,depend-Lipids&ProteinsUndergoTurnoveratingonthedosetaken.DifferentRatesinDifferentMembranes(h)Althoughtheabovemodeldescribesnon-clathrin-coatedvesicles,itappearslikelythatmanyofIthasbeenshownthatthehalf-livesofthelipidsofthetheeventsdescribedaboveapply,atleastinprinciple,ERmembranesofratliveraregenerallyshorterthantoclathrin-coatedvesicles.thoseofitsproteins,sothattheturnoverratesoflipidsandproteinsareindependent.Indeed,differ-entlipidshavebeenfoundtohavedifferenthalf-lives.THEASSEMBLYOFMEMBRANESFurthermore,thehalf-livesoftheproteinsofthesemembranesvaryquitewidely,someexhibitingshortISCOMPLEX(hours)andotherslong(days)half-lives.Thus,individ-Therearemanycellularmembranes,eachwithitsownuallipidsandproteinsoftheERmembranesappeartospecificfeatures.Nosatisfactoryschemedescribingthebeinsertedintoitrelativelyindependently;thisistheassemblyofanyoneofthesemembranesisavailable.caseformanyothermembranes.HowvariousproteinsareinitiallyinsertedintotheThebiogenesisofmembranesisthusacomplexmembraneoftheERhasbeendiscussedabove.Theprocessaboutwhichmuchremainstobelearned.Onetransportofproteins,includingmembraneproteins,toindicationofthecomplexityinvolvedistoconsiderthevariouspartsofthecellinsidevesicleshasalsobeende-numberofposttranslationalmodificationsthatmem-scribed.Somegeneralpointsaboutmembraneassemblybraneproteinsmaybesubjectedtopriortoattainingremaintobeaddressed.theirmaturestate.Theseincludeproteolysis,assembly
510512/CHAPTER46MembraneproteinExteriorsurfaceTable46–8.Majorfeaturesofmembraneassembly.•Lipidsandproteinsareinsertedindependentlyintomem-branes.•Individualmembranelipidsandproteinsturnoverindepen-Cdentlyandatdifferentrates.Plasmamembrane•Topogenicsequences(eg,signal[aminoterminalorinter-Lumennal]andstop-transfer)areimportantindeterminingthein-NNCytoplasmsertionanddispositionofproteinsinmembranes.Integral•Membraneproteinsinsidetransportvesiclesbudofftheen-proteindoplasmicreticulumontheirwaytotheGolgi;finalsortingofmanymembraneproteinsoccursinthetrans-Golginet-Vesiclework.membraneC•Specificsortingsequencesguideproteinstoparticularorganellessuchaslysosomes,peroxisomes,andmitochon-dria.intomultimers,glycosylation,additionofaglycophos-phatidylinositol(GPI)anchor,sulfationontyrosineorcarbohydratemoieties,phosphorylation,acylation,andNprenylation—alistthatisundoubtedlynotcomplete.NNevertheless,significantprogresshasbeenmade;TableC46–8summarizessomeofthemajorfeaturesofmem-braneassemblythathaveemergedtodate.Table46–9.SomedisordersduetomutationsingenesencodingproteinsinvolvedinintracellularNN1membranetransport.2DisorderProteinInvolvedCChédiak-Higashisyndrome,Lysosomaltraffickingregula-C214500torFigure46–8.FusionofavesiclewiththeplasmaCombineddeficiencyoffactorsERGIC-53,amannose-membranepreservestheorientationofanyintegralVandVIII,227300bindinglectinproteinsembeddedinthevesiclebilayer.Initially,theHermansky-Pudlaksyndrome,AP-3adaptorcomplexβ3Aaminoterminaloftheproteinfacesthelumen,orinner203300subunitcavity,ofsuchavesicle.Afterfusion,theaminotermi-nalisontheexteriorsurfaceoftheplasmamembrane.I-celldisease,252500N-AcetylglucosamineThattheorientationoftheproteinhasnotbeenre-1-phosphotransferaseversedcanbeperceivedbynotingthattheotherendOculocerebrorenalsyndrome,OCRL-1,aninositolpoly-ofthemolecule,thecarboxylterminal,isalwaysim-30900phosphate5-phosphatasemersedinthecytoplasm.Thelumenofavesicleand1ModifiedfromOlkonnenVM,IkonenE:Geneticdefectsofintra-theoutsideofthecellaretopologicallyequivalent.(Re-cellular-membranetransport.NEngJMed2000;343:1095.Certaindrawnandmodified,withpermission,fromLodishHF,relatedconditionsnotlistedherearealsodescribedinthispubli-RothmanJE:Theassemblyofcellmembranes.SciAmcation.I-celldiseaseisdescribedinChapter47.Themajorityof[Jan]1979;240:43.)thedisorderslistedaboveaffectlysosomalfunction;readersshouldconsultatextbookofmedicineforinformationontheclinicalmanifestationsoftheseconditions.2ThenumbersaftereachdisorderaretheOMIMnumbers.
511INTRACELLULARTRAFFIC&SORTINGOFPROTEINS/513VariousDisordersResultFromMutationsandattachmentoftransportvesiclestoatargetmem-inGenesEncodingProteinsInvolvedbraneissummarized.inIntracellularTransport•Membraneassemblyisdiscussedandshowntobecomplex.AsymmetryofbothlipidsandproteinsisSomeofthesearelistedinTable46–9;themajorityaf-maintainedduringmembraneassembly.fectlysosomalfunction.Anumberofothermutations•Anumberofdisordershavebeenshowntobeduetoaffectingintracellularproteintransporthavebeenre-mutationsingenesencodingproteinsinvolvedinportedbutarenotincludedhere.variousaspectsofproteintrafficandsorting.SUMMARYREFERENCES•ManyproteinsaretargetedtotheirdestinationsbyFullerGM,ShieldsDL:MolecularBasisofMedicalCellBiology.signalsequences.AmajorsortingdecisionismadeMcGraw-Hill,1998.whenproteinsarepartitionedbetweencytosolicandGouldSJetal:Theperoxisomebiogenesisdisorders.In:TheMeta-membrane-boundpolyribosomesbyvirtueoftheab-bolicandMolecularBasesofInheritedDisease,8thed.Scriversenceorpresenceofasignalpeptide.CRetal(editors).McGraw-Hill,2001.•Thepathwaysofproteinimportintomitochondria,GrahamJM,HigginsJA:MembraneAnalysis.BIOSScientific,nuclei,peroxisomes,andtheendoplasmicreticulum1997.aredescribed.GriffithJ,SansomC:TheTransporterFactsBook.AcademicPress,1998.•Manyproteinssynthesizedonmembrane-boundLodishHetal:MolecularCellBiology,4thed.Freeman,2000.polyribosomesproceedtotheGolgiapparatusand(Chapter17containscomprehensivecoverageofproteinsort-theplasmamembraneintransportvesicles.ingandorganellebiogenesis.)•Anumberofglycosylationreactionsoccurincom-OlkkonenVM,IkonenE:Geneticdefectsofintracellular-mem-partmentsoftheGolgi,andproteinsarefurtherbranetransport.NEnglJMed2000;343:1095.sortedinthetrans-Golginetwork.ReithmeierRAF:Assemblyofproteinsintomembranes.In:Bio-•MostproteinsdestinedfortheplasmamembranechemistryofLipids,LipoproteinsandMembranes.VanceDE,VanceJE(editors).Elsevier,1996.andforsecretionappeartolackspecificsignals—aSabatiniDD,AdesnikMB:Thebiogenesisofmembranesandor-defaultmechanism.ganelles.In:TheMetabolicandMolecularBasesofInherited•Theroleofchaperoneproteinsinthefoldingofpro-Disease,8thed.ScriverCRetal(editors).McGraw-Hill,teinsispresented,andamodeldescribingbudding2001.
512Glycoproteins47RobertK.Murray,MD,PhDBIOMEDICALIMPORTANCEarefirmlyestablished;othersarestillunderinvesti-gation.Glycoproteinsareproteinsthatcontainoligosaccha-ride(glycan)chainscovalentlyattachedtotheirpolypeptidebackbones.Theyareoneclassofglycocon-OLIGOSACCHARIDECHAINSENCODEjugateorcomplexcarbohydrates—equivalenttermsBIOLOGICINFORMATIONusedtodenotemoleculescontainingoneormorecar-Anenormousnumberofglycosidiclinkagescanbegen-bohydratechainscovalentlylinkedtoprotein(toformeratedbetweensugars.Forexample,threedifferenthex-glycoproteinsorproteoglycans)orlipid(toformglyco-osesmaybelinkedtoeachothertoformover1000dif-lipids).(ProteoglycansarediscussedinChapter48andferenttrisaccharides.TheconformationsofthesugarsinglycolipidsinChapter14).Almostalltheplasmapro-oligosaccharidechainsvarydependingontheirlinkagesteinsofhumans—exceptalbumin—areglycoproteins.andproximitytoothermoleculeswithwhichtheoligo-Manyproteinsofcellularmembranes(Chapter41)saccharidesmayinteract.Itisnowestablishedthatcertaincontainsubstantialamountsofcarbohydrate.Anum-oligosaccharidechainsencodeconsiderablebiologicin-berofthebloodgroupsubstancesareglycoproteins,formationandthatthisdependsupontheirconstituentwhereasothersareglycosphingolipids.Certainhor-sugars,theirsequences,andtheirlinkages.Forinstance,mones(eg,chorionicgonadotropin)areglycoproteins.mannose6-phosphateresiduestargetnewlysynthesizedAmajorproblemincancerismetastasis,thephenome-lysosomalenzymestothatorganelle(seebelow).nonwherebycancercellsleavetheirtissueoforigin(eg,thebreast),migratethroughthebloodstreamtosomedistantsiteinthebody(eg,thebrain),andgrowthereTECHNIQUESAREAVAILABLEinanunregulatedmanner,withcatastrophicresultsforFORDETECTION,PURIFICATION,theaffectedindividual.Manycancerresearchersthink&STRUCTURALANALYSISthatalterationsinthestructuresofglycoproteinsandOFGLYCOPROTEINSotherglycoconjugatesonthesurfacesofcancercellsareimportantinthephenomenonofmetastasis.Avarietyofmethodsusedinthedetection,purifica-tion,andstructuralanalysisofglycoproteinsarelistedGLYCOPROTEINSOCCURWIDELYinTable47–3.Theconventionalmethodsusedtopu-&PERFORMNUMEROUSFUNCTIONSrifyproteinsandenzymesarealsoapplicabletothepu-rificationofglycoproteins.OnceaglycoproteinhasGlycoproteinsoccurinmostorganisms,frombacteriabeenpurified,theuseofmassspectrometryandhigh-tohumans.Manyvirusesalsocontainglycoproteins,resolutionNMRspectroscopycanoftenidentifythesomeofwhichhavebeenmuchinvestigated,inpartbe-structuresofitsglycanchains.Analysisofglycoproteinscausetheyareverysuitableforbiosyntheticstudies.canbecomplicatedbythefactthattheyoftenexistasNumerousproteinswithdiversefunctionsareglycopro-glycoforms;theseareproteinswithidenticalaminoteins(Table47–1);theircarbohydratecontentrangesacidsequencesbutsomewhatdifferentoligosaccharidefrom1%toover85%byweight.compositions.AlthoughlinkagedetailsarenotstressedManystudieshavebeenconductedinanattempttointhischapter,itiscriticaltoappreciatethattheprecisedefinethepreciserolesoligosaccharidechainsplayinnaturesofthelinkagesbetweenthesugarsofglycopro-thefunctionsofglycoproteins.Table47–2summarizesteinsareoffundamentalimportanceindeterminingtheresultsfromsuchstudies.Someofthefunctionslistedstructuresandfunctionsofthesemolecules.514
513GLYCOPROTEINS/515Table47–1.SomefunctionsservedTable47–3.Someimportantmethodsusedtobyglycoproteins.studyglycoproteins.FunctionGlycoproteinsMethodUseStructuralmoleculeCollagensPeriodicacid-SchiffreagentDetectsglycoproteinsaspinkbandsafterelectrophoreticsep-LubricantandMucinsaration.protectiveagentIncubationofculturedcellsLeadstodetectionofaradio-TransportmoleculeTransferrin,ceruloplasminwithglycoproteinsasactivesugarafterelectropho-ImmunologicmoleculeImmunoglobulins,histocompatibil-radioactivebandsreticseparation.ityantigensTreatmentwithappropriateResultantshiftsinelectropho-HormoneChorionicgonadotropin,thyroid-endo-orexoglycosidasereticmigrationhelpdistinguishstimulatinghormone(TSH)orphospholipasesamongproteinswithN-glycan,O-glycan,orGPIlinkagesandEnzymeVarious,eg,alkalinephosphatasealsobetweenhighmannoseCellattachment-Variousproteinsinvolvedincell-cellandcomplexN-glycans.recognitionsite(eg,sperm-oocyte),virus-cell,Sepharose-lectincolumnTopurifyglycoproteinsorgly-bacterium-cell,andhormone-cellchromatographycopeptidesthatbindthepar-interactionsticularlectinused.AntifreezeCertainplasmaproteinsofcoldCompositionalanalysisIdentifiessugarsthatthegly-waterfishfollowingacidhydrolysiscoproteincontainsandtheirInteractwithspecificLectins,selectins(celladhesionstoichiometry.carbohydrateslectins),antibodiesMassspectrometryProvidesinformationonmolec-ReceptorVariousproteinsinvolvedinhor-ularmass,composition,se-moneanddrugactionquence,andsometimesbranch-ingofaglycanchain.AffectfoldingofCalnexin,calreticulincertainproteinsNMRspectroscopyToidentifyspecificsugars,theirsequence,linkages,andtheanomericnatureofglycosidiclinkages.Methylation(linkage)Todeterminelinkagesbetweenanalysissugars.AminoacidorcDNADeterminationofaminoacidTable47–2.Somefunctionsofthe1sequencingsequence.oligosaccharidechainsofglycoproteins.•Modulatephysicochemicalproperties,eg,solubility,vis-cosity,charge,conformation,denaturation,andbindingsitesforbacteriaandvirusesEIGHTSUGARSPREDOMINATE•Protectagainstproteolysis,frominsideandoutsideofcellINHUMANGLYCOPROTEINS•AffectproteolyticprocessingofprecursorproteinstosmallerproductsAbout200monosaccharidesarefoundinnature;how-•Areinvolvedinbiologicactivity,eg,ofhumanchorionicever,onlyeightarecommonlyfoundintheoligosac-gonadotropin(hCG)charidechainsofglycoproteins(Table47–4).Mostof•Affectinsertionintomembranes,intracellularmigration,thesesugarsweredescribedinChapter13.N-Acetyl-sortingandsecretionneuraminicacid(NeuAc)isusuallyfoundattheter-•Affectembryonicdevelopmentanddifferentiationminiofoligosaccharidechains,attachedtosubterminal•Mayaffectsitesofmetastasesselectedbycancercellsgalactose(Gal)orN-acetylgalactosamine(GalNAc)1AdaptedfromSchachterH:Biosyntheticcontrolsthatdetermineresidues.Theothersugarslistedaregenerallyfoundinthebranchingandheterogeneityofprotein-boundoligosaccha-moreinternalpositions.Sulfateisoftenfoundinglyco-rides.BiochemCellBiol1986;64:163.proteins,usuallyattachedtoGal,GalNAc,orGlcNAc.
514516/CHAPTER47Table47–4.Theprincipalsugarsfoundinhumanglycoproteins.TheirstructuresareillustratedinChapter13.NucleotideSugarTypeAbbreviationSugarCommentsGalactoseHexoseGalUDP-GalOftenfoundsubterminaltoNeuAcinN-linkedgly-coproteins.Alsofoundincoretrisaccharideofpro-teoglycans.GlucoseHexoseGlcUDP-GlcPresentduringthebiosynthesisofN-linkedglyco-proteinsbutnotusuallypresentinmatureglyco-proteins.Presentinsomeclottingfactors.MannoseHexoseManGDP-ManCommonsugarinN-linkedglycoproteins.N-Acetylneur-Sialicacid(nineNeuAcCMP-NeuAcOftentheterminalsugarinbothN-andO-linkedaminicacidCatoms)glycoproteins.Othertypesofsialicacidarealsofound,butNeuAcisthemajorspeciesfoundinhu-mans.AcetylgroupsmayalsooccurasO-acetylspeciesaswellasN-acetyl.FucoseDeoxyhexoseFucGDP-FucMaybeexternalinbothN-andO-linkedglycopro-teinsorinternal,linkedtotheGlcNAcresidueat-tachedtoAsninN-linkedspecies.CanalsooccurinternallyattachedtotheOHofSer(eg,int-PAandcertainclottingfactors).N-AcetylgalactosamineAminohexoseGalNAcUDP-GalNAcPresentinbothN-andO-linkedglycoproteins.N-AcetylglucosamineAminohexoseGlcNAcUDP-GlcNAcThesugarattachedtothepolypeptidechainviaAsninN-linkedglycoproteins;alsofoundatothersitesintheoligosaccharidesoftheseproteins.ManynuclearproteinshaveGlcNAcattachedtotheOHofSerorThrasasinglesugar.XylosePentoseXylUDP-XylXylisattachedtotheOHofSerinmanyproteogly-cans.XylinturnisattachedtotwoGalresidues,formingalinktrisaccharide.Xylisalsofoundint-PAandcertainclottingfactors.NUCLEOTIDESUGARSACTsuitableacceptorsprovidedappropriatetransferasesareASSUGARDONORSINMANYavailable.BIOSYNTHETICREACTIONSMostnucleotidesugarsareformedinthecytosol,generallyfromreactionsinvolvingthecorrespondingThefirstnucleotidesugartobereportedwasuridinenucleosidetriphosphate.CMP-sialicacidsareformeddiphosphateglucose(UDP-Glc);itsstructureisshowninthenucleus.Formationofuridinediphosphategalac-inFigure18–2.Thecommonnucleotidesugarsin-tose(UDP-Gal)requiresthefollowingtworeactionsinvolvedinthebiosynthesisofglycoproteinsarelistedinmammaliantissues:Table47–4;thereasonssomecontainUDPandothersguanosinediphosphate(GDP)orcytidinemonophos-UDP-GlcPYROPHOS-phate(CMP)areobscure.Manyoftheglycosylationre-PHORYLASEactionsinvolvedinthebiosynthesisofglycoproteinsUTP+Glucose1-phosphateutilizethesecompounds(seebelow).Theanhydrona-UDP-Glc+Pyrophosphatetureofthelinkagebetweenthephosphategroupandthesugarsisofthehigh-energy,high-group-transfer-UDP-Glcpotentialtype(Chapter10).Thesugarsofthesecom-EPIMERASEpoundsarethus“activated”andcanbetransferredtoUDP-GlcUDP-Gal
515GLYCOPROTEINS/517Becausemanyglycosylationreactionsoccurwithintidebackbone(ie,atinternalsites;Figure47–5)andarethelumenoftheGolgiapparatus,carriersystems(per-thususefulinreleasinglargeoligosaccharidechainsformeases,transporters)arenecessarytotransportnu-structuralanalyses.AglycoproteincanbetreatedwithcleotidesugarsacrosstheGolgimembrane.SystemsoneormoreoftheaboveglycosidasestoanalyzethetransportingUDP-Gal,GDP-Man,andCMP-NeuAceffectsonitsbiologicbehaviorofremovalofspecificintothecisternaeoftheGolgiapparatushavebeende-sugars.scribed.Theyareantiportsystems;ie,theinfluxofonemoleculeofnucleotidesugarisbalancedbytheeffluxTHEMAMMALIANofonemoleculeofthecorrespondingnucleotide(eg,ASIALOGLYCOPROTEINRECEPTORUMP,GMP,orCMP)formedfromthenucleotidesugars.Thismechanismensuresanadequateconcentra-ISINVOLVEDINCLEARANCEtionofeachnucleotidesugarinsidetheGolgiappara-OFCERTAINGLYCOPROTEINStus.UMPisformedfromUDP-GalintheaboveFROMPLASMABYHEPATOCYTESprocessasfollows:ExperimentsperformedbyAshwellandhiscolleaguesintheearly1970splayedanimportantroleinfocusingGALACTOSYL-TRANSFERASEattentiononthefunctionalsignificanceoftheoligosac-UDP-Gal+ProteinProteinGal+UDPcharidechainsofglycoproteins.Theytreatedrabbitceruloplasmin(aplasmaprotein;seeChapter50)withneuraminidaseinvitro.Thisprocedureexposedsubter-NUCLEOSIDEDIPHOSPHATEminalGalresiduesthatwerenormallymaskedbyter-PHOSPHATASEminalNeuAcresidues.Neuraminidase-treatedradioac-UDPUMP+Pitiveceruloplasminwasfoundtodisappearrapidlyfromthecirculation,incontrasttotheslowclearanceoftheuntreatedprotein.Verysignificantly,whentheGalEXO-&ENDOGLYCOSIDASESresiduesexposedtotreatmentwithneuraminidasewereFACILITATESTUDYremovedbytreatmentwithagalactosidase,theclear-OFGLYCOPROTEINSancerateoftheproteinreturnedtonormal.Furtherstudiesdemonstratedthatlivercellscontainamam-AnumberofglycosidasesofdefinedspecificityhavemalianasialoglycoproteinreceptorthatrecognizesprovedusefulinexaminingstructuralandfunctionaltheGalmoietyofmanydesialylatedplasmaproteinsaspectsofglycoproteins(Table47–5).Theseenzymesandleadstotheirendocytosis.Thisworkindicatedthatactateitherexternal(exoglycosidases)orinternal(en-anindividualsugar,suchasGal,couldplayanimpor-doglycosidases)positionsofoligosaccharidechains.Ex-tantroleingoverningatleastoneofthebiologicprop-amplesofexoglycosidasesareneuraminidasesanderties(ie,timeofresidenceinthecirculation)ofcertaingalactosidases;theirsequentialuseremovesterminalglycoproteins.ThisgreatlystrengthenedtheconceptNeuAcandsubterminalGalresiduesfrommostglyco-thatoligosaccharidechainscouldcontainbiologicin-proteins.EndoglycosidasesFandHareexamplesofformation.thelatterclass;theseenzymescleavetheoligosaccharidechainsatspecificGlcNAcresiduesclosetothepolypep-LECTINSCANBEUSEDTOPURIFYGLYCOPROTEINSTable47–5.Someglycosidasesusedtostudythe&TOPROBETHEIRFUNCTIONS1structureandfunctionofglycoproteins.Lectinsarecarbohydrate-bindingproteinsthataggluti-natecellsorprecipitateglycoconjugates;anumberofEnzymesTypelectinsarethemselvesglycoproteins.ImmunoglobulinsNeuraminidasesExoglycosidasethatreactwithsugarsarenotconsideredlectins.LectinsGalactosidasesExo-orendoglycosidasecontainatleasttwosugar-bindingsites;proteinswithaEndoglycosidaseFEndoglycosidasesinglesugar-bindingsitewillnotagglutinatecellsorEndoglycosidaseHEndoglycosidaseprecipitateglycoconjugates.Thespecificityofalectinis1usuallydefinedbythesugarsthatarebestatinhibitingTheenzymesareavailablefromavarietyofsourcesandareoftenitsabilitytocauseagglutinationorprecipitation.En-specificforcertaintypesofglycosidiclinkagesandalsofortheiranomericnatures.ThesitesofactionofendoglycosidasesFandHzymes,toxins,andtransportproteinscanbeclassifiedareshowninFigure47–5.Factsonbothhigh-mannoseandcom-aslectinsiftheybindcarbohydrate.Lectinswerefirstplexoligosaccharides,whereasHactsontheformer.discoveredinplantsandmicrobes,butmanylectinsof
516518/CHAPTER47animaloriginarenowknown.Themammalianasialo-O-linked),involvingthehydroxylsidechainofserineglycoproteinreceptordescribedaboveisanimportantorthreonineandasugarsuchasN-acetylgalactosamineexampleofananimallectin.Someimportantlectinsare(GalNAc-Ser[Thr]);(2)thosecontaininganN-glyco-listedinTable47–6.Muchcurrentresearchiscenteredsidiclinkage(ie,N-linked),involvingtheamidenitro-ontherolesofvariousanimallectins(eg,theselectins)genofasparagineandN-acetylglucosamine(GlcNAc-incell-cellinteractionsthatoccurinpathologiccondi-Asn);and(3)thoselinkedtothecarboxylterminaltionssuchasinflammationandcancermetastasis(seeaminoacidofaproteinviaaphosphoryl-ethanolaminebelow).moietyjoinedtoanoligosaccharide(glycan),whichinNumerouslectinshavebeenpurifiedandarecom-turnislinkedviaglucosaminetophosphatidylinositolmerciallyavailable;threeplantlectinsthathavebeen(PI).Thislatterclassisreferredtoasglycosylphos-widelyusedexperimentallyarelistedinTable47–7.phatidylinositol-anchored(GPI-anchored,orGPI-Amongmanyuses,lectinshavebeenemployedtopu-linked)glycoproteins.Otherminorclassesofglycopro-rifyspecificglycoproteins,astoolsforprobingthegly-teinsalsoexist.coproteinprofilesofcellsurfaces,andasreagentsforThenumberofoligosaccharidechainsattachedtogeneratingmutantcellsdeficientincertainenzymesin-oneproteincanvaryfromoneto30ormore,withthevolvedinthebiosynthesisofoligosaccharidechains.sugarchainsrangingfromoneortworesiduesinlengthtomuchlargerstructures.Manyproteinscon-THEREARETHREEMAJORCLASSEStainmorethanonetypeoflinkage;forinstance,gly-OFGLYCOPROTEINScophorin,animportantredcellmembraneglycopro-tein(Chapter52),containsbothO-andN-linkedBasedonthenatureofthelinkagebetweentheirpoly-oligosaccharides.peptidechainsandtheiroligosaccharidechains,glyco-proteinscanbedividedintothreemajorclasses(FigureGLYCOPROTEINSCONTAINSEVERAL47–1):(1)thosecontaininganO-glycosidiclinkage(ie,TYPESOFO-GLYCOSIDICLINKAGESAtleastfoursubclassesofO-glycosidiclinkagesarefoundinhumanglycoproteins:(1)TheGalNAc-Table47–6.Someimportantlectins.Ser(Thr)linkageshowninFigure47–1isthepredomi-nantlinkage.TwotypicaloligosaccharidechainsfoundinmembersofthissubclassareshowninFigure47–2.LectinsExamplesorCommentsUsuallyaGaloraNeuAcresidueisattachedtotheLegumelectinsConcanavalinA,pealectinGalNAc,butmanyvariationsinthesugarcompositionsWheatgermWidelyusedinstudiesofsurfacesofnor-andlengthsofsucholigosaccharidechainsarefound.agglutininmalcellsandcancercellsThistypeoflinkageisfoundinmucins(seebelow).(2)ProteoglycanscontainaGal-Gal-Xyl-Sertrisac-RicinCytotoxicglycoproteinderivedfromseedscharide(theso-calledlinktrisaccharide).(3)CollagensofthecastorplantcontainaGal-hydroxylysine(Hyl)linkage.(SubclassesBacterialtoxinsHeat-labileenterotoxinofEcoliand[2]and[3]arediscussedfurtherinChapter48.)choleratoxin(4)Manynuclearproteins(eg,certaintranscriptionfactors)andcytosolicproteinscontainsidechainscon-InfluenzavirusResponsibleforhost-cellattachmentandsistingofasingleGlcNAcattachedtoaserineorthreo-hemagglutininmembranefusionnineresidue(GlcNAc-Ser[Thr]).2+C-typelectinsCharacterizedbyaCa-dependentcarbo-hydraterecognitiondomain(CRD);in-cludesthemammalianasialoglycoproteinreceptor,theselectins,andthemannose-Table47–7.Threeplantlectinsandthesugarsbindingprotein1withwhichtheyinteract.S-typelectinsβ-Galactoside-bindinganimallectinswithrolesincell-cellandcell-matrixinterac-LectinAbbreviationSugarstionsConcanavalinAConAManandGlcP-typelectinsMannose6-PreceptorSoybeanlectinGalandGalNAcl-typelectinsMembersoftheimmunoglobulinsuper-WheatgermagglutininWGAGlcandNeuAcfamily,eg,sialoadhesinmediatingadhe-1Inmostcases,lectinsshowspecificityfortheanomericnatureofsionofmacrophagestovariouscellstheglycosidiclinkage(αorβ);thisisnotindicatedinthetable.
517GLYCOPROTEINS/519ACCH2OHNH2COOHαProteinHCHCOCH2COHGlycineHCCHSerEthanolamineHHNPCOMannoseCHEthanolaminePMannose3MannoseBCH2OHGlucosamineHCOHHOInositolβPI-PLCPAdditionalfattyacidCOHHCNCCH2CPlasmaHOCCAsnmembraneHHNCOCH3Figure47–1.Depictionsof(A)anO-linkage(N-acetylgalactosaminetoserine);(B)anN-linkage(N-acetylglu-cosaminetoasparagine)and(C)aglycosylphosphatidylinositol(GPI)linkage.TheGPIstructureshownisthatlinkingacetylcholinesterasetotheplasmamembraneofthehumanredbloodcell.ThecarboxylterminalaminoacidisglycinejoinedinamidelinkageviaitsCOOHgrouptotheNH2groupofphosphorylethanolamine,whichinturnisjoinedtoamannoseresidue.Thecoreglycancontainsthreemannoseandoneglucosamineresidues.Theglucosamineislinkedtoinositol,whichisattachedtophosphatidicacid.ThesiteofactionofPI-phospholi-paseC(PI-PLC)isindicated.Thestructureofthecoreglycanisshowninthetext.ThisparticularGPIcontainsanextrafattyacidattachedtoinositolandalsoanextraphosphorylethanolaminemoietyattachedtothemiddleofthethreemannoseresidues.VariationsfoundamongdifferentGPIstructuresincludetheidentityofthecarboxylterminalaminoacid,themoleculesattachedtothemannoseresidues,andtheprecisenatureofthelipidmoiety.MucinsHaveaHighContentofO-LinkedOligosaccharides&ExhibitRepeatingAAminoAcidSequencesα2,6NeuAcGalNAcSer(Thr)Mucinsareglycoproteinswithtwomajorcharacteris-tics:(1)ahighcontentofO-linkedoligosaccharides(thecarbohydratecontentofmucinsisgenerallymoreBthan50%);and(2)thepresenceofrepeatingaminoβ1,3GalGalNAcSer(Thr)acidsequences(tandemrepeats)inthecenteroftheirα2,3α2,6polypeptidebackbones,towhichtheO-glycanchainsNeuAcNeuAcareattachedinclusters(Figure47–3).Thesesequencesarerichinserine,threonine,andproline.AlthoughO-Figure47–2.StructuresoftwoO-linkedoligosac-glycanspredominate,mucinsoftencontainanumbercharidesfoundin(A)submaxillarymucinsand(B)fe-ofN-glycanchains.Bothsecretoryandmembrane-tuinandinthesialoglycoproteinofthemembraneofboundmucinsoccur.Theformerarefoundinthehumanredbloodcells.(Modifiedandreproduced,withmucuspresentinthesecretionsofthegastrointestinal,permission,fromLennarzWJ:TheBiochemistryofGlyco-respiratory,andreproductivetracts.MucusconsistsofproteinsandProteoglycans.PlenumPress,1980.)about94%waterand5%mucins,withtheremainder
518520/CHAPTER47O-glycanchainepitopeshavebeenusedtostimulateanimmunere-N-glycanchainsponseagainstcancercells.Thegenesencodingthepolypeptidebackbonesofanumberofmucinsderivedfromvarioustissues(eg,pancreas,smallintestine,tracheaandbronchi,stomach,andsalivaryglands)havebeenclonedandsequenced.NCThesestudieshaverevealednewinformationaboutthepolypeptidebackbonesofmucins(sizeoftandemre-peats,potentialsitesofN-glycosylation,etc)andulti-Tandemrepeatsequencematelyshouldrevealaspectsoftheirgeneticcontrol.SomeimportantpropertiesofmucinsaresummarizedFigure47–3.Schematicdiagramofamucin.O-gly-inTable47–8.canchainsareshownattachedtothecentralregionoftheextendedpolypeptidechainandN-glycanchainstoTheBiosynthesisofO-Linkedthecarboxylterminalregion.ThenarrowrectanglesGlycoproteinsUsesNucleotideSugarsrepresentaseriesoftandemrepeataminoacidse-quences.ManymucinscontaincysteineresidueswhoseThepolypeptidechainsofO-linkedandotherglyco-proteinsareencodedbymRNAspecies;becausemostSHgroupsforminterchainlinkages;thesearenotglycoproteinsaremembrane-boundorsecreted,theyshowninthefigure.(AdaptedfromStrousGJ,DekkerJ:aregenerallytranslatedonmembrane-boundpolyribo-Mucin-typeglycoproteins.CritRevBiochemMolBiolsomes(Chapter38).Hundredsofdifferentoligosaccha-1992;27:57.)ridechainsoftheO-glycosidictypeexist.Theseglyco-proteinsarebuiltupbythestepwisedonationofsugarsfromnucleotidesugars,suchasUDP-GalNAc,beingamixtureofvariouscellmolecules,electrolytes,UDP-Gal,andCMP-NeuAc.Theenzymescatalyzingandremnantsofcells.Secretorymucinsgenerallyhavethistypeofreactionaremembrane-boundglycopro-anoligomericstructureandthusoftenhaveaveryhighteinglycosyltransferases.Generally,synthesisofonemolecularmass.Theoligomersarecomposedofspecifictypeoflinkagerequirestheactivityofacorre-monomerslinkedbydisulfidebonds.Mucusexhibitsaspondinglyspecifictransferase.Thefactorsthatdeter-highviscosityandoftenformsagel.Thesequalitiesareminewhichspecificserineandthreonineresiduesarefunctionsofitscontentofmucins.ThehighcontentofglycosylatedhavenotbeenidentifiedbutareprobablyO-glycansconfersanextendedstructureonmucins.foundinthepeptidestructuresurroundingtheglycosy-Thisisinpartexplainedbystericinteractionsbetweenlationsite.TheenzymesassemblingO-linkedchainsaretheirGalNAcmoietiesandadjacentaminoacids,re-locatedintheGolgiapparatus,sequentiallyarrangedinsultinginachain-stiffeningeffectsothattheconforma-anassemblylinewithterminalreactionsoccurringintionsofmucinsoftenbecomethoseofrigidrods.Inter-thetrans-Golgicompartments.molecularnoncovalentinteractionsbetweenvariousThemajorfeaturesofthebiosynthesisofO-linkedsugarsonneighboringglycanchainscontributetogelglycoproteinsaresummarizedinTable47–9.formation.ThehighcontentofNeuAcandsulfateresiduesfoundinmanymucinsconfersanegativechargeonthem.Withregardtofunction,mucinshelplubricateandformaprotectivephysicalbarrieronTable47–8.Somepropertiesofmucins.epithelialsurfaces.Membrane-boundmucinspartici-pateinvariouscell-cellinteractions(eg,involvingse-•Foundinsecretionsofthegastrointestinal,respiratory,lectins;seebelow).Thedensityofoligosaccharideandreproductivetractsandalsoinmembranesofvariouschainsmakesitdifficultforproteasestoapproachtheircells.polypeptidebackbones,sothatmucinsareoftenresis-•ExhibithighcontentofO-glycanchains,usuallycontainingtanttotheiraction.Mucinsalsotendto“mask”certainNeuAc.surfaceantigens.Manycancercellsformexcessive•Containrepeatingaminoacidsequencesrichinserine,thre-amountsofmucins;perhapsthemucinsmaymaskcer-onine,andproline.tainsurfaceantigensonsuchcellsandthusprotectthe•Extendedstructurecontributestotheirhighvisco-cellsfromimmunesurveillance.Mucinsalsocarrycan-elasticity.cer-specificpeptideandcarbohydrateepitopes(anepi-•Formprotectivephysicalbarrieronepithelialsurfaces,aretopeisasiteonanantigenrecognizedbyanantibody,involvedincell-cellinteractions,andmaycontainormaskalsocalledanantigenicdeterminant).Someofthesecertainsurfaceantigens.
519GLYCOPROTEINS/521Table47–9.Summaryofmainfeaturespenta-antennarystructuresmayallbefound.Abewil-ofO-glycosylation.deringnumberofchainsofthecomplextypeexist,andthatindicatedinFigure47–4isonlyoneofmany.OthercomplexchainsmayterminateinGalorFuc.•Involvesabatteryofmembrane-boundglycoproteinglyco-High-mannoseoligosaccharidestypicallyhavetwotosyltransferasesactinginastepwisemanner;eachtrans-sixadditionalManresidueslinkedtothepentasaccha-feraseisgenerallyspecificforaparticulartypeoflinkage.•Theenzymesinvolvedarelocatedinvarioussubcompart-ridecore.HybridmoleculescontainfeaturesofbothofmentsoftheGolgiapparatus.thetwootherclasses.•Eachglycosylationreactioninvolvestheappropriatenucleotide-sugar.TheBiosynthesisofN-Linked•Dolichol-P-P-oligosaccharideisnotinvolved,noraregly-GlycoproteinsInvolvescosidases;andthereactionsarenotinhibitedbytuni-Dolichol-P-P-Oligosaccharidecamycin.•O-GlycosylationoccursposttranslationallyatcertainSerandLeloirandhiscolleaguesdescribedtheoccurrenceofThrresidues.adolichol-pyrophosphate-oligosaccharide(Dol-P-P-oligosaccharide),whichsubsequentresearchshowedtoplayakeyroleinthebiosynthesisofN-linkedglyco-N-LINKEDGLYCOPROTEINSCONTAINproteins.TheoligosaccharidechainofthiscompoundANAsn-GlcNAcLINKAGEgenerallyhasthestructureR-GlcNAc2Man9Glc3(R=Dol-P-P).Thesugarsofthiscompoundarefirstassem-N-Linkedglycoproteinsaredistinguishedbythepres-bledontheDol-P-Pbackbone,andtheoligosaccharideenceoftheAsn-GlcNAclinkage(Figure47–1).ItisthechainisthentransferredenbloctosuitableAsnresi-majorclassofglycoproteinsandhasbeenmuchstud-duesofacceptorapoglycoproteinsduringtheirsynthe-ied,sincethemostreadilyaccessibleglycoproteins(eg,sisonmembrane-boundpolyribosomes.AllN-glycansplasmaproteins)mainlybelongtothisgroup.Itin-haveacommonpentasaccharidecorestructure(Fig-cludesbothmembrane-boundandcirculatingglyco-ure47–5).proteins.TheprincipaldifferencebetweenthisandtheToformhigh-mannosechains,onlytheGlcpreviousclass,apartfromthenatureoftheaminoacidresiduespluscertainoftheperipheralManresiduesaretowhichtheoligosaccharidechainisattached(Asnver-removed.Toformanoligosaccharidechainofthecom-susSerorThr),concernstheirbiosynthesis.plextype,theGlcresiduesandfouroftheManresiduesareremovedbyglycosidasesintheendoplas-Complex,Hybrid,&High-MannosemicreticulumandGolgi.ThesugarscharacteristicofAretheThreeMajorClassescomplexchains(GlcNAc,Gal,NeuAc)areaddedbytheactionofindividualglycosyltransferaseslocatedinofN-LinkedOligosaccharidestheGolgiapparatus.ThephenomenonwherebytheTherearethreemajorclassesofN-linkedoligosaccha-glycanchainsofN-linkedglycoproteinsarefirstpar-rides:complex,hybrid,andhigh-mannose(Figuretiallydegradedandtheninsomecasesrebuiltisreferred47–4).Eachtypesharesacommonpentasaccharide,toasoligosaccharideprocessing.HybridchainsareMan3GlcNAc2—shownwithintheboxedareainFigureformedbypartialprocessing,formingcomplexchains47–4anddepictedalsoinFigure47–5—buttheydifferononearmandManstructuresontheotherarm.intheirouterbranches.ThepresenceofthecommonThus,theinitialstepsinvolvedinthebiosynthesisofpentasaccharideisexplainedbythefactthatallthreetheN-linkedglycoproteinsdiffermarkedlyfromthoseclassesshareaninitialcommonmechanismofbiosyn-involvedinthebiosynthesisoftheO-linkedglycopro-thesis.Glycoproteinsofthecomplextypegenerallyteins.TheformerinvolvesDol-P-P-oligosaccharide;thecontainterminalNeuAcresiduesandunderlyingGallatter,asdescribedearlier,doesnot.andGlcNAcresidues,thelatteroftenconstitutingtheTheprocessofN-glycosylationcanbebrokendowndisaccharideN-acetyllactosamine.RepeatingN-acetyl-intotwostages:(1)assemblyofDol-P-P-oligosaccha-lactosamineunits—[Galβ1–3/4GlcNAcβ1–3]n(poly-rideandtransferoftheoligosaccharide;and(2)pro-N-acetyllactosaminoglycans)—areoftenfoundonN-cessingoftheoligosaccharidechain.linkedglycanchains.I/ibloodgroupsubstancesbelongtothisclass.Themajorityofcomplex-typeoligosaccha-A.ASSEMBLY&TRANSFEROFridescontaintwo,three,orfourouterbranches(FigureDOLICHOL-P-P-OLIGOSACCHARIDE47–4),butstructurescontainingfivebrancheshavealsoPolyisoprenolcompoundsexistinbothbacteriaandbeendescribed.Theoligosaccharidebranchesareofteneukaryoticcells.Theyparticipateinthesynthesisofreferredtoasantennae,sothatbi-,tri-,tetra-,andbacterialpolysaccharidesandinthebiosynthesisofN-
520522/CHAPTER47SialicacidSialicacidα2,3or2,6α2,3or2,6GalGalGalManManManβ1,4β1,4β1,4α1,2α1,2α1,2GlcNAcGlcNAcGlcNAcManManManManManβ1,2β1,2β1,2α1,3α1,6α1,2α1,3α1,6ManManManManManManα1,6β1,4α1,3α1,6α1,3β1,4α1,3α1,6±GlcNAcManManGlcNAcManβ1,4β1,4β1,4GlcNAcGlcNAcGlcNAcβ1,4β1,4β1,4α1,6±FucGlcNAcGlcNAcGlcNAcAsnAsnAsnComplexHybridHigh-mannoseFigure47–4.Structuresofthemajortypesofasparagine-linkedoligosaccharides.Theboxedareaen-closesthepentasaccharidecorecommontoallN-linkedglycoproteins.(Reproduced,withpermission,fromKornfeldR,KornfeldS:Assemblyofasparagine-linkedoligosaccharides.AnnuRevBiochem1985;54:631.)linkedglycoproteinsandGPIanchors.Thepolyiso-inthemembranesoftheendoplasmicreticulumfromprenolusedineukaryotictissuesisdolichol,whichis,Dol-PandUDP-GlcNAcinthefollowingreaction,nexttorubber,thelongestnaturallyoccurringhydro-catalyzedbyGlcNAc-Ptransferase:carbonmadeupofasinglerepeatingunit.Dolicholiscomposedof17–20repeatingisoprenoidunits(FigureDol-P+UDP-GlcNAc→Dol-P-P-GlcNAc+UMP47–6).BeforeitparticipatesinthebiosynthesisofDol-P-P-oligosaccharide,dolicholmustfirstbephosphorylatedTheabovereaction—whichisthefirststepintheas-toformdolicholphosphate(Dol-P)inareactioncat-semblyofDol-P-P-oligosaccharide—andtheotherlateralyzedbydolicholkinaseandusingATPasthephos-reactionsaresummarizedinFigure47–7.Theessentialphatedonor.featuresofthesubsequentstepsintheassemblyofDol-Dolichol-P-P-GlcNAc(Dol-P-P-GlcNAc)istheP-P-oligosaccharideareasfollows:keylipidthatactsasanacceptorforothersugarsinthe(1)AsecondGlcNAcresidueisaddedtothefirst,assemblyofDol-P-P-oligosaccharide.ItissynthesizedagainusingUDP-GlcNAcasthedonor.(2)FiveManresiduesareadded,usingGDP-man-noseasthedonor.EndoglycosidaseFMan(3)FouradditionalManresiduesarenextadded,α1,6β1,4β1,4usingDol-P-Manasthedonor.Dol-P-ManisManGlcNAcGlcNAcAsnformedbythefollowingreaction:α1,3ManEndoglycosidaseHDolPGDPMan--+→+DolPManGDP--Figure47–5.Schematicdiagramofthepentasac-charidecorecommontoallN-linkedglycoproteinsand(4)Finally,thethreeperipheralglucoseresiduesaretowhichvariousouterchainsofoligosaccharidesmaydonatedbyDol-P-Glc,whichisformedinareac-beattached.ThesitesofactionofendoglycosidasesFtionanalogoustothatjustpresentedexceptthatandHarealsoindicated.Dol-PandUDP-Glcarethesubstrates.
521GLYCOPROTEINS/523Figure47–6.Thestructureofdolichol.HCH3CH3Thephosphateindolicholphosphateisat-tachedtotheprimaryalcoholgroupattheHOCH2CH2CCH2CH2CHCCH2CH2CHCCH3left-handendofthemolecule.ThegroupCH3withinthebracketsisanisopreneunit(n=n17–20isoprenoidunits).Itshouldbenotedthatthefirstsevensugars(twopreferencefortheDol-P-P-GlcNAc2Man9Glc3struc-GlcNAcandfiveManresidues)aredonatedbynu-ture.GlycosylationoccursattheAsnresidueofanAsn-cleotidesugars,whereasthelastsevensugars(fourManX-Ser/Thrtripeptidesequence,whereXisanyaminoandthreeGlcresidues)addedaredonatedbydolichol-acidexceptproline,asparticacid,orglutamicacid.AP-sugars.Thenetresultisassemblyofthecompoundtripeptidesitecontainedwithinaβturnisfavored.illustratedinFigure47–8andreferredtoinshorthandOnlyaboutone-thirdoftheAsnresiduesthatarepo-asDol-P-P-GlcNAc2Man9Glc3.tentialacceptorsitesareactuallyglycosylated,suggest-Theoligosaccharidelinkedtodolichol-P-Pistrans-ingthatfactorsotherthanthetripeptidearealsoim-ferredenbloctoformanN-glycosidicbondwithoneportant.TheacceptorproteinsareofboththesecretoryormorespecificAsnresiduesofanacceptorproteinandintegralmembraneclass.Cytosolicproteinsareemergingfromtheluminalsurfaceofthemembraneofrarelyglycosylated.Thetransferreactionandsubse-theendoplasmicreticulum.ThereactioniscatalyzedbyquentprocessesintheglycosylationofN-linkedglyco-oligosaccharide:proteintransferase,amembrane-proteins,alongwiththeirsubcellularlocations,arede-associatedenzymecomplex.Thetransferasewillrecog-pictedinFigure47–9.Theotherproductofthenizeandtransferanysubstratewiththegeneralstruc-oligosaccharide:proteintransferasereactionisdolichol-tureDol-P-P-(GlcNAc)2-R,butithasastrongP-P,whichissubsequentlyconvertedtodolichol-PbyaUDP-GIcNAcDol-PTunicamycinUMPMMGIcNAcPPDolMUDP-GIcNAcMMMP(GIcNAc)2PDolUDPGGGMMMPDolGIcNAcGIcNAcPPDolGDP-MMPDolandGPDolGDP(M)6(GIcNAc)2PPDolPDolMGIcNAcGIcNAcPPDolMMPDolMP(GIcNAc)2PDol(GDP-M)(GDP)MMM44Figure47–7.Pathwayofbiosynthesisofdolichol-P-P-oligosaccharide.Thespecificlinkagesformedareindi-catedinFigure47–8.NotethatthefirstfiveinternalmannoseresiduesaredonatedbyGDP-mannose,whereasthemoreexternalmannoseresiduesandtheglucoseresiduesaredonatedbydolichol-P-mannoseanddolichol-P-glucose.(UDP,uridinediphosphate;Dol,dolichol;P,phosphate;UMP,uridinemonophosphate;GDP,guanosinediphosphate;M,mannose;G,glucose.)
522524/CHAPTER47α1,2ManManα1,6Manα1,6α1,3α1,2ManManβ1,4β1,4αManGlcNAcGlcNAcPPDolicholα1,2α1,3α1,3α1,2α1,2α1,3GlcGlcGlcManManManFigure47–8.Structureofdolichol-P-P-oligosaccharide.(Reproduced,withpermission,fromLennarzWJ:TheBiochemistryofGlycoproteinsandProteoglycans.PlenumPress,1980.)phosphatase.Thedolichol-Pcanserveagainasanac-PHOSPHO-ceptorforthesynthesisofanothermoleculeofDol-P-DIESTERASEP-oligosaccharide.GlcNAc-1-P-6-ManProteinP-6-ManProtein+GlcNAcB.PROCESSINGOFTHEOLIGOSACCHARIDECHAIN1.Earlyphase—Thevariousreactionsinvolvedarein-Man6-Preceptors,locatedintheGolgiapparatus,dicatedinFigure47–9.Theoligosaccharide:proteinbindtheMan6-Presidueoftheseenzymesanddirecttransferasecatalyzesreaction1(seeabove).Reactions2themtothelysosomes.Fibroblastsfrompatientswithand3involvetheremovaloftheterminalGlcresidueI-celldisease(seebelow)areseverelydeficientinthebyglucosidaseIandofthenexttwoGlcresiduesbyactivityoftheGlcNAcphosphotransferase.glucosidaseII,respectively.Inthecaseofhigh-2.Latephase—Toassembleatypicalcomplexmannoseglycoproteins,theprocessmaystophere,oroligosaccharidechain,additionalsugarsmustbeaddeduptofourManresiduesmayalsoberemoved.How-tothestructureformedinreaction7.Hence,inreac-ever,toformcomplexchains,additionalstepsarenec-tion8,asecondGlcNAcisaddedtotheperipheralessary,asfollows.FourexternalManresiduesarere-Manresidueoftheotherarmofthebi-antennarystruc-movedinreactions4and5byatleasttwodifferenttureshowninFigure47–9;theenzymecatalyzingthismannosidases.Inreaction6,aGlcNAcresidueisaddedstepisGlcNActransferaseII.Reactions9,10,and11totheManresidueoftheManα1–3armbyGlcNAcinvolvetheadditionofFuc,Gal,andNeuAcresiduesattransferaseI.Theactionofthislatterenzymepermitsthesitesindicated,inreactionscatalyzedbyfucosyl,theoccurrenceofreaction7,areactioncatalyzedbyyetgalactosyl,andsialyltransferases,respectively.Theas-anothermannosidase(Golgiα-mannosidaseII)andsemblyofpoly-N-acetyllactosaminechainsrequiresad-whichresultsinareductionoftheManresiduestotheditionalGlcNActransferases.corenumberofthree(Figure47–5).Animportantadditionalpathwayisindicatedinre-TheEndoplasmicReticulumactionsIandIIofFigure47–9.Thisinvolvesenzymes&GolgiApparatusArethedestinedforlysosomes.Suchenzymesaretargetedtothelysosomesbyaspecificchemicalmarker.InreactionMajorSitesofGlycosylationI,aresidueofGlcNAc-1-Pisaddedtocarbon6ofoneAsindicatedinFigure47–9,theendoplasmicreticulumormorespecificManresiduesoftheseenzymes.TheandtheGolgiapparatusarethemajorsitesinvolvedinreactioniscatalyzedbyaGlcNAcphosphotransferase,glycosylationprocesses.TheassemblyofDol-P-P-whichusesUDP-GlcNAcasthedonorandgeneratesoligosaccharideoccursonboththecytoplasmicandlu-UMPastheotherproduct:minalsurfacesoftheERmembranes.Additionoftheoligosaccharidetoproteinoccursintheroughendo-GlcNAcplasmicreticulumduringoraftertranslation.RemovalPHOSPHO-TRANSFERASEoftheGlcandsomeoftheperipheralManresiduesalsoUDP-GlcNAc+ManProteinoccursintheendoplasmicreticulum.TheGolgiappa-GlcNAc-1-P-6-ManProtein+UMPratusiscomposedofcis,medial,andtranscisternae;thesecanbeseparatedbyappropriatecentrifugationInreactionII,theGlcNAcisremovedbytheactionofprocedures.Vesiclescontainingglycoproteinsappeartoaphosphodiesterase,leavingtheManresiduesphos-budoffintheendoplasmicreticulumandaretrans-phorylatedinthe6position:portedtothecisGolgi.Variousstudieshaveshown
523GLYCOPROTEINS/525ROUGHENDOPLASMICRETICULUM1234PPDolGOLGIAPPARATUSP––PII–P––P–I5cisUDP-6789medialUDP-UDP-GDP-1011ExittransUDP-CMP-Figure47–9.Schematicpathwayofoligosaccharideprocessing.Thereactionsarecat-alyzedbythefollowingenzymes:1,oligosaccharide:proteintransferase;2,α-glucosidaseI;3,α-glucosidaseII;4,endoplasmicreticulumα1,2-mannosidase;l,N-acetylglu-cosaminylphosphotransferase;ll,N-acetylglucosamine-1-phosphodiesterα-N-acetylglu-cosaminidase;5,Golgiapparatusα-mannosidaseI;6,N-acetylglucosaminyltransferaseI;7,Golgiapparatusα-mannosidaseII;8,N-acetylglucosaminyltransferaseII;9,fucosyltrans-ferase;10,galactosyltransferase;11,sialyltransferase.Thethickarrowsindicatevariousnu-cleotidesugarsinvolvedintheoveallscheme.(Solidsquare,N-acetylglucosamine;opencir-cle,mannose;solidtriangle,glucose;opentriangle,fucose;solidcircle,galactose;soliddiamond,sialicacid.)(Reproduced,withpermission,fromKornfeldR,KornfeldS:Assemblyofasparagine-linkedoligosaccharides.AnnuRevBiochem1985;54:631.)thattheenzymesinvolvedinglycoproteinprocessingthefucosyl,galactosyl,andsialyltransferases(catalyzingshowdifferentiallocationsinthecisternaeoftheGolgi.reactions9,10,and11)arelocatedmainlyinthetransAsindicatedinFigure47–9,Golgiα-mannosidaseIGolgi.ThemajorfeaturesofthebiosynthesisofN-(catalyzingreaction5)islocatedmainlyinthecislinkedglycoproteinsaresummarizedinTable47–10Golgi,whereasGlcNActransferaseI(catalyzingreac-andshouldbecontrastedwiththosepreviouslylistedtion6)appearstobelocatedinthemedialGolgi,and(Table47–9)forO-linkedglycoproteins.
524526/CHAPTER47Table47–10.SummaryofmainfeaturesofinginthelumenoftheER.Thesolubleproteincal-N-glycosylation.reticulinappearstoplayasimilarfunction.•TheoligosaccharideGlc3Man9(GIcNAc)2istransferredfromSeveralFactorsRegulatetheGlycosylationdolichol-P-P-oligosaccharideinareactioncatalyzedbyofGlycoproteinsoligosaccharide:proteintransferase,whichisinhibitedbyItisevidentthatglycosylationofglycoproteinsisatunicamycin.complexprocessinvolvingalargenumberofenzymes.•TransferoccurstospecificAsnresiduesinthesequenceOneindexofitscomplexityisthatmorethantendis-Asn-X-Ser/Thr,whereXisanyresidueexceptPro,Asp,ortinctGlcNActransferasesinvolvedinglycoproteinGlu.•Transfercanoccurcotranslationallyintheendoplasmicbiosynthesishavebeenreported,andmanyothersarereticulum.theoreticallypossible.Multiplespeciesoftheothergly-•Theprotein-boundoligosaccharideisthenpartiallycosyltransferases(eg,sialyltransferases)alsoexist.Con-processedbyglucosidasesandmannosidases;ifnoaddi-trollingfactorsofthefirststageofN-linkedglycopro-tionalsugarsareadded,thisresultsinahigh-mannoseteinbiosynthesis(ie,oligosaccharideassemblyandchain.transfer)include(1)thepresenceofsuitableacceptor•Ifprocessingoccursdowntothecoreheptasaccharidesitesinproteins,(2)thetissuelevelofDol-P,and(3)the(Man5[GlcNAc]2),complexchainsaresynthesizedbythead-activityoftheoligosaccharide:proteintransferase.ditionofGlcNAc,removaloftwoMan,andthestepwisead-SomefactorsknowntobeinvolvedintheregulationditionofindividualsugarsinreactionscatalyzedbyspecificofoligosaccharideprocessingarelistedinTabletransferases(eg,GlcNAc,Gal,NeuActransferases)thatem-47–11.Twoofthepointslistedmeritfurthercom-ployappropriatenucleotidesugars.ment.First,speciesvariationsamongprocessingen-zymeshaveassumedimportanceinrelationtoproduc-tionofglycoproteinsoftherapeuticusebymeansofrecombinantDNAtechnology.Forinstance,recombi-SomeGlycanIntermediatesnanterythropoietin(epoetinalfa;EPO)issometimesFormedDuringN-Glycosylationadministeredtopatientswithcertaintypesofchronicanemiainordertostimulateerythropoiesis.Thehalf-HaveSpecificFunctionslifeofEPOinplasmaisinfluencedbythenatureofitsThefollowingareanumberofspecificfunctionsofN-glycosylationpattern,withcertainpatternsbeingasso-glycanchainsthathavebeenestablishedorareunderciatedwithashorthalf-life,appreciablylimitingitspe-investigation.(1)Theinvolvementofthemannose6-Priodoftherapeuticeffectiveness.ItisthusimportanttosignalintargetingofcertainlysosomalenzymesisclearharvestEPOfromhostcellsthatconferapatternof(seeaboveanddiscussionofI-celldisease,below).(2)Itglycosylationconsistentwithanormalhalf-lifeinislikelythatthelargeN-glycanchainspresentonnewlyplasma.Second,thereisgreatinterestinanalysisofthesynthesizedglycoproteinsmayassistinkeepingtheseactivitiesofglycoprotein-processingenzymesinvariousproteinsinasolublestateinsidethelumenoftheendo-typesofcancercells.Thesecellshaveoftenbeenfoundplasmicreticulum.(3)OnespeciesofN-glycanchainstosynthesizedifferentoligosaccharidechains(eg,theyhasbeenshowntoplayaroleinthefoldingandreten-oftenexhibitgreaterbranching)fromthosemadeintionofcertainglycoproteinsinthelumenoftheendo-controlcells.Thiscouldbeduetocancercellscontain-plasmicreticulum.Calnexinisaproteinpresentintheingdifferentpatternsofglycosyltransferasesfromthoseendoplasmicreticulummembranethatactsasa“chap-exhibitedbycorrespondingnormalcells,duetospeci-erone”(Chapter46).Ithasbeenfoundthatcalnexinficgeneactivationorrepression.Thedifferencesinwillbindspecificallytoanumberofglycoproteins(eg,oligosaccharidechainscouldaffectadhesiveinteractionstheinfluenzavirushemagglutinin[HA])thatpossessthebetweencancercellsandtheirnormalparenttissuemonoglycosylatedcorestructure.Thisspeciesisthecells,contributingtometastasis.Ifacorrelationcouldproductofreaction2showninFigure47–9butfrombefoundbetweentheactivityofparticularprocessingwhichtheterminalglucoseresiduehasbeenremoved,enzymesandthemetastaticpropertiesofcancercells,leavingonlytheinnermostglucoseattached.There-thiscouldbeimportantasitmightpermitsynthesisofleaseoffullyfoldedHAfromcalnexinrequirestheen-drugstoinhibittheseenzymesand,secondarily,metas-zymaticremovalofthislastglucosylresiduebyα-glu-tasis.cosidaseII.Inthisway,calnexinretainscertainpartlyThegenesencodingmanyglycosyltransferaseshavefolded(ormisfolded)glycoproteinsandreleasesthemalreadybeencloned,andothersareunderstudy.whenproperfoldinghasoccurred;itisthusanimpor-Cloninghasrevealednewinformationonbothproteintantcomponentofthequalitycontrolsystemsoperat-andgenestructures.Thelattershouldalsocastlighton
525GLYCOPROTEINS/527Table47–11.SomefactorsaffectingtheactivitiesTable47–12.Threeinhibitorsofenzymesofglycoproteinprocessingenzymes.involvedintheglycosylationofglycoproteinsandtheirsitesofaction.FactorCommentCelltypeDifferentcelltypescontaindifferentpro-InhibitorSiteofActionfilesofprocessingenzymes.TunicamycinInhibitsGlcNAc-Ptransferase,theenzymePreviousenzymeCertainglycosyltransferaseswillonlyactcatalyzingadditionofGlcNActodolichol-onanoligosaccharidechainifithasal-P,thefirststepinthebiosynthesisofreadybeenacteduponbyanotherpro-oligosaccharide-P-P-dolichol1cessingenzyme.DeoxynojirimycinInhibitorofglucosidasesIandIIDevelopmentThecellularprofileofprocessingenzymesSwainsonineInhibitorofmannosidaseIImaychangeduringdevelopmentiftheirgenesareturnedonoroff.IntracellularForinstance,ifanenzymeisdestinedforlocationinsertionintothemembraneoftheERincreasethesusceptibilityoftheseproteinstoproteoly-(eg,HMG-CoAreductase),itmayneversis.InhibitionofglycosylationdoesnotappeartohaveencounterGolgi-locatedprocessingenzymes.aconsistenteffectuponthesecretionofglycoproteinsthatarenormallysecreted.Theinhibitorsofglycopro-ProteinDifferencesinconformationofdifferentteinprocessinglistedinTable47–12donotaffecttheconformationproteinsmayfacilitateorhinderaccessofbiosynthesisofO-linkedglycoproteins.Theextensionprocessingenzymestoidenticaloligosac-ofO-linkedchainscanbepreventedbyGalNAc-charidechains.benzyl.Thiscompoundcompeteswithnaturalglyco-SpeciesSamecells(eg,fibroblasts)fromdifferentproteinsubstratesandthuspreventschaingrowthbe-speciesmayexhibitdifferentpatternsofyondGalNAc.processingenzymes.CancerCancercellsmayexhibitprocessingen-SOMEPROTEINSAREANCHOREDzymesdifferentfromthoseofcorrespond-TOTHEPLASMAMEMBRANEingnormalcells.BYGLYCOSYLPHOSPHATIDYL-1Forexample,prioractionofGlcNActransferaseIisnecessaryforINOSITOLSTRUCTUREStheactionofGolgiα-mannosidaseII.Glycosylphosphatidylinositol(GPI)-linkedglycopro-teinscomprisethethirdmajorclassofglycoprotein.themechanismsinvolvedintheirtranscriptionalcon-TheGPIstructure(sometimescalleda“stickyfoot”)trol,andgeneknockoutstudiesarebeingusedtoevalu-involvedinlinkageoftheenzymeacetylcholinesteraseatethebiologicimportanceofvariousglycosyltrans-(AChesterase)totheplasmamembraneoftheredferases.bloodcellisshowninFigure47–1.GPI-linkedpro-teinsareanchoredtotheouterleafletoftheplasmamembranebythefattyacidsofphosphatidylinositolTunicamycinInhibits(PI).ThePIislinkedviaaGlcNH2moietytoaglycanN-butNotO-Glycosylationchainthatcontainsvarioussugars(eg,Man,GlcNH2).Inturn,theoligosaccharidechainislinkedviaphos-Anumberofcompoundsareknowntoinhibitvariousphorylethanolamineinanamidelinkagetothecar-reactionsinvolvedinglycoproteinprocessing.Tuni-boxylterminalaminoacidoftheattachedprotein.Thecamycin,deoxynojirimycin,andswainsoninearecoreofmostGPIstructurescontainsonemoleculeofthreesuchagents.Thereactionstheyinhibitareindi-phosphorylethanolamine,threeManresidues,onemol-catedinTable47–12.Theseagentscanbeusedexperi-eculeofGlcNH2,andonemoleculeofphosphatidyl-mentallytoinhibitvariousstagesofglycoproteininositol,asfollows:biosynthesisandtostudytheeffectsofspecificalter-ationsupontheprocess.Forinstance,ifcellsaregrownEthanolamine-phospho→→6Man1αinthepresenceoftunicamycin,noglycosylationoftheirnormallyN-linkedglycoproteinswilloccur.In2Man1ααα→→→6Man1GlcN1certaincases,lackofglycosylationhasbeenshownto6—myo-inositol-1-phospholipid
526528/CHAPTER47AdditionalconstituentsarefoundinmanyGPIstruc-text(eg,transportmolecules,immunologicmolecules,tures;forexample,thatshowninFigure47–1containsandhormones).Here,theirinvolvementintwospecificanextraphosphorylethanolamineattachedtothemid-processes—fertilizationandinflammation—willbedleofthethreeManmoietiesoftheglycanandanextrabrieflydescribed.Inaddition,thebasesofanumberoffattyacidattachedtoGlcNH2.Thefunctionalsignifi-diseasesthatareduetoabnormalitiesinthesynthesiscanceofthesevariationsamongstructuresisnotunder-anddegradationofglycoproteinswillbesummarized.stood.ThistypeoflinkagewasfirstdetectedbytheuseofbacterialPI-specificphospholipaseC(PI-PLC),GlycoproteinsAreImportantwhichwasfoundtoreleasecertainproteinsfromtheinFertilizationplasmamembraneofcellsbysplittingthebondindi-catedinFigure47–1.ExamplesofsomeproteinsthatToreachtheplasmamembraneofanoocyte,aspermareanchoredbythistypeoflinkagearegiveninTablehastotraversethezonapellucida(ZP),athick,trans-47–13.Atleastthreepossiblefunctionsofthistypeofparent,noncellularenvelopethatsurroundstheoocyte.linkagehavebeensuggested:(1)TheGPIanchormayThezonapellucidacontainsthreeglycoproteinsofinter-allowgreatlyenhancedmobilityofaproteinintheest,ZP1–3.OfparticularnoteisZP3,anO-linkedgly-plasmamembranecomparedwiththatobservedforacoproteinthatfunctionsasareceptorforthesperm.Aproteinthatcontainstransmembranesequences.Thisisproteinonthespermsurface,possiblygalactosyltrans-perhapsnotsurprising,astheGPIanchorisattachedferase,interactsspecificallywitholigosaccharidechainsofonlytotheouterleafletofthelipidbilayer,sothatitisZP3;inatleastcertainspecies(eg,themouse),thisinter-freertodiffusethanaproteinanchoredviabothleafletsaction,bytransmembranesignaling,inducestheacroso-ofthebilayer.Increasedmobilitymaybeimportantinfa-malreaction,inwhichenzymessuchasproteasesandcilitatingrapidresponsestoappropriatestimuli.(2)SomehyaluronidaseandothercontentsoftheacrosomeoftheGPIanchorsmayconnectwithsignaltransductionspermarereleased.Liberationoftheseenzymeshelpspathways.(3)IthasbeenshownthatGPIstructurescanthespermtopassthroughthezonapellucidaandreachtargetcertainproteinstoapicaldomainsoftheplasmatheplasmamembrane(PM)oftheoocyte.Inhamsters,membraneofcertainepithelialcells.Thebiosynthesisofithasbeenshownthatanotherglycoprotein,PH-30,isGPIanchorsiscomplexandbeginsintheendoplasmicimportantinboththebindingofthePMofthespermtoreticulum.TheGPIanchorisassembledindependentlythePMoftheoocyteandalsointhesubsequentfusionbyaseriesofenzyme-catalyzedreactionsandthentrans-ofthetwomembranes.Theseinteractionsenabletheferredtothecarboxylterminalendofitsacceptorpro-spermtoenterandthusfertilizetheoocyte.Itmaybetein,accompaniedbycleavageofthepreexistingcar-possibletoinhibitfertilizationbydevelopingdrugsorboxylterminalhydrophobicpeptidefromthatprotein.antibodiesthatinterferewiththenormalfunctionsofThisprocessissometimescalledglypiation.Anac-ZP3andPH-30andwhichwouldthusactascontracep-quireddefectinanearlystageofthebiosynthesisofthetiveagents.GPIstructurehasbeenimplicatedinthecausationofparoxysmalnocturnalhemoglobinuria(seebelow).SelectinsPlayKeyRolesinInflammation&inLymphocyteHomingGLYCOPROTEINSAREINVOLVEDINMANYBIOLOGICPROCESSESLeukocytesplayimportantrolesinmanyinflammatoryandimmunologicphenomena.Thefirststepsinmany&INMANYDISEASESofthesephenomenaareinteractionsbetweencirculat-AslistedinTable47–1,glycoproteinshavemanydif-ingleukocytesandendothelialcellspriortopassageofferentfunctions;somehavealreadybeenaddressedintheformeroutofthecirculation.Workdonetoiden-thischapterandothersaredescribedelsewhereinthistifyspecificmoleculesonthesurfacesofthecellsin-volvedinsuchinteractionshasrevealedthatleukocytesandendothelialcellscontainontheirsurfacesspecificlectins,calledselectins,thatparticipateintheirinter-Table47–13.SomeGPI-linkedproteins.cellularadhesion.FeaturesofthethreemajorclassesofselectinsaresummarizedinTable47–14.Selectinsare•Acetylcholinesterase(redcellmembrane)single-chainCa2+-bindingtransmembraneproteinsthat•Alkalinephosphatase(intestinal,placental)containanumberofdomains(Figure47–10).Their•Decay-acceleratingfactor(redcellmembrane)aminoterminalendscontainthelectindomain,which•5′-Nucleotidase(Tlymphocytes,othercells)isinvolvedinbindingtospecificcarbohydrateligands.•Thy-1antigen(brain,Tlymphocytes)Theadhesionofneutrophilstoendothelialcellsof•Variablesurfaceglycoprotein(Trypanosomabrucei)postcapillaryvenulescanbeconsideredtooccurinfour
527GLYCOPROTEINS/5291Table47–14.Somemoleculesinvolvedinleukocyte-endothelialcellinteractions.MoleculeCellLigandsSelectins2L-selectinPMN,lymphsCD34,Gly-CAM-1xSialyl-LewisandothersP-selectinEC,plateletsP-selectinglycoproteinligand-1(PSGL-1)xSialyl-LewisandothersxE-selectinECSialyl-LewisandothersIntegrinsLFA-1PMN,lymphsICAM-1,ICAM-2(CD11a/CD18)Mac-1PMNICAM-1andothers(CD11b/CD18)ImmunoglobulinsuperfamilyICAM-1Lymphs,ECLFA-1,Mac-1ICAM-2Lymphs,ECLFA-1PECAM-1EC,PMN,lymphsVariousplatelets1ModifiedfromAlbeldaSM,SmithCW,WardPA:Adhesionmoleculesandinflammatoryinjury.FASEBJ1994;8:504.2TheseareligandsforlymphocyteL-selectin;theligandsforneutrophilL-selectinhavenotbeenidentified.Key:PMN,polymorphonuclearleukocytes;EC,endothelialcell;lymphs,lymphocytes;CD,clusterofdifferentiation;ICAM,intercellularad-hesionmolecule;LFA-1,lymphocytefunction-associatedantigen-1;PECAM-1,plateletendothelialcelladhesioncellmolecule-1.stages,asshowninFigure47–11.Theinitialbaselinethisstage,activationoftheneutrophilsbyvariousstageissucceededbyslowingorrollingoftheneu-chemicalmediators(discussedbelow)occurs,resultingtrophils,mediatedbyselectins.Interactionsbetweeninachangeofshapeoftheneutrophilsandfirmadhe-L-selectinontheneutrophilsurfaceandCD34andsionofthesecellstotheendothelium.AnadditionalsetGlyCAM-1orotherglycoproteinsontheendothelialofadhesionmoleculesisinvolvedinfirmadhesion,surfaceareinvolved.Theseparticularinteractionsarenamely,LFA-1andMac-1ontheneutrophilsandinitiallyshort-lived,andtheoverallbindingisofrela-ICAM-1andICAM-2onendothelialcells.LFA-1andtivelylowaffinity,permittingrolling.However,duringMac-1areCD11/CD18integrins(seeChapter52foradiscussionofintegrins),whereasICAM-1andICAM-2aremembersoftheimmunoglobulinsuperfamily.TheL-selectinfourthstageistransmigrationoftheneutrophilsacrosstheendothelialwall.Forthistooccur,theneutrophilsNH2LectinEGF12COOHinsertpseudopodsintothejunctionsbetweenendothe-Figure47–10.Schematicdiagramofthestructurelialcells,squeezethroughthesejunctions,crossthebasementmembrane,andthenarefreetomigrateinofhumanL-selectin.Theextracellularportioncontainstheextravascularspace.Platelet-endothelialcelladhe-anaminoterminaldomainhomologoustoC-typesionmolecule-1(PECAM-1)hasbeenfoundtobelo-lectinsandanadjacentepidermalgrowthfactor-likecalizedatthejunctionsofendothelialcellsandthusdomain.Thesearefollowedbyavariablenumberofmayhavearoleintransmigration.Avarietyofbiomol-complementregulatory-likemodules(numberedcir-eculeshavebeenfoundtobeinvolvedinactivationofcles)andatransmembranesequence(blackdiamond).neutrophilandendothelialcells,includingtumorAshortcytoplasmicsequence(openrectangle)isatthenecrosisfactorα,variousinterleukins,plateletactivat-carboxylterminal.ThestructuresofP-andE-selectiningfactor(PAF),leukotrieneB4,andcertaincomple-aresimilartothatshownexceptthattheycontainmorementfragments.Thesecompoundsstimulatevariouscomplement-regulatorymodules.Thenumbersofsignalingpathways,resultinginchangesincellshapeaminoacidsinL-,P-,andE-selectins,asdeducedfromandfunction,andsomearealsochemotactic.Oneim-thecDNAsequences,are385,789,and589,respec-portantfunctionalchangeisrecruitmentofselectinstotively.(Reproduced,withpermission,fromBevilacquaMP,thecellsurface,asinsomecasesselectinsarestoredinNelsonRM:Selectins.JClinInvest1993;91:370.)granules(eg,inendothelialcellsandplatelets).
528530/CHAPTER47Alished.Sulfatedmolecules,suchasthesulfatides(Chap-ter14),maybeligandsincertaininstances.ThisbasicBaselineknowledgeisbeingusedinattemptstosynthesizecom-poundsthatblockselectin-ligandinteractionsandthusmayinhibittheinflammatoryresponse.Approachesin-cludeadministrationofspecificmonoclonalantibodiesBXorofchemicallysynthesizedanalogsofsialyl-Lewis,Rollingbothofwhichbindselectins.CancercellsoftenexhibitXsialyl-Lewisandotherselectinligandsontheirsur-faces.Itisthoughtthattheseligandsplayaroleintheinvasionandmetastasisofcancercells.CActivationandAbnormalitiesintheSynthesisoffirmadhesionGlycoproteinsUnderlieCertainDiseasesTable47–15listsanumberofconditionsinwhichab-Dnormalitiesinthesynthesisofglycoproteinsareofim-TransmigrationTable47–15.SomediseasesduetoorinvolvingFigure47–11.Schematicdiagramofneutrophil-abnormalitiesinthebiosynthesisofendothelialcellinteractions.A:Baselineconditions:glycoproteins.Neutrophilsdonotadheretothevesselwall.B:ThefirsteventistheslowingorrollingoftheneutrophilswithinDiseaseAbnormalitythevessel(venule)mediatedbyselectins.C:Activationoccurs,resultinginneutrophilsfirmlyadheringtotheCancerIncreasedbranchingofcellsurfacesurfacesofendothelialcellsandalsoassumingaflat-glycansorpresentationofselectinli-gandsmaybeimportantinmetastasis.tenedshape.ThisrequiresinteractionofactivatedCD18integrinsonneutrophilswithICAM-1ontheen-CongenitaldisordersSeeTable47–16.1dothelium.D:Theneutrophilsthenmigratethroughofglycosylationthejunctionsofendothelialcellsintotheinterstitialtis-HEMPAS2(MIMAbnormalitiesincertainenzymes(eg,sue;thisrequiresinvolvementofPECAM-1.Chemotaxis224100)mannosidaseIIandothers)involvedinisalsoinvolvedinthislatterstage.(Reproduced,withthebiosynthesisofN-glycans,particu-permission,fromAlbeldaSM,SmithCW,WardPA:Adhe-larlyaffectingtheredbloodcellmem-sionmoleculesandinflammatoryinjury.FASEBJbrane.1994;8;504.)LeukocyteadhesionProbablymutationsaffectingaGolgi-deficiency,typeIIlocatedGDP-fucosetransporter,re-(MIM266265)sultingindefectivefucosylation.TheprecisechemicalnatureofsomeoftheligandsParoxysmalnocturnalAcquireddefectinbiosynthesisoftheinvolvedinselectin-ligandinteractionshasbeendeter-hemoglobinuriaGPI3structuresofdecayacceleratingmined.Allthreeselectinsbindsialylatedandfucosy-(MIM311770)factor(DAF)andCD59.latedoligosaccharides,andinparticularallthreebindsialyl-LewisX(Figure47–12),astructurepresentonI-celldiseaseDeficiencyofGlcNAcphosphotrans-(MIM252500)ferase,resultinginabnormaltargetingbothglycoproteinsandglycolipids.Whetherthiscom-ofcertainlysosomalenzymes.poundistheactualligandinvolvedinvivoisnotestab-1TheMIMnumberforcongenitaldisorderofglycosylationtypeIais212065.2HereditaryerythroblasticmultinuclearitywithapositiveacidifiedNeuAcα23Galβ14GlcNAcserumlysistest(congenitaldyserythropoieticanemiatypeII).Thisα1–3isarelativelymildformofanemia.ItreflectsatleastinparttheFucpresenceintheredcellmembranesofvariousglycoproteinswithabnormalN-glycanchains,whichcontributetothesusceptibilityFigure47–12.Schematicrepresentationofthetolysis.X3Glycosylphosphatidylinositol.structureofsialyl-Lewis.
529GLYCOPROTEINS/531portance.Asmentionedabove,manycancercellsex-ofsomaticmutationsinthePIG-A(forphosphatidyl-hibitdifferentprofilesofoligosaccharidechainsoninositolglycanclassA)geneofcertainhematopoietictheirsurfaces,someofwhichmaycontributetometas-cells.Theproductofthisgeneappearstobetheen-tasis.Thecongenitaldisordersofglycosylationzymethatlinksglucosaminetophosphatidylinositolin(CDG)areagroupofdisordersofconsiderablecurrenttheGPIstructure(Figure47–1).Thus,proteinsthatinterest.ThemajorfeaturesoftheseconditionsareareanchoredbyaGPIlinkagearedeficientintheredsummarizedinTable47–16.Leukocyteadhesionde-cellmembrane.Twoproteinsareofparticularinterest:ficiency(LAD)IIisarareconditionprobablyduetodecayacceleratingfactor(DAF)andanotherproteinmutationsaffectingtheactivityofaGolgi-locateddesignatedCD59.TheynormallyinteractwithcertainGDP-fucosetransporter.Itcanbeconsideredacongen-componentsofthecomplementsystem(Chapter50)toitaldisorderofglycosylation.Theabsenceoffucosy-preventthehemolyticactionsofthelatter.However,latedligandsforselectinsleadstoamarkeddecreaseinwhentheyaredeficient,thecomplementsystemcanactneutrophilrolling.Subjectssufferlife-threatening,re-ontheredcellmembranetocausehemolysis.Paroxys-currentbacterialinfectionsandalsopsychomotorandmalnocturnalhemoglobinuriacanbediagnosedrela-mentalretardation.Theconditionappearstorespondtivelysimply,astheredcellsaremuchmoresensitivetotooralfucose.Hereditaryerythroblasticmultinuclear-hemolysisinnormalserumacidifiedtopH6.2(Ham’sitywithapositiveacidifiedlysistest(HEMPAS)—test);thecomplementsystemisactivatedunderthesecongenitaldyserythropoieticanemiatypeII—isan-conditions,butnormalcellsarenotaffected.Figureotherdisorderduetoabnormalitiesintheprocessingof47–13summarizestheetiologyofparoxysmalnoctur-N-glycans.Somecaseshavebeenclaimedtobeduetonalhemoglobinuria.defectsinalpha–mannosidaseII.I-celldiseaseisdis-cussedfurtherbelow.Paroxysmalnocturnalhemo-I-CellDiseaseResultsFromFaultyglobinuriaisanacquiredmildanemiacharacterizedbyTargetingofLysosomalEnzymesthepresenceofhemoglobininurineduetohemolysisofredcells,particularlyduringsleep.Thislatterphe-Asindicatedabove,Man6-PservesasachemicalnomenonmayreflectaslightdropinplasmapHdur-markertotargetcertainlysosomalenzymestothator-ingsleep,whichincreasessusceptibilitytolysisbytheganelle.Analysisofculturedfibroblastsderivedfromcomplementsystem(Chapter50).ThebasicdefectinpatientswithI-cell(inclusioncell)diseaseplayedalargeparoxysmalnocturnalhemoglobinuriaistheacquisitionpartinrevealingtheaboveroleofMan6-P.I-celldis-easeisanuncommonconditioncharacterizedbysevereprogressivepsychomotorretardationandavarietyofphysicalsigns,withdeathoftenoccurringinthefirstTable47–16.Majorfeaturesofthecongenitaldecade.CulturedcellsfrompatientswithI-celldiseasedisordersofglycosylation.werefoundtolackalmostallofthenormallysosomalenzymes;thelysosomesthusaccumulatemanydifferent•Autosomalrecessivedisorders•Multisystemdisordersthathaveprobablynotbeenrecog-nizedinthepast•Generallyaffectthecentralnervoussystem,resultinginAcquiredmutationsinthePIG-Agenepsychomotorretardationandotherfeaturesofcertainhematopoieticcells•TypeIdisordersareduetomutationsingenesencodingen-zymes(eg,phosphomannomutase-2[PMM-2],causingCDGDefectivesynthesisoftheGlcNH-PI2Ia)involvedinthesynthesisofdolichol-P-P-oligo-linkageofGPIanchorssaccharide•TypeIIdisordersareduetomutationsingenesencodingenzymes(eg,GlcNActransferase-2,causingCDGIIa)in-DecreasedamountsintheredbloodmembraneofvolvedintheprocessingofN-glycanchainsGPI-anchoredproteins,withdecayacceleratingfactor•About11distinctdisordershavebeenrecognized(DAF)andCD59beingofespecialimportance•Isoelectricfocusingoftransferrinisausefulbiochemicaltestforassistinginthediagnosisoftheseconditions;trun-CertaincomponentsofthecomplementsystemcationoftheoligosaccharidechainsofthisproteinaltersitsarenotopposedbyDAFandCD59,resultingisolectricfocusingpatternincomplement-mediatedlysisofredcells•OralmannosehasprovedofbenefitinthetreatmentofCDGIaFigure47–13.SchemeofcausationofparoxysmalKey:CDG,congenitaldisorderofglycosylation.nocturnalhemoglobinuria(MIM311770).
530532/CHAPTER47typesofundegradedmolecules,forminginclusionbod-MutationsinDNAies.Samplesofplasmafrompatientswiththediseasewereobservedtocontainveryhighactivitiesoflysoso-malenzymes;thissuggestedthattheenzymeswereMutantGlcNAcphosphotransferasebeingsynthesizedbutwerefailingtoreachtheirproperintracellulardestinationandwereinsteadbeingse-LackofnormaltransferofGlcNAc1-Pcreted.Culturedcellsfrompatientswiththediseasetospecificmannoseresiduesofcertainenzymeswerenotedtotakeupexogenouslyaddedlysosomalen-destinedforlysosomeszymesobtainedfromnormalsubjects,indicatingthatthecellscontainedanormalreceptorontheirsurfacesTheseenzymesconsequentlylackMan6-Pforendocyticuptakeoflysosomalenzymes.Inaddition,andaresecretedfromcells(eg,intotheplasma)thisfindingsuggestedthatlysosomalenzymesfrompa-ratherthantargetedtolysosomestientswithI-celldiseasemightlackarecognitionmarker.Furtherstudiesrevealedthatlysosomalen-zymesfromnormalindividualscarriedtheMan6-PLysosomesarethusdeficientincertainhydrolases,dorecognitionmarkerdescribedabove,whichinteractednotfunctionproperly,andaccumulatepartlydigestedwithaspecificintracellularprotein,theMan6-Precep-cellularmaterial,manifestingasinclusionbodiestor.CulturedcellsfrompatientswithI-celldiseasewereFigure47–14.SummaryofthecausationofI-cellthenfoundtobedeficientintheactivityofthecisdisease(MIM252500).Golgi-locatedGlcNAcphosphotransferase,explaininghowtheirlysosomalenzymesfailedtoacquiretheMan6-Pmarker.ItisnowknownthattherearetwoMan6-Preceptorproteins,oneofhigh(275kDa)andonecallyrecognizesandinteractswithlysosomalenzymes.oflow(46kDa)molecularmass.TheseproteinsareIthasbeenproposedthatthedefectinpseudo-Hurlerlectins,recognizingMan6-P.Theformeriscation-polydystrophyliesinthelatterdomain,andthereten-independentandalsobindsIGF-II(henceitisnamedtionofsomecatalyticactivityresultsinamildercondi-theMan6-P–IGF-IIreceptor),whereasthelatteristion.cation-dependentinsomespeciesanddoesnotbindIGF-II.ItappearsthatbothreceptorsfunctionintheGeneticDeficienciesofGlycoproteinintracellularsortingoflysosomalenzymesintoclathrin-LysosomalHydrolasesCauseDiseasescoatedvesicles,whichoccursinthetransGolgisubse-Suchasα-MannosidosisquenttosynthesisofMan6-PinthecisGolgi.ThesevesiclesthenleavetheGolgiandfusewithaprelysoso-Glycoproteins,likemostotherbiomolecules,undergomalcompartment.ThelowpHinthiscompartmentbothsynthesisanddegradation(ie,turnover).Degrada-causesthelysosomalenzymestodissociatefromtheirtionoftheoligosaccharidechainsofglycoproteinsin-receptorsandsubsequentlyenterintolysosomes.Thevolvesabatteryoflysosomalhydrolases,includingreceptorsarerecycledandreused.Onlythesmallerre-α-neuraminidase,β-galactosidase,β-hexosaminidase,ceptorfunctionsintheendocytosisofextracellularlyso-α-andβ-mannosidases,α-N-acetylgalactosaminidase,somalenzymes,whichisaminorpathwayforlysosomalα-fucosidase,endo-β-N-acetylglucosaminidase,andas-location.NotallcellsemploytheMan6-Preceptortopartylglucosaminidase.Thesitesofactionofthelasttargettheirlysosomalenzymes(eg,hepatocytesuseatwoenzymesareindicatedinthelegendtoFiguredifferentbutundefinedpathway);furthermore,notall47–5.Geneticallydetermineddefectsoftheactivitiesoflysosomalenzymesaretargetedbythismechanism.theseenzymescanoccur,resultinginabnormaldegra-Thus,biochemicalinvestigationsofI-celldiseasenotdationofglycoproteins.Theaccumulationintissuesofonlyledtoelucidationofitsbasisbutalsocontributedsuchabnormallydegradedglycoproteinscanleadtosignificantlytoknowledgeofhownewlysynthesizedvariousdiseases.Amongthebest-recognizedoftheseproteinsaretargetedtospecificorganelles,inthiscasediseasesaremannosidosis,fucosidosis,sialidosis,as-thelysosome.Figure47–14summarizesthecausationpartylglycosaminuria,andSchindlerdisease,duere-ofI-celldisease.spectivelytodeficienciesofα-mannosidase,α-fucosi-Pseudo-Hurlerpolydystrophyisanothergeneticdase,α-neuraminidase,aspartylglucosaminidase,anddiseasecloselyrelatedtoI-celldisease.Itisamilderα-N-acetyl-galactosaminidase.Thesediseases,whichcondition,andpatientsmaysurvivetoadulthood.arerelativelyuncommon,haveavarietyofmanifesta-StudieshaverevealedthattheGlcNAcphosphotrans-tions;someoftheirmajorfeaturesarelistedinTableferaseinvolvedinI-celldiseasehasseveraldomains,in-47–17.Thefactthatpatientsaffectedbythesedisor-cludingacatalyticdomainandadomainthatspecifi-dersallshowsignsreferabletothecentralnervoussys-
531GLYCOPROTEINS/533Table47–17.MajorfeaturesofsomediseasesItcanthusbindtheagalactosylIgGmolecules,which(eg,α-mannosidosis,β-mannosidosis,fucosidosis,subsequentlyactivatethecomplementsystem,con-sialidosis,aspartylglycosaminuria,andSchindlertributingtochronicinflammationinthesynovialmem-branesofjoints.Thisproteincanalsobindtheabovedisease)duetodeficienciesofglycoprotein1sugarswhentheyarepresentonthesurfacesofcertainhydrolases.bacteria,fungi,andviruses,preparingthesepathogensforopsonizationorfordestructionbythecomplement•Usuallyexhibitmentalretardationorotherneurologicab-system.Thisisanexampleofinnateimmunity,notin-normalities,andinsomedisorderscoarsefeaturesorvis-volvingimmunoglobulins.Deficiencyofthisproteininceromegaly(orboth)younginfants,duetomutation,rendersthemverysus-•Variationsinseverityfrommildtorapidlyprogressiveceptibletorecurrentinfections.•Autosomalrecessiveinheritance•Mayshowethnicdistribution(eg,aspartylglycosaminuriaisOtherdisordersinwhichglycoproteinshavebeencommoninFinland)implicatedincludehepatitisBandC,Creutzfeldt-•VacuolizationofcellsobservedbymicroscopyinsomeJakobdisease,anddiarrheasduetoanumberofbacter-disordersialenterotoxins.Itishopedthatbasicstudiesofglyco-•Presenceofabnormaldegradationproducts(eg,oligo-proteinsandotherglycoconjugates(ie,thefieldofsaccharidesthataccumulatebecauseoftheenzymeglycobiology)willleadtoeffectivetreatmentsfordis-deficiency)inurine,detectablebyTLCandcharacterizableeasesinwhichthesemoleculesareinvolved.Already,atbyGLC-MSleasttwodisordershavebeenfoundtorespondtooral•Definitivediagnosismadebyassayofappropriateenzyme,supplementsofsugars.oftenusingleukocytesThefantasticprogressmadeinrelationtothe•Possibilityofprenataldiagnosisbyappropriateenzymehumangenomehasstimulatedintenseinterestinbothassaysgenomicsandproteomics.Itisanticipatedthatthepace•Nodefinitivetreatmentatpresentofresearchinglycomics—characterizationoftheentire1MIMnumbers:α-mannosidosis,248500;β-mannosidosis,complementofsugarchainsfoundincells(thegly-248510;fucosidosis,230000;sialidosis,256550;aspartylgly-come)—willalsoacceleratemarkedly.Foranumberofcosaminuria,208400;Schindlerdisease,104170.reasons,thisfieldwillprovemorechallengingthanei-thergenomicsorproteomics.Thesereasonsincludethecomplexityofthestructuresofoligosaccharidechainsduetolinkagevariations—incontrasttothegenerallytemreflectstheimportanceofglycoproteinsinthede-uniformnatureofthelinkagesbetweennucleotidesandvelopmentandnormalfunctionofthatsystem.betweenaminoacids.Therearealsosignificantvaria-Fromtheabove,itshouldbeapparentthatglyco-tionsinoligosaccharidestructuresamongcellsandatproteinsareinvolvedinawidevarietyofbiologicdifferentstagesofdevelopment.Inaddition,nosimpleprocessesanddiseases.Glycoproteinsplaydirectorin-techniqueexistsforamplifyingoligosaccharides,com-directrolesinanumberofotherdiseases,asshowninparabletothePCRreaction.Despitetheseandotherthefollowingexamples.problems,itseemscertainthatresearchinthisareawilluncovermanynewimportantbiologicinteractionsthat(1)Theinfluenzaviruspossessesaneuraminidasearesugar-dependentandwillprovidetargetsfordrugthatplaysakeyroleinelutionofnewlysynthesizedandothertherapies.progenyfrominfectedcells.Ifthisprocessisinhibited,spreadofthevirusismarkedlydiminished.Inhibitorsofthisenzymearenowavailableforuseintreatingpa-SUMMARYtientswithinfluenza.•Glycoproteinsarewidelydistributedproteins—with(2)HIV-1,thoughtbymanytobethecausativediversefunctions—thatcontainoneormorecova-agentofAIDS,attachestocellsviaoneofitssurfacelentlylinkedcarbohydratechains.glycoproteins,gp120.(3)Rheumatoidarthritisisassociatedwithanal-•Thecarbohydratecomponentsofaglycoproteinterationintheglycosylationofcirculatingim-rangefrom1%tomorethan85%ofitsweightandmunoglobulin-γ(IgG)molecules(Chapter50),suchmaybesimpleorverycomplexinstructure.thattheylackgalactoseintheirFcregionsandtermi-•Atleastcertainoftheoligosaccharidechainsofglyco-nateinGlcNAc.Mannose-bindingprotein(nottobeproteinsencodebiologicinformation;theyarealsoconfusedwiththemannose-6-Preceptor),aC-lectinimportanttoglycoproteinsinmodulatingtheirsolu-synthesizedbylivercellsandsecretedintothecircula-bilityandviscosity,inprotectingthemagainstprote-tion,bindsmannose,GlcNAc,andcertainothersugars.olysis,andintheirbiologicactions.
532534/CHAPTER47•Thestructuresofmanyoligosaccharidechainscanbe•Developmentsinthenewfieldofglycomicsarelikelyelucidatedbygas-liquidchromatography,massspec-toprovidemuchnewinformationontherolesoftrometry,andhigh-resolutionNMRspectrometry.sugarsinhealthanddiseaseandalsoindicatetargets•Glycosidaseshydrolyzespecificlinkagesinoligosac-fordrugandothertypesoftherapies.charidesandareusedtoexploreboththestructuresandfunctionsofglycoproteins.•Lectinsarecarbohydrate-bindingproteinsinvolvedincelladhesionandotherbiologicprocesses.REFERENCES•ThemajorclassesofglycoproteinsareO-linked(in-BrockhausenI,KuhnsW:GlycoproteinsandHumanDisease.Chap-volvinganOHofserineorthreonine),N-linked(in-man&Hall,1997.volvingtheNoftheamidegroupofasparagine),andKornfeldR,KornfeldS:Assemblyofasparagine-linkedoligosaccha-glycosylphosphatidylinositol(GPI)-linked.rides.AnnuRevBiochem1985;54:631.•MucinsareaclassofO-linkedglycoproteinsthatareLehrmanMAOligosaccharide-basedinformationinendoplasmicdistributedonthesurfacesofepithelialcellsofthereticulumqualitycontrolandotherbiologicalsystems.JBiolrespiratory,gastrointestinal,andreproductivetracts.Chem.2001;276:8623.PerkelJM:Glycobiologygoestotheball.TheScientist2002;•TheGolgiapparatusplaysamajorroleinglycosyla-16:32.tionreactionsinvolvedinthebiosynthesisofglyco-RosemanS:Reflectionsonglycobiology.JBiolChem2001;276:proteins.41527.•TheoligosaccharidechainsofO-linkedglycoproteinsSchachterH:Theclinicalrelevanceofglycobiology.JClinInvestaresynthesizedbythestepwiseadditionofsugarsdo-2001;108:1579.natedbynucleotidesugarsinreactionscatalyzedbySchwartzNB,DomowiczM:Chondrodysplasiasduetoproteogly-individualspecificglycoproteinglycosyltransferases.candefects.Glycobiology2002;12:57R.•Incontrast,thebiosynthesisofN-linkedglycopro-Science2001;21(5512):2263.(Thisissuecontainsaspecialsectionteinsinvolvesaspecificdolichol-P-P-oligosaccharideentitledCarbohydratesandGlycobiology.Itcontainsarticlesonthesynthesis,structuraldetermination,andfunctionsofandvariousglycosidases.Dependingontheglycosi-sugar-containingmoleculesandtherolesofglycosylationindasesandprecursorproteinssynthesizedbyatissue,theimmunesystem).itcansynthesizecomplex,hybrid,orhigh-mannoseScriverCRetal(editors):TheMetabolicandMolecularBasesofIn-typesofN-linkedoligosaccharides.heritedDisease,8thed.McGraw-Hill,2001.(Variouschapters•Glycoproteinsareimplicatedinmanybiologicinthistextgivein-depthcoverageoftopicssuchasI-celldis-processes.Forinstance,theyhavebeenfoundtoplayeaseanddisordersofglycoproteindegradation.)keyrolesinfertilizationandinflammation.SpiroRG:Proteinglycosylation:nature,distribution,enzymaticformation,anddiseaseimplicationsofglycopeptidebonds.•AnumberofdiseasesinvolvingabnormalitiesintheGlycobiology2002;12:43R.synthesisanddegradationofglycoproteinshavebeenVarkiAetal(editors):EssentialsofGlycobiology.ColdSpringHar-recognized.GlycoproteinsarealsoinvolvedinmanyborLaboratoryPress,1999.otherdiseases,includinginfluenza,AIDS,andVestweberW,BlanksJE:Mechanismsthatregulatethefunctionofrheumatoidarthritis.theselectinsandtheirligands.PhysiolRev1999;79:181.
533TheExtracellularMatrix48RobertK.Murray,MD,PhD,&FrederickW.Keeley,PhDBIOMEDICALIMPORTANCEcollagen-likedomainsintheirstructures;theseproteinsaresometimesreferredtoas“noncollagencollagens.”MostmammaliancellsarelocatedintissueswheretheyTable48–1summarizesthetypesofcollagensfoundaresurroundedbyacomplexextracellularmatrixinhumantissues;thenomenclatureusedtodesignate(ECM)oftenreferredtoas“connectivetissue.”ThetypesofcollagenandtheirgenesisdescribedintheECMcontainsthreemajorclassesofbiomolecules:(1)footnote.thestructuralproteins,collagen,elastin,andfibrillin;The19typesofcollagenmentionedabovecanbe(2)certainspecializedproteinssuchasfibrillin,fi-subdividedintoanumberofclassesbasedprimarilyonbronectin,andlaminin;and(3)proteoglycans,whosethestructurestheyform(Table48–2).Inthischapter,chemicalnaturesaredescribedbelow.TheECMhasweshallbeprimarilyconcernedwiththefibril-formingbeenfoundtobeinvolvedinmanynormalandpatho-collagensIandII,themajorcollagensofskinandbonelogicprocesses—eg,itplaysimportantrolesindevelop-andofcartilage,respectively.However,mentionwillbement,ininflammatorystates,andinthespreadofcan-madeofsomeoftheothercollagens.cercells.InvolvementofcertaincomponentsoftheECMhasbeendocumentedinbothrheumatoidarthri-tisandosteoarthritis.Severaldiseases(eg,osteogenesisCOLLAGENTYPEIISCOMPOSEDimperfectaandanumberoftypesoftheEhlers-DanlosOFATRIPLEHELIXSTRUCTUREsyndrome)areduetogeneticdisturbancesofthesyn-&FORMSFIBRILSthesisofcollagen.Specificcomponentsofproteogly-cans(theglycosaminoglycans;GAGs)areaffectedinAllcollagentypeshaveatriplehelicalstructure.Inthegroupofgeneticdisordersknownasthemu-somecollagens,theentiremoleculeistriplehelical,copolysaccharidoses.ChangesoccurintheECMdur-whereasinothersthetriplehelixmayinvolveonlyaingtheagingprocess.Thischapterdescribesthebasicfractionofthestructure.MaturecollagentypeI,con-biochemistryofthethreemajorclassesofbiomoleculestainingapproximately1000aminoacids,belongstothefoundintheECMandillustratestheirbiomedicalsig-formertype;init,eachpolypeptidesubunitoralphanificance.Majorbiochemicalfeaturesoftwospecializedchainistwistedintoaleft-handedhelixofthreeformsofECM—boneandcartilage—andofanumberresiduesperturn(Figure48–1).Threeofthesealphaofdiseasesinvolvingthemarealsobrieflyconsidered.chainsarethenwoundintoaright-handedsuperhelix,formingarod-likemolecule1.4nmindiameterandCOLLAGENISTHEMOSTABUNDANTabout300nmlong.AstrikingcharacteristicofcollagenPROTEININTHEANIMALWORLDistheoccurrenceofglycineresiduesateverythirdposi-tionofthetriplehelicalportionofthealphachain.Collagen,themajorcomponentofmostconnectivetis-Thisisnecessarybecauseglycineistheonlyaminoacidsues,constitutesapproximately25%oftheproteinofsmallenoughtobeaccommodatedinthelimitedspacemammals.Itprovidesanextracellularframeworkforallavailabledownthecentralcoreofthetriplehelix.Thismetazoananimalsandexistsinvirtuallyeveryanimalrepeatingstructure,representedas(Gly-X-Y)n,isanab-tissue.Atleast19distincttypesofcollagenmadeupofsoluterequirementfortheformationofthetriplehelix.30distinctpolypeptidechains(eachencodedbyasepa-WhileXandYcanbeanyotheraminoacids,aboutrategene)havebeenidentifiedinhumantissues.Al-100oftheXpositionsareprolineandabout100ofthethoughseveralofthesearepresentonlyinsmallpro-Ypositionsarehydroxyproline.Prolineandhydroxy-portions,theymayplayimportantrolesindeterminingprolineconferrigidityonthecollagenmolecule.Hy-thephysicalpropertiesofspecifictissues.Inaddition,adroxyprolineisformedbytheposttranslationalhy-numberofproteins(eg,theC1qcomponentofthedroxylationofpeptide-boundprolineresiduescatalyzedcomplementsystem,pulmonarysurfactantproteinsbytheenzymeprolylhydroxylase,whosecofactorsareSP-AandSP-D)thatarenotclassifiedascollagenshaveascorbicacid(vitaminC)andα-ketoglutarate.Lysines535
534536/CHAPTER48Table48–1.Typesofcollagenandtheirgenes.1,2Table48–2.Classificationofcollagens,based1primarilyonthestructuresthattheyform.TypeGenesTissueClassTypeICOL1A1,COL1A2Mostconnectivetissues,includingboneFibril-formingI,II,III,V,andXIIICOL2A1Cartilage,vitreoushumorNetwork-likeIV,VIII,XIIICOL3A1Extensibleconnectivetissues2FACITsIX,XII,XIV,XVI,XIXsuchasskin,lung,andtheBeadedfilamentsVIvascularsystemAnchoringfibrilsVIIIVCOL4A1–COL4A6BasementmembranesTransmembranedomainXIII,XVIIVCOL5A1–COL5A3MinorcomponentintissuescontainingcollagenIOthersXV,XVIII1VICOL6A1–COL6A3MostconnectivetissuesBasedonProckopDJ,KivirrikkoKI:Collagens:molecularbiology,diseases,andpotentialsfortherapy.AnnuRevBiochemVIICOL7A1Anchoringfibrils1995;64:403.2VIIICOL8A1–COL8A2Endothelium,othertissuesFACITs=fibril-associatedcollagenswithinterruptedtriplehelices.IXCOL9A1–COL9A3TissuescontainingcollagenIIXCOL10A1HypertrophiccartilageXICOL11A1,COL11A2,TissuescontainingcollagenIICOL2A1XIICOL12A1TissuescontainingcollagenIXIIICOL13A1Manytissues67nmXIVCOL14A1TissuescontainingcollagenIFibrilXVCOL15A1ManytissuesXVICOL16A1ManytissuesXVIICOL17A1Skinhemidesmosomes300nmXVIIICOL18A1Manytissues(eg,liver,kidney)XIXCOL19A1RhabdomyosarcomacellsMolecule1AdaptedslightlyfromProckopDJ,KivirrikkoKI:Collagens:mole-cularbiology,diseases,andpotentialsfortherapy.AnnuRevBiochem1995;64:403.2ThetypesofcollagenaredesignatedbyRomannumerals.Con-stituentprocollagenchains,calledproαchains,arenumberedTriplehelix1.4nmusingArabicnumerals,followedbythecollagentypeinparen-theses.Forinstance,typeIprocollagenisassembledfromtwoproα1(I)andoneproα2(I)chain.Itisthusaheterotrimer,whereastype2procollagenisassembledfromthreeproα1(II)chainsandAlphachainisthusahomotrimer.Thecollagengenesarenamedaccordingtothecollagentype,writteninArabicnumeralsforthegenesym-bol,followedbyanAandthenumberoftheproαchainthattheyAminoacidsequence–Gly–X–Y–Gly–X–Y–Gly–X–Y–encode.Thus,theCOL1A1andCOL1A2genesencodetheα1andα2chainsoftypeIcollagen,respectively.Figure48–1.Molecularfeaturesofcollagenstruc-turefromprimarysequenceuptothefibril.(Slightlymodifiedandreproduced,withpermission,fromEyreDR:Collagen:Moleculardiversityinthebody’sproteinscaf-fold.Science1980;207:1315.Copyright©1980bytheAmericanAssociationfortheAdvancementofScience.)
535THEEXTRACELLULARMATRIX/537intheYpositionmayalsobeposttranslationallymodi-Table48–3.Orderandlocationofprocessingoffiedtohydroxylysinethroughtheactionoflysylhy-thefibrillarcollagenprecursor.droxylase,anenzymewithsimilarcofactors.SomeofthesehydroxylysinesmaybefurthermodifiedbytheIntracellularadditionofgalactoseorgalactosyl-glucosethroughan1.CleavageofsignalpeptideO-glycosidiclinkage,aglycosylationsitethatis2.Hydroxylationofprolylresiduesandsomelysyluniquetocollagen.residues;glycosylationofsomehydroxylysylresiduesCollagentypesthatformlongrod-likefibersintis-3.FormationofintrachainandinterchainS–Sbondsinex-suesareassembledbylateralassociationofthesetripletensionpeptideshelicalunitsintoa“quarterstaggered”alignmentsuch4.FormationoftriplehelixthateachisdisplacedlongitudinallyfromitsneighborExtracellularbyslightlylessthanone-quarterofitslength(Figure1.Cleavageofaminoandcarboxylterminalpropeptides48–1,upperpart).Thisarrangementisresponsiblefor2.Assemblyofcollagenfibersinquarter-staggeredalign-thebandedappearanceofthesefibersinconnectivetis-mentsues.Collagenfibersarefurtherstabilizedbytheforma-3.Oxidativedeaminationofε-aminogroupsoflysylandtionofcovalentcross-links,bothwithinandbetweenhydroxylysylresiduestoaldehydesthetriplehelicalunits.Thesecross-linksformthrough4.Formationofintra-andinterchaincross-linksviaSchifftheactionoflysyloxidase,acopper-dependenten-basesandaldolcondensationproductszymethatoxidativelydeaminatestheε-aminogroupsofcertainlysineandhydroxylysineresidues,yieldingreac-tivealdehydes.Suchaldehydescanformaldolconden-sationproductswithotherlysine-orhydroxylysine-polypeptideextensions(extensionpeptides)of20–35derivedaldehydesorformSchiffbaseswiththekDaatbothitsaminoandcarboxylterminalends,nei-ε-aminogroupsofunoxidizedlysinesorhydroxy-therofwhichispresentinmaturecollagen.Bothexten-lysines.Thesereactions,afterfurtherchemicalre-sionpeptidescontaincysteineresidues.Whilethearrangements,resultinthestablecovalentcross-linksaminoterminalpropeptideformsonlyintrachaindisul-thatareimportantforthetensilestrengthofthefibers.fidebonds,thecarboxylterminalpropeptidesformHistidinemayalsobeinvolvedincertaincross-links.bothintrachainandinterchaindisulfidebonds.Forma-Severalcollagentypesdonotformfibrilsintissuestionofthesedisulfidebondsassistsintheregistrationof(Table48–2).Theyarecharacterizedbyinterruptionsthethreecollagenmoleculestoformthetriplehelix,ofthetriplehelixwithstretchesofproteinlackingGly-windingfromthecarboxylterminalend.Afterforma-X-Yrepeatsequences.Thesenon-Gly-X-Ysequencestionofthetriplehelix,nofurtherhydroxylationofpro-resultinareasofglobularstructureinterspersedinthelineorlysineorglycosylationofhydroxylysinescantriplehelicalstructure.takeplace.Self-assemblyisacardinalprincipleintheTypeIVcollagen,thebest-characterizedexampleofbiosynthesisofcollagen.acollagenwithdiscontinuoustriplehelices,isanim-Followingsecretionfromthecellbywayoftheportantcomponentofbasementmembranes,whereitGolgiapparatus,extracellularenzymescalledprocolla-formsamesh-likenetwork.genaminoproteinaseandprocollagencarboxypro-teinaseremovetheextensionpeptidesattheaminoandCollagenUndergoesExtensivecarboxylterminalends,respectively.CleavageofthesepropeptidesmayoccurwithincryptsorfoldsinthecellPosttranslationalModificationsmembrane.Oncethepropeptidesareremoved,theNewlysynthesizedcollagenundergoesextensivepost-triplehelicalcollagenmolecules,containingapproxi-translationalmodificationbeforebecomingpartofamately1000aminoacidsperchain,spontaneouslyas-matureextracellularcollagenfiber(Table48–3).Likesembleintocollagenfibers.Thesearefurtherstabilizedmostsecretedproteins,collagenissynthesizedonribo-bytheformationofinter-andintrachaincross-linkssomesinaprecursorform,preprocollagen,whichcon-throughtheactionoflysyloxidase,asdescribedprevi-tainsaleaderorsignalsequencethatdirectstheously.polypeptidechainintothelumenoftheendoplasmicThesamecellsthatsecretecollagenalsosecretefi-reticulum.Asitenterstheendoplasmicreticulum,thisbronectin,alargeglycoproteinpresentoncellsurfaces,leadersequenceisenzymaticallyremoved.Hydroxyla-intheextracellularmatrix,andinblood(seebelow).Fi-tionofprolineandlysineresiduesandglycosylationofbronectinbindstoaggregatingprecollagenfibersandhydroxylysinesintheprocollagenmoleculealsotakealtersthekineticsoffiberformationinthepericellularplaceatthissite.Theprocollagenmoleculecontainsmatrix.Associatedwithfibronectinandprocollagenin
536538/CHAPTER48thismatrixaretheproteoglycansheparansulfateandTable48–4.Diseasescausedbymutationsinchondroitinsulfate(seebelow).Infact,typeIXcolla-collagengenesorbydeficienciesintheactivitiesgen,aminorcollagentypefromcartilage,containsat-ofposttranslationalenzymesinvolvedinthetachedproteoglycanchains.Suchinteractionsmay1biosynthesisofcollagen.servetoregulatetheformationofcollagenfibersandtodeterminetheirorientationintissues.2Onceformed,collagenisrelativelymetabolicallysta-GeneorEnzymeDiseaseble.However,itsbreakdownisincreasedduringstarva-COL1A1,COL1A2Osteogenesisimperfecta,type13(MIMtionandvariousinflammatorystates.Excessiveproduc-1566200)tionofcollagenoccursinanumberofconditions,eg,4Osteoporosis(MIM166710)hepaticcirrhosis.Ehlers-DanlossyndrometypeVIIauto-somaldominant(130060)ANumberofGeneticDiseasesResultFromCOL2A1Severechondrodysplasias4AbnormalitiesintheSynthesisofCollagenOsteoarthritis(MIM120140)About30genesencodecollagen,anditspathwayofCOL3A1Ehlers-DanlossyndrometypeIV(MIMbiosynthesisiscomplex,involvingatleasteighten-130050)zyme-catalyzedposttranslationalsteps.Thus,itisnotCOL4A3–COL4A6Alportsyndrome(includingbothauto-surprisingthatanumberofdiseases(Table48–4)aresomalandX-linkedforms)(MIM104200)duetomutationsincollagengenesoringenesen-COL7A1Epidermolysisbullosa,dystrophic(MIMcodingsomeoftheenzymesinvolvedinthesepost-131750)translationalmodifications.Thediseasesaffectingbone(eg,osteogenesisimperfecta)andcartilage(eg,theCOL10A1Schmidmetaphysialchondrodysplasiachondrodysplasias)willbediscussedlaterinthischap-(MIM156500)ter.LysylhydroxylaseEhlers-DanlossyndrometypeVI(MIMEhlers-Danlossyndromecomprisesagroupofin-225400)heriteddisorderswhoseprincipalclinicalfeaturesareProcollagenEhlers-DanlossyndrometypeVIIauto-hyperextensibilityoftheskin,abnormaltissuefragility,N-proteinasesomalrecessive(MIM225410)andincreasedjointmobility.Theclinicalpictureis5variable,reflectingunderlyingextensivegenetichetero-LysylhydroxylaseMenkesdisease(MIM309400)geneity.Atleast10typeshavebeenrecognized,most1AdaptedfromProckopDJ,KivirrikkoKI:Collagens:molecularbi-butnotallofwhichreflectavarietyoflesionsintheology,diseases,andpotentialsfortherapy.AnnuRevBiochemsynthesisofcollagen.TypeIVisthemostseriousbe-1995;64:403.causeofitstendencyforspontaneousruptureofarteries2Geneticlinkagetocollagengeneshasbeenshownforafeworthebowel,reflectingabnormalitiesintypeIIIcolla-otherconditionsnotlistedhere.3gen.PatientswithtypeVI,duetoadeficiencyoflysylAtleastfourtypesofosteogenesisimperfectaarerecognized;hydroxylase,exhibitmarkedjointhypermobilityandathegreatmajorityofmutationsinalltypesareintheCOL1A1andtendencytoocularrupture.AdeficiencyofprocollagenCOL1A2genes.4AtpresentappliestoonlyarelativelysmallnumberofsuchN-proteinase,causingformationofabnormalthin,ir-patients.regularcollagenfibrils,resultsintypeVIIC,manifested5Secondarytoadeficiencyofcopper(Chapter50).bymarkedjointhypermobilityandsoftskin.Alportsyndromeisthedesignationappliedtoanumberofgeneticdisorders(bothX-linkedandautoso-mal)affectingthestructureoftypeIVcollagenfibers,duetomutationsinCOL7A1,affectingthestructureofthemajorcollagenfoundinthebasementmembranestypeVIIcollagen.Thiscollagenformsdelicatefibrilsoftherenalglomeruli(seediscussionoflaminin,thatanchorthebasallaminatocollagenfibrilsinthebelow).MutationsinseveralgenesencodingtypeIVdermis.Theseanchoringfibrilshavebeenshowntobecollagenfibershavebeendemonstrated.Thepresentingmarkedlyreducedinthisformofthedisease,probablysignishematuria,andpatientsmayeventuallydevelopresultingintheblistering.Epidermolysisbullosasim-end-stagerenaldisease.Electronmicroscopyrevealsplex,anothervariant,isduetomutationsinkeratin5characteristicabnormalitiesofthestructureofthebase-(Chapter49).mentmembraneandlaminadensa.Scurvyaffectsthestructureofcollagen.However,itInepidermolysisbullosa,theskinbreaksandblis-isduetoadeficiencyofascorbicacid(Chapter45)andtersasaresultofminortrauma.Thedystrophicformisisnotageneticdisease.Itsmajorsignsarebleeding
537THEEXTRACELLULARMATRIX/539gums,subcutaneoushemorrhages,andpoorwoundTable48–5.Majordifferencesbetweencollagenhealing.Thesesignsreflectimpairedsynthesisofcolla-andelastin.genduetodeficienciesofprolylandlysylhydroxylases,bothofwhichrequireascorbicacidasacofactor.CollagenElastinELASTINCONFERSEXTENSIBILITY1.ManydifferentgeneticOnegenetictypetypes&RECOILONLUNG,BLOOD2.TriplehelixNotriplehelix;randomcoilVESSELS,&LIGAMENTSconformationspermittingstretchingElastinisaconnectivetissueproteinthatisresponsible3.(Gly-X-Y)nrepeatingNo(Gly-X-Y)nrepeatingforpropertiesofextensibilityandelasticrecoilintis-structurestructuresues.Althoughnotaswidespreadascollagen,elastinis4.PresenceofhydroxylysineNohydroxylysinepresentinlargeamounts,particularlyintissuesthatre-5.Carbohydrate-containingNocarbohydratequirethesephysicalproperties,eg,lung,largearterial6.IntramolecularaldolIntramoleculardesmosinebloodvessels,andsomeelasticligaments.Smallerquan-cross-linkscross-linkstitiesofelastinarealsofoundinskin,earcartilage,and7.PresenceofextensionNoextensionpeptidespresentseveralothertissues.Incontrasttocollagen,thereap-peptidesduringbio-duringbiosynthesispearstobeonlyonegenetictypeofelastin,althoughsynthesisvariantsarisebyalternativesplicing(Chapter37)ofthehnRNAforelastin.Elastinissynthesizedasasolublemonomerof70kDacalledtropoelastin.Someoftheprolinesoftropoelastinarehydroxylatedtohydroxy-MARFANSYNDROMEISDUETOprolinebyprolylhydroxylase,thoughhydroxylysineMUTATIONSINTHEGENEFORFIBRILLIN,andglycosylatedhydroxylysinearenotpresent.UnlikeAPROTEINPRESENTINMICROFIBRILScollagen,tropoelastinisnotsynthesizedinapro-formwithextensionpeptides.Furthermore,elastindoesnotMarfansyndromeisarelativelyprevalentinheriteddis-containrepeatGly-X-Ysequences,triplehelicalstruc-easeaffectingconnectivetissue;itisinheritedasanau-ture,orcarbohydratemoieties.tosomaldominanttrait.Itaffectstheeyes(eg,causingAftersecretionfromthecell,certainlysylresiduesofdislocationofthelens,knownasectopialentis),thetropoelastinareoxidativelydeaminatedtoaldehydesbyskeletalsystem(mostpatientsaretallandexhibitlonglysyloxidase,thesameenzymeinvolvedinthisprocessdigits[arachnodactyly]andhyperextensibilityoftheincollagen.However,themajorcross-linksformedinjoints),andthecardiovascularsystem(eg,causingelastinarethedesmosines,whichresultfromthecon-weaknessoftheaorticmedia,leadingtodilationofthedensationofthreeoftheselysine-derivedaldehydeswithascendingaorta).AbrahamLincolnmayhavehadthisanunmodifiedlysinetoformatetrafunctionalcross-condition.Mostcasesarecausedbymutationsinthelinkuniquetoelastin.Oncecross-linkedinitsmature,gene(onchromosome15)forfibrillin;missensemuta-extracellularform,elastinishighlyinsolubleandex-tionshavebeendetectedinseveralpatientswithMar-tremelystableandhasaverylowturnoverrate.Elastinfansyndrome.exhibitsavarietyofrandomcoilconformationsthatper-Fibrillinisalargeglycoprotein(about350kDa)mittheproteintostretchandsubsequentlyrecoilduringthatisastructuralcomponentofmicrofibrils,10-totheperformanceofitsphysiologicfunctions.12-nmfibersfoundinmanytissues.FibrillinissecretedTable48–5summarizesthemaindifferencesbe-(subsequenttoaproteolyticcleavage)intotheextracel-tweencollagenandelastin.lularmatrixbyfibroblastsandbecomesincorporatedDeletionsintheelastingene(locatedat7q11.23)intotheinsolublemicrofibrils,whichappeartoprovidehavebeenfoundinapproximately90%ofsubjectswithascaffoldfordepositionofelastin.OfspecialrelevanceWilliamssyndrome,adevelopmentaldisorderaffect-toMarfansyndrome,fibrillinisfoundinthezonularingconnectivetissueandthecentralnervoussystem.fibersofthelens,intheperiosteum,andassociatedThemutations,byaffectingsynthesisofelastin,proba-withelastinfibersintheaorta(andelsewhere);theselo-blyplayacausativeroleinthesupravalvularaorticcationsrespectivelyexplaintheectopialentis,arach-stenosisoftenfoundinthiscondition.Anumberofnodactyly,andcardiovascularproblemsfoundintheskindiseases(eg,scleroderma)areassociatedwithaccu-syndrome.Otherproteins(eg,emelinandtwomi-mulationofelastin.Fragmentationor,alternatively,acrofibril-associatedproteins)arealsopresentinthesedecreaseofelastinisfoundinconditionssuchaspul-microfibrils,anditappearslikelythatabnormalitiesofmonaryemphysema,cutislaxa,andagingoftheskin.themmaycauseotherconnectivetissuedisorders.An-
538540/CHAPTER48othergeneforfibrillinexistsonchromosome5;muta-ECM,withatypicalcell(eg,fibroblast)presentinthetionsinthisgenearelinkedtocausationofcongenitalmatrix.contracturalarachnodactylybutnottoMarfansyn-Thefibronectinreceptorinteractsindirectlywithdrome.Theprobablesequenceofeventsleadingtoactinmicrofilaments(Chapter49)presentinthecy-MarfansyndromeissummarizedinFigure48–2.tosol(Figure48–5).Anumberofproteins,collectivelyknownasattachmentproteins,areinvolved;thesein-FIBRONECTINISANIMPORTANTcludetalin,vinculin,anactin-filamentcappingprotein,GLYCOPROTEININVOLVEDINCELLandα-actinin.Talininteractswiththereceptorandvinculin,whereasthelattertwointeractwithactin.TheADHESION&MIGRATIONinteractionoffibronectinwithitsreceptorprovidesoneFibronectinisamajorglycoproteinoftheextracellularroutewherebytheexteriorofthecellcancommunicatematrix,alsofoundinasolubleforminplasma.Itcon-withtheinteriorandthusaffectcellbehavior.Viathesistsoftwoidenticalsubunits,eachofabout230kDa,interactionwithitscellreceptor,fibronectinplaysanjoinedbytwodisulfidebridgesneartheircarboxylter-importantroleintheadhesionofcellstotheECM.Itisminals.Thegeneencodingfibronectinisverylarge,alsoinvolvedincellmigrationbyprovidingabindingcontainingsome50exons;theRNAproducedbyitssiteforcellsandthushelpingthemtosteertheirwaytranscriptionissubjecttoconsiderablealternativesplic-throughtheECM.Theamountoffibronectinarounding,andasmanyas20differentmRNAshavebeende-manytransformedcellsissharplyreduced,partlyex-tectedinvarioustissues.FibronectincontainsthreeplainingtheirfaultyinteractionwiththeECM.typesofrepeatingmotifs(I,II,andIII),whichareorga-nizedintofunctionaldomains(atleastseven);func-tionsofthesedomainsincludebindingheparin(seeLAMININISAMAJORPROTEINbelow)andfibrin,collagen,DNA,andcellsurfacesCOMPONENTOFRENALGLOMERULAR(Figure48–3).Theaminoacidsequenceofthefi-&OTHERBASALLAMINASbronectinreceptoroffibroblastshasbeenderived,andtheproteinisamemberofthetransmembraneintegrinBasallaminasarespecializedareasoftheECMthatsur-classofproteins(Chapter51).Theintegrinsarehet-roundepithelialandsomeothercells(eg,musclecells);erodimers,containingvarioustypesofαandβherewediscussonlythelaminasfoundintherenalpolypeptidechains.FibronectincontainsanArg-Gly-glomerulus.Inthatstructure,thebasallaminaiscon-Asp(RGD)sequencethatbindstothereceptor.Thetributedbytwoseparatesheetsofcells(oneendothelialRGDsequenceissharedbyanumberofotherproteinsandoneepithelial),eachdisposedonoppositesidesofpresentintheECMthatbindtointegrinspresentinthelamina;thesethreelayersmakeuptheglomerularcellsurfaces.SyntheticpeptidescontainingtheRGDmembrane.Theprimarycomponentsofthebasallam-sequenceinhibitthebindingoffibronectintocellsur-inaarethreeproteins—laminin,entactin,andtypeIVfaces.Figure48–4illustratestheinteractionofcollagen,collagen—andtheGAGheparinorheparansulfate.fibronectin,andlaminin,allmajorproteinsoftheThesecomponentsaresynthesizedbytheunderlyingcells.Laminin(about850kDa,70nmlong)consistsofthreedistinctelongatedpolypeptidechains(A,B1,andMutationsingene(onchromosome15)B2)linkedtogethertoformanelongatedcruciformforfibrillin,alargeglycoproteinpresentinshape.IthasbindingsitesfortypeIVcollagen,heparin,elastin-associatedmicrofibrilsandintegrinsoncellsurfaces.Thecollageninteractswithlaminin(ratherthandirectlywiththecellsurface),Abnormalitiesofthestructureoffibrillinwhichinturninteractswithintegrinsorotherlamininreceptorproteins,thusanchoringthelaminatothecells.Entactin,alsoknownas“nidogen,”isaglycopro-Structuresofthesuspensoryligamentoftheeye,teincontaininganRGDsequence;itbindstolaminintheperiosteum,andthemediaoftheaortaaffectedandisamajorcellattachmentfactor.Therelativelythickbasallaminaoftherenalglomerulushasanim-Ectopialentis,arachnodactyly,portantroleinglomerularfiltration,regulatingtheanddilationoftheascendingaortapassageoflargemolecules(mostplasmaproteins)acrosstheglomerulusintotherenaltubule.TheglomerularFigure48–2.Probablesequenceofeventsinthemembraneallowssmallmolecules,suchasinulin(5.2causationofthemajorsignsexhibitedbypatientswithkDa),topassthroughaseasilyaswater.OntheotherMarfansyndrome(MIM154700).hand,onlyasmallamountoftheproteinalbumin(69
539THEEXTRACELLULARMATRIX/541RGDHeparinACollagenDNACellAHeparinBCellBFibrinBFibrinAFigure48–3.Schematicrepresentationoffibronectin.Sevenfunctionaldomainsoffibronectinarerepresented;twodifferenttypesofdomainforheparin,cell-binding,andfibrinareshown.Thedomainsarecomposedofvariouscombinationsofthreestructuralmotifs(I,II,andIII),notdepictedinthefigure.Alsonotshownisthefactthatfibronectinisadimerjoinedbydisulfidebridgesnearthecarboxylterminalsofthemonomers.Theap-proximatelocationoftheRGDsequenceoffibronectin,whichinteractswithavarietyoffibronectinintegrinreceptorsoncellsurfaces,isindicatedbythearrow.(RedrawnafterYamadaKM:Adhesiverecognitionsequences.JBiolChem1991;266:12809.)kDa),themajorplasmaprotein,passesthroughthenormalglomerulus.Thisisexplainedbytwosetsoffacts:(1)Theporesintheglomerularmembranearelargeenoughtoallowmoleculesuptoabout8nmtopassthrough.(2)Albuminissmallerthanthisporesize,butitispreventedfrompassingthrougheasilybythenegativechargesofheparansulfateandofcertainsialicCollagenacid-containingglycoproteinspresentinthelamina.Thesenegativechargesrepelalbuminandmostplasmaproteins,whicharenegativelychargedatthepHofHeparinFibronectinblood.Thenormalstructureoftheglomerulusmaybeseverelydamagedincertaintypesofglomerulonephri-OUTSIDEtis(eg,causedbyantibodiesdirectedagainstvariousS-Scomponentsoftheglomerularmembrane).ThisaltersS-Stheporesandtheamountsanddispositionsofthenega-αβtivelychargedmacromoleculesreferredtoabove,andrelativelymassiveamountsofalbumin(andofcertainIntegrinreceptorPlasmamembraneCollagenFibronectinTalinαβαβVinculinINSIDECappingproteinα-ActinαβActinLamininFigure48–5.Schematicrepresentationoffibro-Figure48–4.Schematicrepresentationofacellin-nectininteractingwithanintegrinfibronectinreceptorteractingthroughvariousintegrinreceptorswithcolla-situatedintheexterioroftheplasmamembraneofagen,fibronectin,andlamininpresentintheECM.(Spe-celloftheECMandofvariousattachmentproteinsin-cificsubunitsarenotindicated.)(RedrawnafterYamadateractingindirectlyordirectlywithanactinmicrofila-KM:Adhesiverecognitionsequences.JBiolChemmentinthecytosol.Forsimplicity,theattachmentpro-1991;266:12809.)teinsarerepresentedasacomplex.
540542/CHAPTER48otherplasmaproteins)canpassthroughintotheurine,resultinginseverealbuminuria.PROTEOGLYCANS&GLYCOSAMINOGLYCANSTheGlycosaminoglycansFoundinProteoglycansAreBuiltUpofRepeatingDisaccharidesProteoglycansareproteinsthatcontaincovalentlylinkedglycosaminoglycans.Anumberofthemhavebeencharacterizedandgivennamessuchassyndecan,betaglycan,serglycin,perlecan,aggrecan,versican,decorin,biglycan,andfibromodulin.Theyvaryintis-suedistribution,natureofthecoreprotein,attachedglycosaminoglycans,andfunction.Theproteinsboundcovalentlytoglycosaminoglycansarecalled“corepro-teins”;theyhaveproveddifficulttoisolateandcharac-terize,buttheuseofrecombinantDNAtechnologyisbeginningtoyieldimportantinformationabouttheirstructures.Theamountofcarbohydrateinaproteogly-canisusuallymuchgreaterthanisfoundinaglycopro-teinandmaycompriseupto95%ofitsweight.Figures48–6and48–7showthegeneralstructureofonepar-ticularproteoglycan,aggrecan,themajortypefoundin3cartilage.Itisverylarge(about2×10kDa),withitsoverallstructureresemblingthatofabottlebrush.Itcontainsalongstrandofhyaluronicacid(onetypeofFigure48–6.DarkfieldelectronmicrographofaGAG)towhichlinkproteinsareattachednoncova-proteoglycanaggregateinwhichtheproteoglycanlently.Inturn,theselatterinteractnoncovalentlywithsubunitsandfilamentousbackboneareparticularlycoreproteinmoleculesfromwhichchainsofotherwellextended.(Reproduced,withpermission,fromGAGs(keratansulfateandchondroitinsulfateinthisRosenbergL,HellmanW,KleinschmidtAK:Electronmicro-case)project.Moredetailsonthismacromoleculearegivenwhencartilageisdiscussedbelow.scopicstudiesofproteoglycanaggregatesfrombovineThereareatleastsevenglycosaminoglycansarticularcartilage.JBiolChem1975;250:1877.)(GAGs):hyaluronicacid,chondroitinsulfate,keratansulfatesIandII,heparin,heparansulfate,andder-matansulfate.AGAGisanunbranchedpolysaccharidemadeupofrepeatingdisaccharides,onecomponentofbiologicroles;andtheyareinvolvedinanumberofdis-whichisalwaysanaminosugar(hencethenameeaseprocesses—sothatinterestinthemisincreasingGAG),eitherD-glucosamineorD-galactosamine.Therapidly.othercomponentoftherepeatingdisaccharide(exceptinthecaseofkeratansulfate)isauronicacid,eitherBiosynthesisofGlycosaminoglycansL-glucuronicacid(GlcUA)orits5′-epimer,L-iduronicInvolvesAttachmenttoCoreProteins,acid(IdUA).Withtheexceptionofhyaluronicacid,allChainElongation,&ChainTerminationtheGAGscontainsulfategroups,eitherasO-estersorasN-sulfate(inheparinandheparansulfate).A.ATTACHMENTTOCOREPROTEINSHyaluronicacidaffordsanotherexceptionbecauseThelinkagebetweenGAGsandtheircoreproteinsisthereisnoclearevidencethatitisattachedcovalentlygenerallyoneofthreetypes.toprotein,asthedefinitionofaproteoglycangivenabovespecifies.BothGAGsandproteoglycanshave1.AnO-glycosidicbondbetweenxylose(Xyl)andproveddifficulttoworkwith,partlybecauseoftheirSer,abondthatisuniquetoproteoglycans.Thislink-complexity.However,theyaremajorcomponentsofageisformedbytransferofaXylresiduetoSerfromthegroundsubstance;theyhaveanumberofimportantUDP-xylose.TworesiduesofGalarethenaddedtothe
541THEEXTRACELLULARMATRIX/543asinthecaseofcertaintypesoflinkagesfoundingly-coproteins.Theenzymesystemsinvolvedinchainelon-Hyaluronicacidgationarecapableofhigh-fidelityreproductionofcom-LinkproteinplexGAGs.KeratansulfateC.CHAINTERMINATIONChondroitinsulfateThisappearstoresultfrom(1)sulfation,particularlyatcertainpositionsofthesugars,and(2)theprogressionCoreproteinofthegrowingGAGchainawayfromthemembranesitewherecatalysisoccurs.D.FURTHERMODIFICATIONSAfterformationoftheGAGchain,numerouschemicalSubunitsmodificationsoccur,suchastheintroductionofsulfategroupsontoGalNAcandothermoietiesandtheepimerizationofGlcUAtoIdUAresidues.Theenzymescatalyzingsulfationaredesignatedsulfotransferasesanduse3′-phosphoadenosine-5′-phosphosulfate(PAPS;ac-tivesulfate)asthesulfatedonor.TheseGolgi-locatedenzymesarehighlyspecific,anddistinctenzymescat-alyzesulfationatdifferentpositions(eg,carbons2,3,4,and6)ontheacceptorsugars.Anepimerasecatalyzesconversionsofglucuronyltoiduronylresidues.Figure48–7.Schematicrepresentationofthepro-TheVariousGlycosaminoglycansExhibitteoglycanaggrecan.(Reproduced,withpermission,fromDifferencesinStructure&HaveLennarzWJ:TheBiochemistryofGlycoproteinsandProteo-CharacteristicDistributionsglycans.PlenumPress,1980.)ThesevenGAGsnamedabovedifferfromeachotherinanumberofthefollowingproperties:aminosugarcom-position,uronicacidcomposition,linkagesbetweenthesecomponents,chainlengthofthedisaccharides,theXylresidue,formingalinktrisaccharide,Gal-Gal-Xyl-presenceorabsenceofsulfategroupsandtheirpositionsSer.FurtherchaingrowthoftheGAGoccursontheofattachmenttotheconstituentsugars,thenatureofterminalGal.thecoreproteinstowhichtheyareattached,thenature2.AnO-glycosidicbondformsbetweenGalNAcofthelinkagetocoreprotein,theirtissueandsubcellu-(N-acetylgalactosamine)andSer(Thr)(Figure47–lardistribution,andtheirbiologicfunctions.1[a]),presentinkeratansulfateII.ThisbondisformedThestructures(Figure48–8)andthedistributionsbydonationtoSer(orThr)ofaGalNAcresidue,em-ofeachoftheGAGswillnowbebrieflydiscussed.TheployingUDP-GalNAcasitsdonor.majorfeaturesofthesevenGAGsaresummarizedin3.AnN-glycosylaminebondbetweenGlcNAcTable48–6.(N-acetylglucosamine)andtheamidenitrogenofAsn,asfoundinN-linkedglycoproteins(Figure47–1[b]).A.HYALURONICACIDItssynthesisisbelievedtoinvolvedolichol-P-P-Hyaluronicacidconsistsofanunbranchedchainofre-oligosaccharide.peatingdisaccharideunitscontainingGlcUAandGlc-Thesynthesisofthecoreproteinsoccursintheen-NAc.Hyaluronicacidispresentinbacteriaandisdoplasmicreticulum,andformationofatleastsomewidelydistributedamongvariousanimalsandtissues,oftheabovelinkagesalsooccursthere.Mostofthelaterincludingsynovialfluid,thevitreousbodyoftheeye,stepsinthebiosynthesisofGAGchainsandtheirsub-cartilage,andlooseconnectivetissues.sequentmodificationsoccurintheGolgiapparatus.B.CHONDROITINSULFATES(CHONDROITINB.CHAINELONGATION4-SULFATE&CHONDROITIN6-SULFATE)AppropriatenucleotidesugarsandhighlyspecificProteoglycanslinkedtochondroitinsulfatebytheXyl-Golgi-locatedglycosyltransferasesareemployedtosyn-SerO-glycosidicbondareprominentcomponentsofthesizetheoligosaccharidechainsofGAGs.The“onecartilage(seebelow).Therepeatingdisaccharideisenzyme,onelinkage”relationshipappearstoholdhere,similartothatfoundinhyaluronicacid,containing
542544/CHAPTER48β1,4β1,3β1,4β1,3β1,4Hyaluronicacid:GlcUAGlcNAcGlcUAGlcNAcβ1,4β1,3β1,4β1,3β1,3β1,4βChondroitinsulfates:GlcUAGalNAcGlcUAGalGalXylSer4-or6-SulfateβGlcNAcAsn(keratansulfateI)Keratansulfates(GlcNAc,Man)β1,4β1,3β1,4β1,3IandII:GlcNAcGalGlcNAcGal1,6α6-Sulfate6-SulfateGalNAcThr(Ser)(keratansulfateII)Gal-NeuAc6-SulfateHeparinandα1,4α1,4α1,4β1,4α1,4β1,3β1,3β1,4βheparansulfate:IdUAGlcNGlcUAGlcNAcGlcUAGalGalXylSer2-SulfateSO–orAc3β1,4α1,3β1,4β1,3β1,4β1,3β1,3β1,4βDermatansulfate:IdUAGalNAcGlcUAGalNAcGlcUAGalGalXylSer2-Sulfate4-SulfateFigure48–8.Summaryofstructuresofglycosaminoglycansandtheirattachmentstocoreproteins.(GlcUA,D-glucuronicacid;IdUA,L-iduronicacid;GlcN,D-glucosamine;GalN,D-galactosamine;Ac,acetyl;Gal,D-galac-tose;Xyl,D-xylose;Ser,L-serine;Thr,L-threonine;Asn,L-asparagine;Man,D-mannose;NeuAc,N-acetylneu-raminicacid.)Thesummarystructuresarequalitativerepresentationsonlyanddonotreflect,forexample,theuronicacidcompositionofhybridglycosaminoglycanssuchasheparinanddermatansulfate,whichcontainbothL-iduronicandD-glucuronicacid.Neithershoulditbeassumedthattheindicatedsubstituentsarealwayspresent,eg,whereasmostiduronicacidresiduesinheparincarrya2′-sulfategroup,amuchsmallerproportionoftheseresiduesaresulfatedindermatansulfate.Thepresenceoflinktrisaccharides(Gal-Gal-Xyl)inthechon-droitinsulfates,heparin,andheparananddermatansulfatesisshown.(Slightlymodifiedandreproduced,withpermission,fromLennarzWJ:TheBiochemistryofGlycoproteinsandProteoglycans.PlenumPress,1980.)Table48–6.Majorpropertiesoftheglycosaminoglycans.1GAGSugarsSulfateLinkageofProteinLocationHAGIcNAc,GlcUANilNofirmevidenceSynovialfluid,vitreoushumor,looseconnectivetissueCSGaINAc,GlcUAGalNAcXyl-Ser;associatedwithHAvialinkproteinsCartilage,bone,corneaKSIGlcNAc,GalGlcNAcGlcNAc-AsnCorneaKSIIGlcNAc,GalSameasKSIGalNAc-ThrLooseconnectivetissueHeparinGlcN,IdUAGlcNSerMastcellsGlcNIdUAHeparansulfateGlcN,GlcUAGlcNXyl-SerSkinfibroblasts,aorticwallDermatanGalNAc,IdUA,GaINAcXyl-SerWidedistributionsulfate(GlcUA)IdUa1Thesulfateisattachedtovariouspositionsofthesugarsindicated(seeFigure48–7).
543THEEXTRACELLULARMATRIX/545GlcUAbutwithGalNAcreplacingGlcNAc.TheceptthatinplaceofaGlcUAinβ-1,3linkagetoGalNAcissubstitutedwithsulfateateitherits4′oritsGalNAcitcontainsanIdUAinanα-1,3linkageto6′position,withapproximatelyonesulfatebeingpre-GalNAc.FormationoftheIdUAoccurs,asinheparinsentperdisaccharideunit.andheparansulfate,by5′-epimerizationofGlcUA.Be-causethisisregulatedbythedegreeofsulfationandbe-C.KERATANSULFATESI&IIcausesulfationisincomplete,dermatansulfatecontainsAsshowninFigure48–8,thekeratansulfatesconsistofbothIdUA-GalNAcandGlcUA-GalNAcdisaccha-repeatingGal-GlcNAcdisaccharideunitscontainingrides.sulfateattachedtothe6′positionofGlcNAcorocca-sionallyofGal.TypeIisabundantincornea,andtypeIIisfoundalongwithchondroitinsulfateattachedtohyaluronicacidinlooseconnectivetissue.TypesIandDeficienciesofEnzymesThatDegradeIIhavedifferentattachmentstoprotein(Figure48–8).GlycosaminoglycansResultinMucopolysaccharidosesD.HEPARINTherepeatingdisaccharidecontainsglucosamineBothexo-andendoglycosidasesdegradeGAGs.Like(GlcN)andeitherofthetwouronicacids(Figuremostotherbiomolecules,GAGsaresubjectto48–9).MostoftheaminogroupsoftheGlcNresiduesturnover,beingbothsynthesizedanddegraded.InareN-sulfated,butafewareacetylated.TheGlcNalsoadulttissues,GAGsgenerallyexhibitrelativelyslowcarriesaC6sulfateester.turnover,theirhalf-livesbeingdaystoweeks.Approximately90%oftheuronicacidresiduesareUnderstandingofthedegradativepathwaysforIdUA.Initially,alloftheuronicacidsareGlcUA,butaGAGs,asinthecaseofglycoproteins(Chapter47)and5′-epimeraseconvertsapproximately90%oftheglycosphingolipids(Chapter24),hasbeengreatlyaidedGlcUAresiduestoIdUAafterthepolysaccharidechainbyelucidationofthespecificenzymedeficienciesthatisformed.Theproteinmoleculeoftheheparinproteo-occurincertaininbornerrorsofmetabolism.Whenglycanisunique,consistingexclusivelyofserineandGAGsareinvolved,theseinbornerrorsarecalledmu-glycineresidues.Approximatelytwo-thirdsoftheserinecopolysaccharidoses(Table48–7).residuescontainGAGchains,usuallyof5–15kDabutDegradationofGAGsiscarriedoutbyabatteryofoccasionallymuchlarger.Heparinisfoundinthegran-lysosomalhydrolases.Theseincludecertainendogly-ulesofmastcellsandalsoinliver,lung,andskin.cosidases,variousexoglycosidases,andsulfatases,gener-allyactinginsequencetodegradethevariousGAGs.AE.HEPARANSULFATEnumberofthemareindicatedinTable48–7.ThismoleculeispresentonmanycellsurfacesasaThemucopolysaccharidosesshareacommonproteoglycanandisextracellular.ItcontainsGlcNwithmechanismofcausation,asillustratedinFigure48–10.fewerN-sulfatesthanheparin,and,unlikeheparin,itsTheyareinheritedinanautosomalrecessivemanner,predominanturonicacidisGlcUA.withHurlerandHuntersyndromesbeingperhapsthemostwidelystudied.Nonearecommon.Insomecases,F.DERMATANSULFATEafamilyhistoryofamucopolysaccharidosisisobtained.Thissubstanceiswidelydistributedinanimaltissues.Specificlaboratoryinvestigationsofhelpintheirdiag-Itsstructureissimilartothatofchondroitinsulfate,ex-nosisareurinetestingforthepresenceofincreasedCHOSO–CHOSO–CHOSO–CO–CHOSO–232323223OOOOOOOCO–CO–22OOOOOHOHOHOHOHOHOHOOOOHNSO–OSO–HNSO–OHHNSO–OHHNAc3333GlcNIdUAGlcNIdUAGlcNGlcUAGlcNAcFigure48–9.Structureofheparin.Thepolymersectionillustratesstructuralfeaturestypicalofheparin;however,thesequenceofvariouslysubstitutedrepeatingdisaccharideunitshasbeenarbitrarilyselected.Inaddition,non-O-sulfatedor3-O-sulfatedglucosamineresiduesmayalsooccur.(Modified,redrawn,andrepro-duced,withpermission,fromLindahlUetal:Structureandbiosynthesisofheparin-likepolysaccharides.FedProc1977;36:19.)
544546/CHAPTER48Table48–7.Biochemicaldefectsanddiagnostictestsinmucopolysaccharidoses(MPS)and1mucolipidoses(ML).AlternativeUrinary2,3NameDesignationEnzymaticDefectMetabolitesMucopolysaccharidosesHurler,Scheie,MPSIα-L-IduronidaseDermatansulfate,heparansulfateHurler-Scheie(MIM252800)Hunter(MIM309900)MPSIIIduronatesulfataseDermatansulfate,heparansulfateSanfilippoAMPSIIIAHeparansulfateN-sulfataseHeparansulfate(MIM252900)(sulfamidase)SanfilippoBMPSIIIBα-N-AcetylglucosaminidaseHeparansulfate(MIM252920)SanfilippoCMPSIIICAcetyltransferaseHeparansulfate(MIM252930)SanfilippoDMPSIIIDN-AcetylglucosamineHeparansulfate(MIM252940)6-sulfataseMorquioAMPSIVAGalactosamine6-sulfataseKeratansulfate,chondroitin6-sulfate(MIM253000)MorquioBMPSIVBβ-GalactosidaseKeratansulfate(MIM253010)Maroteaux-LamyMPSVIN-Acetylgalactosamine4-Dermatansulfate(MIM253200)sulfatase(arylsulfataseB)Sly(MIM253220)MPSVIIβ-GlucuronidaseDermatansulfate,heparansulfate,chondroitin4-sulfate,chondroitin6-sulfateMucolipidosesSialidosisMLISialidase(neuraminidase)Glycoproteinfragments(MIM256550)I-celldiseaseMLIIUDP-N-acetylglucosamine:Glycoproteinfragments(MIM252500)glycoproteinN-acetylglu-cosamininylphosphotrans-ferase.(Acidhydrolasesthuslackphosphoman-nosylresidues.)Pseudo-HurlerMLIIIAsforMLIIbutdeficiencyGlycoproteinfragmentspolydystrophyisincomplete(MIM252600)1Modifiedandreproduced,withpermission,fromDiNataleP,NeufeldEF:Thebiochemicaldiagnosisofmucopolysaccharidoses,mucolipidosesandrelateddisorders.In:PerspectivesinInheritedMetabolicDiseases,vol2.BarrBetal(editors).EditionesErmes(Milan),1979.2Fibroblasts,leukocytes,tissues,amnioticfluidcells,orserumcanbeusedfortheassayofmanyoftheaboveenzymes.Patientswiththesedisordersexhibitavarietyofclinicalfindingsthatmayincludecloudycorneas,mentalretardation,stiffjoints,cardiacab-normalities,hepatosplenomegaly,andshortstature,dependingonthespecificdiseaseanditsseverity.3ThetermMPSVisnolongerused.TheexistenceofMPSVIII(suspectedglucosamine6-sulfatasedeficiency:MIM253230)hasnotbeenconfirmed.Atleastonecaseofhyaluronidasedeficiency(MPSIX;MIM601492)hasbeenreported.amountsofGAGsandassaysofsuspectedenzymesinTheterm“mucolipidosis”wasintroducedtode-whitecells,fibroblasts,orsometimesinserum.Incer-notediseasesthatcombinedfeaturescommontobothtaincases,atissuebiopsyisperformedandtheGAGmucopolysaccharidosesandsphingolipidoses(Chapterthathasaccumulatedcanbedeterminedbyelec-24).ThreemucolipidosesarelistedinTable48–7.Introphoresis.DNAtestsareincreasinglyavailable.Pre-sialidosis(mucolipidosisI,ML-I),variousoligosaccha-nataldiagnosiscanbemadeusingamnioticcellsorridesderivedfromglycoproteinsandcertainganglio-chorionicvillusbiopsy.sidescanaccumulateintissues.I-celldisease(ML-II)
545THEEXTRACELLULARMATRIX/547TGF-β,modulatingtheireffectsoncells.Inaddition,Mutation(s)inageneencodingalysosomalhydrolaseinvolvedinthedegradationofoneormoreGAGssomeoftheminteractwithcertainadhesiveproteinssuchasfibronectinandlaminin(seeabove),alsofoundinthematrix.TheGAGspresentintheproteoglycansDefectivelysosomalhydrolasearepolyanionsandhencebindpolycationsandcationssuchasNa+andK+.ThislatterabilityattractswaterbyAccumulationofsubstrateinvarioustissues,includingosmoticpressureintotheextracellularmatrixandcon-liver,spleen,bone,skin,andcentralnervoussystemtributestoitsturgor.GAGsalsogelatrelativelylowconcentrations.BecauseofthelongextendednatureofFigure48–10.SimplifiedschemeofcausationofathepolysaccharidechainsofGAGsandtheirabilitytomucopolysaccharidosis,suchasHurlersyndrome(MIMgel,theproteoglycanscanactassieves,restrictingthe252800),inwhichtheaffectedenzymeisα-L-iduroni-passageoflargemacromoleculesintotheECMbutal-dase.MarkedaccumulationoftheGAGsinthetissueslowingrelativelyfreediffusionofsmallmolecules.mentionedinthefigurecouldcausehepatomegaly,Again,becauseoftheirextendedstructuresandthesplenomegaly,disturbancesofgrowth,coarsefacies,hugemacromolecularaggregatestheyoftenform,theyandmentalretardation,respectively.occupyalargevolumeofthematrixrelativetoproteins.A.SOMEFUNCTIONSOFSPECIFICGAGS&PROTEOGLYCANSandpseudo-Hurlerpolydystrophy(ML-III)arede-HyaluronicacidisespeciallyhighinconcentrationinscribedinChapter47.Theterm“mucolipidosis”isre-embryonictissuesandisthoughttoplayanimportanttainedbecauseitisstillinrelativelywidespreadclinicalroleinpermittingcellmigrationduringmorphogenesisusage,butitisnotappropriateforthesetwolatterdis-andwoundrepair.Itsabilitytoattractwaterintotheeasessincethemechanismoftheircausationinvolvesextracellularmatrixandthereby“loosenitup”maybemislocationofcertainlysosomalenzymes.Geneticde-importantinthisregard.Thehighconcentrationsoffectsofthecatabolismoftheoligosaccharidechainsofhyaluronicacidandchondroitinsulfatespresentincar-glycoproteins(eg,mannosidosis,fucosidosis)arealsotilagecontributetoitscompressibility(seebelow).describedinChapter47.Mostofthesedefectsarechar-Chondroitinsulfatesarelocatedatsitesofcalcifica-acterizedbyincreasedexcretionofvariousfragmentsoftioninendochondralboneandarealsofoundincarti-glycoproteinsintheurine,whichaccumulatebecauselage.Theyarealsolocatedinsidecertainneuronsandofthemetabolicblock,asinthecaseofthemucolipi-mayprovideanendoskeletalstructure,helpingtodoses.maintaintheirshape.HyaluronidaseisoneimportantenzymeinvolvedBothkeratansulfateIanddermatansulfateareinthecatabolismofbothhyaluronicacidandchondro-presentinthecornea.Theyliebetweencollagenfibrilsitinsulfate.Itisawidelydistributedendoglycosidaseandplayacriticalroleincornealtransparency.Changesthatcleaveshexosaminidiclinkages.Fromhyaluronicinproteoglycancompositionfoundincornealscarsdis-acid,theenzymewillgenerateatetrasaccharidewithappearwhenthecorneaheals.Thepresenceofder-thestructure(GlcUA-β-1,3-GlcNAc-β-1,4)2,whichmatansulfateinthescleramayalsoplayaroleinmain-canbedegradedfurtherbyaβ-glucuronidaseandβ-N-tainingtheoverallshapeoftheeye.KeratansulfateIisacetylhexosaminidase.Surprisingly,onlyonecaseofanalsopresentincartilage.apparentgeneticdeficiencyofthisenzymeappearstoHeparinisanimportantanticoagulant.Itbindshavebeenreported.withfactorsIXandXI,butitsmostimportantinterac-tioniswithplasmaantithrombinIII(discussedinChapter51).HeparincanalsobindspecificallytoProteoglycansHaveNumerousFunctionslipoproteinlipasepresentincapillarywalls,causingaAsindicatedabove,proteoglycansareremarkablycom-releaseofthisenzymeintothecirculation.plexmoleculesandarefoundineverytissueoftheCertainproteoglycans(eg,heparansulfate)areas-body,mainlyintheECMor“groundsubstance.”sociatedwiththeplasmamembraneofcells,withtheirTheretheyareassociatedwitheachotherandalsowithcoreproteinsactuallyspanningthatmembrane.Inittheothermajorstructuralcomponentsofthematrix,theymayactasreceptorsandmayalsoparticipateincollagenandelastin,inquitespecificmanners.Somethemediationofcellgrowthandcell-cellcommunica-proteoglycansbindtocollagenandotherstoelastin.tion.Theattachmentofcellstotheirsubstratumincul-Theseinteractionsareimportantindeterminingthetureismediatedatleastinpartbyheparansulfate.Thisstructuralorganizationofthematrix.Someproteogly-proteoglycanisalsofoundinthebasementmembranecans(eg,decorin)canalsobindgrowthfactorssuchasofthekidneyalongwithtypeIVcollagenandlaminin
546548/CHAPTER48(seeabove),whereitplaysamajorroleindeterminingInvarioustypesofarthritis,proteoglycansmayactthechargeselectivenessofglomerularfiltration.asautoantigens,thuscontributingtothepathologicProteoglycansarealsofoundinintracellularloca-featuresoftheseconditions.Theamountofchon-tionssuchasthenucleus;theirfunctioninthisor-droitinsulfateincartilagediminisheswithage,whereasganellehasnotbeenelucidated.Theyarepresentintheamountsofkeratansulfateandhyaluronicacidin-somestorageorsecretorygranules,suchasthechromaf-crease.Thesechangesmaycontributetothedevelop-fingranulesoftheadrenalmedulla.Ithasbeenpostu-mentofosteoarthritis.Changesintheamountsofcer-latedthattheyplayaroleinreleaseofthecontentsofsuchgranules.ThevariousfunctionsofGAGsaresum-marizedinTable48–8.B.ASSOCIATIONSWITHMAJORDISEASESTable48–9.Theprincipalproteinsfound1&WITHAGINGinbone.HyaluronicacidmaybeimportantinpermittingtumorcellstomigratethroughtheECM.TumorcellsProteinsCommentscaninducefibroblaststosynthesizegreatlyincreasedCollagensamountsofthisGAG,therebyperhapsfacilitatingtheirCollagentypeIApproximately90%oftotalboneownspread.Sometumorcellshavelessheparansulfateprotein.Composedoftwoα1(I)attheirsurfaces,andthismayplayaroleinthelackofandoneα2(I)chains.adhesivenessthatthesecellsdisplay.CollagentypeVMinorcomponent.Theintimaofthearterialwallcontainshyaluronicacidandchondroitinsulfate,dermatansulfate,andhe-Noncollagenproteinsparansulfateproteoglycans.Oftheseproteoglycans,PlasmaproteinsMixtureofvariousplasmaproteins.dermatansulfatebindsplasmalow-densitylipopro-2Proteoglycansteins.Inaddition,dermatansulfateappearstobetheCS-PGI(biglycan)ContainstwoGAGchains;foundinmajorGAGsynthesizedbyarterialsmoothmuscleothertissues.cells.Becauseitisthesecellsthatproliferateinathero-scleroticlesionsinarteries,dermatansulfatemayplayCS-PGII(decorin)ContainsoneGAGchain;foundinanimportantroleindevelopmentoftheatheroscle-othertissues.roticplaque.CS-PGIIIBone-specific.3BoneSPARCproteinNotbone-specific.(osteonectin)Table48–8.SomefunctionsofOsteocalcin(boneGlaContainsγ-carboxyglutamate1glycosaminoglycansandproteoglycans.protein)residuesthatbindtohydroxyap-atite.Bone-specific.•ActasstructuralcomponentsoftheECMOsteopontinNotbone-specific.Glycosylated•Havespecificinteractionswithcollagen,elastin,fibronectin,andphosphorylated.laminin,andotherproteinssuchasgrowthfactors•Aspolyanions,bindpolycationsandcationsBonesialoproteinBone-specific.Heavilyglycosylated,•Contributetothecharacteristicturgorofvarioustissuesandsulfatedontyrosine.•ActassievesintheECMBonemorphogeneticAfamily(eightormore)ofsecreted•Facilitatecellmigration(HA)proteins(BMPs)proteinswithavarietyofactions•Haveroleincompressibilityofcartilageinweight-bearingonbone;manyinduceectopic(HA,CS)bonegrowth.•Playroleincornealtransparency(KSIandDS)1•Havestructuralroleinsclera(DS)Variousfunctionshavebeenascribedtothenoncollagen•Actasanticoagulant(heparin)proteins,includingrolesinmineralization;however,mostof•Arecomponentsofplasmamembranes,wheretheymaythemarestillspeculative.Itisconsideredunlikelythattheactasreceptorsandparticipateincelladhesionandcell-cellnoncollagenproteinsthatarenotbone-specificplayakeyroleinmineralization.Anumberofotherproteinsarealsointeractions(eg,HS)presentinbone,includingatyrosine-richacidicmatrixpro-•Determinecharge-selectivenessofrenalglomerulus(HS)tein(TRAMP),somegrowthfactors(eg,TGFβ),andenzymes•Arecomponentsofsynapticandothervesicles(eg,HS)involvedincollagensynthesis(eg,lysyloxidase).1ECM,extracellularmatrix;HA,hyaluronicacid;CS,chondroitin2CS-PG,chondroitinsulfate–proteoglycan;thesearesimilartosulfate;KSI,keratansulfateI;DS,dermatansulfate;HS,heparanthedermatansulfatePGs(DS-PGs)ofcartilage(Table48–11).3sulfate.SPARC,secretedproteinacidicandrichincysteine.
547THEEXTRACELLULARMATRIX/549OsteoclastMesenchymeNewlyformedmatrix(osteoid)OsteoblastOsteocyteBonematrixFigure48–11.Schematicillustrationofthemajorcellspresentinmembranousbone.Osteoblasts(lightercolor)aresynthesizingtypeIcollagen,whichformsamatrixthattrapscells.Asthisoccurs,osteoblastsgraduallydifferentiatetobecomeosteo-cytes.(Reproduced,withpermission,fromJunqueiraLC,CarneiroJ:BasicHistology:Text&Atlas,10thed.McGraw-Hill,2003.)tainGAGsintheskinarealsoobservedwithagingandbone(Chapter45).Hydroxyapatiteconfersonbonehelptoaccountforthecharacteristicchangesnotedinthestrengthandresiliencerequiredbyitsphysiologicthisorganintheelderly.roles.AnexcitingnewphaseinproteoglycanresearchisBoneisadynamicstructurethatundergoescontinu-openingupwiththefindingsthatmutationsthataffectingcyclesofremodeling,consistingofresorptionfol-individualproteoglycansortheenzymesneededforlowedbydepositionofnewbonetissue.Thisremodel-theirsynthesisaltertheregulationofspecificsignalingingpermitsbonetoadapttobothphysical(eg,pathwaysindrosophilaandCaenorhabditiselegans,thusincreasesinweight-bearing)andhormonalsignals.affectingdevelopment;italreadyseemslikelythatsimi-Themajorcelltypesinvolvedinboneresorptionlareffectsexistinmiceandhumans.anddepositionareosteoclastsandosteoblasts(Figure48–11).Theformerareassociatedwithresorptionandthelatterwithdepositionofbone.Osteocytesarede-BONEISAMINERALIZEDscendedfromosteoblasts;theyalsoappeartobein-CONNECTIVETISSUEvolvedinmaintenanceofbonematrixbutwillnotbediscussedfurtherhere.Bonecontainsbothorganicandinorganicmaterial.OsteoclastsaremultinucleatedcellsderivedfromTheorganicmatterismainlyprotein.Theprincipalpluripotenthematopoieticstemcells.Osteoclastspos-proteinsofbonearelistedinTable48–9;typeIcolla-sessanapicalmembranedomain,exhibitingaruffledgenisthemajorprotein,comprising90–95%oftheborderthatplaysakeyroleinboneresorption(Figureorganicmaterial.TypeVcollagenisalsopresentin48–12).Aproton-translocatingATPaseexpelsprotonssmallamounts,asareanumberofnoncollagenpro-acrosstheruffledborderintotheresorptionarea,whichteins,someofwhicharerelativelyspecifictobone.isthemicroenvironmentoflowpHshowninthefig-Theinorganicormineralcomponentismainlycrys-ure.ThislowersthelocalpHto4.0orless,thusin-tallinehydroxyapatite—Ca10(PO4)6(OH)2—alongcreasingthesolubilityofhydroxyapatiteandallowingwithsodium,magnesium,carbonate,andfluoride;ap-demineralizationtooccur.Lysosomalacidproteasesareproximately99%ofthebody’scalciumiscontainedinreleasedthatdigestthenowaccessiblematrixproteins.
548550/CHAPTER48BloodcapillaryNucleusOsteoclastGolgiNucleusLysosomesCO+HOH++HCO–223SectionofcircumferentialclearzoneRuffledborderMicroenvironmentoflowpHBonematrixandlysosomalenzymesFigure48–12.Schematicillustrationofsomeaspectsoftheroleoftheosteoclastinboneresorption.Lysosomalenzymesandhydrogenionsarereleasedintotheconfinedmicroenvironmentcreatedbytheattachmentbetweenbonematrixandtheperipheralclearzoneoftheosteoclast.Theacidificationofthisconfinedspacefacilitatesthedis-solutionofcalciumphosphatefromboneandistheoptimalpHfortheactivityoflyso-somalhydrolases.Bonematrixisthusremoved,andtheproductsofboneresorptionaretakenupintothecytoplasmoftheosteoclast,probablydigestedfurther,andtrans-ferredintocapillaries.ThechemicalequationshowninthefigurereferstotheactionofcarbonicanhydraseII,describedinthetext.(Reproduced,withpermission,fromJun-queiraLC,CarneiroJ:BasicHistology:Text&Atlas,10thed.McGraw-Hill,2003.)Osteoblasts—mononuclearcellsderivedfrompluripo-Recentinteresthasfocusedonacidicphosphoproteins,tentmesenchymalprecursors—synthesizemostofthesuchasbonesialoprotein,actingassitesofnucleation.proteinsfoundinbone(Table48–9)aswellasvariousTheseproteinscontainmotifs(eg,poly-Aspandpoly-growthfactorsandcytokines.TheyareresponsibleforGlustretches)thatbindcalciumandmayprovideanthedepositionofnewbonematrix(osteoid)anditsinitialscaffoldformineralization.Somemacromole-subsequentmineralization.Osteoblastscontrolminer-cules,suchascertainproteoglycansandglycoproteins,alizationbyregulatingthepassageofcalciumandphos-canalsoactasinhibitorsofnucleation.phateionsacrosstheirsurfacemembranes.ThelatterItisestimatedthatapproximately4%ofcompactcontainalkalinephosphatase,whichisusedtogenerateboneisrenewedannuallyinthetypicalhealthyadult,phosphateionsfromorganicphosphates.Themecha-whereasapproximately20%oftrabecularboneisre-nismsinvolvedinmineralizationarenotfullyunder-placed.stood,butseveralfactorshavebeenimplicated.AlkalineManyfactorsareinvolvedintheregulationofbonephosphatasecontributestomineralizationbutinitselfmetabolism,onlyafewofwhichwillbementionedisnotsufficient.Smallvesicles(matrixvesicles)contain-here.Somestimulateosteoblasts(eg,parathyroidhor-ingcalciumandphosphatehavebeendescribedatsitesmoneand1,25-dihydroxycholecalciferol)andothersofmineralization,buttheirroleisnotclear.TypeIcol-inhibitthem(eg,corticosteroids).Parathyroidhormonelagenappearstobenecessary,withmineralizationbeingand1,25-dihydroxycholecalciferolalsostimulateosteo-firstevidentinthegapsbetweensuccessivemolecules.clasts,whereascalcitoninandestrogensinhibitthem.
549THEEXTRACELLULARMATRIX/551Table48–10.SomemetabolicandgeneticMANYMETABOLIC&GENETICdiseasesaffectingboneandcartilage.DISORDERSINVOLVEBONEAnumberofthemoreimportantexamplesofmeta-DiseaseCommentsbolicandgeneticdisordersthataffectbonearelistedinDwarfismOftenduetoadeficiencyofgrowthTable48–10.hormone,buthasmanyothercauses.Osteogenesisimperfecta(brittlebones)ischarac-terizedbyabnormalfragilityofbones.ThesclerasareRicketsDuetoadeficiencyofvitaminDoftenabnormallythinandtranslucentandmayappearduringchildhood.blueowingtoadeficiencyofconnectivetissue.FourOsteomalaciaDuetoadeficiencyofvitaminDtypesofthiscondition(mild,extensive,severe,andduringadulthood.variable)havebeenrecognized,ofwhichtheextensiveHyperparathyroidismExcessparathormonecausesbonetypeoccurringinthenewbornisthemostominous.resorption.Affectedinfantsmaybebornwithmultiplefracturesandnotsurvive.Over90%ofpatientswithosteogene-OsteogenesisDuetoavarietyofmutationsinthesisimperfectahavemutationsintheCOL1A1andimperfecta(eg,COL1A1andCOL1A2genesaffectingCOL1A2genes,encodingproα1(I)andproα2(I)MIM166200)thesynthesisandstructureoftypeIchains,respectively.Over100mutationsinthesetwocollagen.geneshavebeendocumentedandincludepartialgeneOsteoporosisCommonlypostmenopausalorindeletionsandduplications.Othermutationsaffectothercasesismoregradualandre-RNAsplicing,andthemostfrequenttyperesultsinthelatedtoage;asmallnumberofcasesreplacementofglycinebyanotherbulkieraminoacid,areduetomutationsintheCOL1A1affectingformationofthetriplehelix.Ingeneral,theseandCOL1A2genesandpossiblyinthemutationsresultindecreasedexpressionofcollagenorvitaminDreceptorgene(MIM166710)OsteoarthritisAsmallnumberofcasesareduetomutationsintheCOL1Agenes.Table48–11.TheprincipalproteinsfoundSeveralchondro-DuetomutationsinCOL2A1genes.incartilage.dysplasias1PfeiffersyndromeMutationsinthegeneencodingfi-ProteinsComments(MIM100600)broblastgrowthreceptor1(FGFR1).CollagenproteinsJackson-WeissMutationsinthegeneencodingCollagentypeII90–98%oftotalarticularcartilage(MIM123150)FGFR2.collagen.ComposedofthreeandCrouzonα1(II)chains.(MIM123500)CollagensV,VI,IX,TypeIXcross-linkstotypeIIcolla-1syndromesX,XIgen.TypeXImayhelpcontroldi-AchondroplasiaMutationsinthegeneencodingameteroftypeIIfibrils.(MIM100800)FGFR3.NoncollagenproteinsandthanatophoricProteoglycans2dysplasiaAggrecanThemajorproteoglycanofcartilage.(MIM187600)Largenon-Foundinsometypesofcartilage.1aggregatingThePfeiffer,Jackson-Weiss,andCrouzonsyndromesarecran-iosynostosissyndromes;craniosynostosisisatermsignifyingpre-proteoglycanmaturefusionofsuturesintheskull.1DS-PGI(biglycan)SimilartoCS-PGIofbone.2Thanatophoric(Gkthanatos“death”+phoros“bearing”)dyspla-DS-PGII(decorin)SimilartoCS-PGIIofbone.siaisthemostcommonneonatallethalskeletaldysplasia,dis-ChondronectinMayplayroleinbindingtypeIIcolla-playingfeaturessimilartothoseofhomozygousachondroplasia.gentosurfaceofcartilage.AnchorinCIIMaybindtypeIIcollagentosurfaceofchondrocyte.1ThecoreproteinsofDS-PGIandDS-PGIIarehomologoustothoseofCS-PGIandCS-PGIIfoundinbone(Table48–9).Apossi-bleexplanationisthatosteoblastslacktheepimeraserequiredtoconvertglucuronicacidtoiduronicacid,thelatterofwhichisfoundindermatansulfate.
550552/CHAPTER48instructurallyabnormalproαchainsthatassembleintoionsarepumpedacrosstheirruffledborders(seeabnormalfibrils,weakeningtheoverallstructureofabove).Thus,ifCAIIisdeficientinactivityinosteo-bone.Whenoneabnormalchainispresent,itmayin-clasts,normalboneresorptiondoesnotoccur,andos-teractwithtwonormalchains,butfoldingmaybepre-teopetrosisresults.Themechanismofthecerebralcalci-vented,resultinginenzymaticdegradationofalloftheficationisnotclear,whereastherenaltubularacidosischains.Thisiscalled“procollagensuicide”andisanex-reflectsdeficientactivityofCAIIintherenaltubules.ampleofadominantnegativemutation,aresultoftenOsteoporosisisageneralizedprogressivereductionseenwhenaproteinconsistsofmultipledifferentsub-inbonetissuemassperunitvolumecausingskeletalunits.weakness.TheratioofmineraltoorganicelementsisOsteopetrosis(marblebonedisease),characterizedunchangedintheremainingnormalbone.Fracturesofbyincreasedbonedensity,isduetoinabilitytoresorbvariousbones,suchastheheadofthefemur,occurverybone.Oneformoccursalongwithrenaltubularacido-easilyandrepresentahugeburdentoboththeaffectedsisandcerebralcalcification.Itisduetomutationsinpatientsandtothehealthcarebudgetofsociety.thegene(locatedonchromosome8q22)encodingcar-Amongotherfactors,estrogensandinterleukins-1andbonicanhydraseII(CAII),oneoffourisozymesofcar--6appeartobeintimatelyinvolvedinthecausationofbonicanhydrasepresentinhumantissues.Thereactionosteoporosis.catalyzedbycarbonicanhydraseisshownbelow:THEMAJORCOMPONENTSOF67nmCARTILAGEARETYPEIICOLLAGENFibril&CERTAINPROTEOGLYCANSTheprincipalproteinsofhyalinecartilage(themajorReactionIIisspontaneous.Inosteoclastsinvolvedintypeofcartilage)arelistedinTable48–11.TypeIIcolla-boneresorption,CAIIapparentlyprovidesprotonstogenistheprincipalprotein(Figure48–13),andanum-neutralizetheOH−ionsleftinsidethecellwhenH+berofotherminortypesofcollagenarealsopresent.InHyaluronicacidTypeIIcollagenfibrilHyaluronicacidLinkproteinChondroitinsulfateProteoglycanCoreproteinCollagen(typeII)Figure48–13.Schematicrepresentationofthemolecularorganizationincartilagematrix.Linkproteinsnoncovalentlybindthecoreprotein(lightercolor)ofproteogly-canstothelinearhyaluronicacidmolecules(darkercolor).Thechondroitinsulfatesidechainsoftheproteoglycanelectrostaticallybindtothecollagenfibrils,formingacross-linkedmatrix.Theovaloutlinestheareaenlargedinthelowerpartofthefigure.(Reproduced,withpermission,fromJunqueiraLC,CarneiroJ:BasicHistology:Text&Atlas,10thed.McGraw-Hill,2003.)
551THEEXTRACELLULARMATRIX/553additiontothesecomponents,elasticcartilagecontainsdegradecollagenandtheotherproteinsfoundincarti-elastinandfibroelasticcartilagecontainstypeIcollagen.lage.Interleukin-1(IL-1)andtumornecrosisfactorαCartilagecontainsanumberofproteoglycans,which(TNFα)appeartostimulatetheproductionofsuchplayanimportantroleinitscompressibility.Aggrecanproteases,whereastransforminggrowthfactorβ3(about2×10kDa)isthemajorproteoglycan.Asshown(TGFβ)andinsulin-likegrowthfactor1(IGF-I)gener-inFigure48–14,ithasaverycomplexstructure,con-allyexertananabolicinfluenceoncartilage.tainingseveralGAGs(hyaluronicacid,chondroitinsul-fate,andkeratansulfate)andbothlinkandcoreproteins.Thecoreproteincontainsthreedomains:A,B,andC.THEMOLECULARBASESOFTHEThehyaluronicacidbindsnoncovalentlytodomainAofCHONDRODYSPLASIASINCLUDEthecoreproteinaswellastothelinkprotein,whichsta-MUTATIONSINGENESENCODINGbilizesthehyaluronate–coreproteininteractions.TheTYPEIICOLLAGEN&FIBROBLASTkeratansulfatechainsarelocatedindomainB,whereasGROWTHFACTORRECEPTORSthechondroitinsulfatechainsarelocatedindomainC;bothofthesetypesofGAGsareboundcovalentlytotheChondrodysplasiasareamixedgroupofhereditarydis-coreprotein.ThecoreproteinalsocontainsbothO-andordersaffectingcartilage.Theyaremanifestedbyshort-N-linkedoligosaccharidechains.limbeddwarfismandnumerousskeletaldeformities.ATheotherproteoglycansfoundincartilagehavenumberofthemareduetoavarietyofmutationsinthesimplerstructuresthanaggrecan.COL2A1gene,leadingtoabnormalformsoftypeIIChondronectinisinvolvedintheattachmentofcollagen.OneexampleisSticklersyndrome,mani-typeIIcollagentochondrocytes.festedbydegenerationofjointcartilageandofthevit-Cartilageisanavasculartissueandobtainsmostofreousbodyoftheeye.itsnutrientsfromsynovialfluid.ItexhibitsslowbutThebest-knownofthechondrodysplasiasisachon-continuousturnover.Variousproteases(eg,collage-droplasia,thecommonestcauseofshort-limbednasesandstromalysin)synthesizedbychondrocytescandwarfism.Affectedindividualshaveshortlimbs,nor-DomainADomainBDomainCHyaluronate-bindingregionCoreN-linkedproteinoligosaccharideLinkproteinKeratanChondroitinO-linkedHyaluronicacidsulfatesulfateoligosaccharideFigure48–14.Schematicdiagramoftheaggrecanfrombovinenasalcartilage.Astrandofhyaluronicacidisshownontheleft.Thecoreprotein(about210kDa)hasthreemajordomains.DomainA,atitsaminoterminalend,interactswithapproxi-matelyfiverepeatingdisaccharidesinhyaluronate.ThelinkproteininteractswithbothhyaluronateanddomainA,stabilizingtheirinteractions.Approximately30ker-atansulfatechainsareattached,viaGalNAc-Serlinkages,todomainB.DomainCcontainsabout100chondroitinsulfatechainsattachedviaGal-Gal-Xyl-Serlinkagesandabout40O-linkedoligosaccharidechains.OneormoreN-linkedglycanchainsarealsofoundnearthecarboxylterminalofthecoreprotein.(Reproduced,withper-mission,fromMoranLAetal:Biochemistry,2nded.NeilPattersonPublishers,1994.)
552554/CHAPTER48maltrunksize,macrocephaly,andavarietyofotherSUMMARYskeletalabnormalities.Theconditionisofteninherited•ThemajorcomponentsoftheECMarethestruc-asanautosomaldominanttrait,butmanycasesaredueturalproteinscollagen,elastin,andfibrillin;anum-tonewmutations.Themolecularbasisofachondropla-berofspecializedproteins(eg,fibronectinandsiaisoutlinedinFigure48–15.Achondroplasiaisnotalaminin);andvariousproteoglycans.collagendisorderbutisduetomutationsinthegeneencodingfibroblastgrowthfactorreceptor3•Collagenisthemostabundantproteinintheanimal(FGFR3).Fibroblastgrowthfactorsareafamilyofatkingdom;approximately19typeshavebeenisolated.leastnineproteinsthataffectthegrowthanddifferenti-Allcollagenscontaingreaterorlesserstretchesofationofcellsofmesenchymalandneuroectodermalori-triplehelixandtherepeatingstructure(Gly-X-Y)n.gin.Theirreceptorsaretransmembraneproteinsand•Thebiosynthesisofcollageniscomplex,featuringformasubgroupofthefamilyofreceptortyrosineki-manyposttranslationalevents,includinghydroxyla-nases.FGFR3isonememberofthissubgroupandme-tionofprolineandlysine.diatestheactionsofFGF3oncartilage.Inalmostall•Diseasesassociatedwithimpairedsynthesisofcolla-casesofachondroplasiathathavebeeninvestigated,thegenincludescurvy,osteogenesisimperfecta,Ehlers-mutationswerefoundtoinvolvenucleotide1138andDanlossyndrome(manytypes),andMenkesdisease.resultedinsubstitutionofarginineforglycine(residue•Elastinconfersextensibilityandelasticrecoilontis-number380)inthetransmembranedomainofthepro-sues.Elastinlackshydroxylysine,Gly-X-Ysequences,tein,renderingitinactive.Nosuchmutationwasfoundtriplehelicalstructure,andsugarsbutcontainsinunaffectedindividuals.AsindicatedinTable48–10,desmosineandisodesmosinecross-linksnotfoundinotherskeletaldysplasias(includingcertaincraniosynos-collagen.tosissyndromes)arealsoduetomutationsingenesen-•Fibrillinislocatedinmicrofibrils.MutationsinthecodingFGFreceptors.Anothertypeofskeletaldyspla-geneforfibrillincauseMarfansyndrome.sia(diastrophicdysplasia)hasbeenfoundtobeduetomutationinasulfatetransporter.Thus,thankstore-•Theglycosaminoglycans(GAGs)aremadeupofre-combinantDNAtechnology,anewerainunderstand-peatingdisaccharidescontainingauronicacid(glu-ingofskeletaldysplasiashasbegun.curonicoriduronic)orhexose(galactose)andahex-osamine(galactosamineorglucosamine).Sulfateisalsofrequentlypresent.•ThemajorGAGsarehyaluronicacid,chondroitin4-and6-sulfates,keratansulfatesIandII,heparin,Mutationsofnucleotide1138inthegeneheparansulfate,anddermatansulfate.encodingFGFR3onchromosome4•TheGAGsaresynthesizedbythesequentialactionsofabatteryofspecificenzymes(glycosyltransferases,ReplacementinFGFR3ofGly(codon380)byArgepimerases,sulfotransferases,etc)andaredegradedbythesequentialactionoflysosomalhydrolases.Ge-DefectivefunctionofFGFR3neticdeficienciesofthelatterresultinmucopolysac-charidoses(eg,Hurlersyndrome).Abnormaldevelopmentandgrowthofcartilage•GAGsoccurintissuesboundtovariousproteinsleadingtoshort-limbeddwarfismandotherfeatures(linkerproteinsandcoreproteins),constitutingpro-teoglycans.ThesestructuresareoftenofveryhighFigure48–15.Simplifiedschemeofthecausationofmolecularweightandservemanyfunctionsintis-achondroplasia(MIM100800).Inmostcasesstudiedsosues.far,themutationhasbeenaGtoAtransitionatnu-•ManycomponentsoftheECMbindtoproteinsofcleotide1138.Inafewcases,themutationwasaGtoCthecellsurfacenamedintegrins;thisconstitutesonetransversionatthesamenucleotide.Thisparticularnu-pathwaybywhichtheexteriorsofcellscancommu-cleotideisareal“hotspot”formutation.Bothmuta-nicatewiththeirinteriors.tionsresultinreplacementofaGlyresiduebyanArg•BoneandcartilagearespecializedformsoftheECM.residueinthetransmembranesegmentofthereceptor.CollagenIandhydroxyapatitearethemajorcon-AfewcasesinvolvingreplacementofGlybyCysatstituentsofbone.CollagenIIandcertainproteogly-codon375havealsobeenreported.cansaremajorconstituentsofcartilage.
553THEEXTRACELLULARMATRIX/555•Themolecularcausesofanumberofheritabledis-ProckopDJ,KivirikkoKI:Collagens:molecularbiology,diseases,easesofbone(eg,osteogenesisimperfecta)andofcar-andpotentialtherapy.AnnuRevBiochem1995;64:403.tilage(eg,thechondrodystrophies)arebeingrevealedPyeritzRE:Ehlers-Danlossyndrome.NEnglJMed2000;342:730.bytheapplicationofrecombinantDNAtechnology.SageE:Regulationofinteractionsbetweencellsandextracellularmatrix:acommandperformanceonseveralstages.JClinIn-vest2001;107:781.(Thisarticleintroducesaseriesofsixarti-REFERENCESclesoncell-matrixinteraction.Thetopicscoveredarecelladhesionandde-adhesion,thrombospondins,syndecans,BandtlowCE,ZimmermannDR:ProteoglycansinthedevelopingSPARC,osteopontin,andEhlers-Danlossyndrome.Allofbrain:newconceptualinsightsforoldproteins.PhysiolRevthearticlescanbeaccessedatwww.jci.org.)2000;80:1267.ScriverCRetal(editors):TheMetabolicandMolecularBasesofIn-BikleDD:Biochemicalmarkersintheassessmentofbonediseases.heritedDisease,8thed.McGraw-Hill,2001(Thiscompre-AmJMed1997;103:427.hensivefour-volumetextcontainschaptersondisordersofBurkeDetal:Fibroblastgrowthfactorreceptors:lessonsfromthecollagenbiosynthesisandstructure,Marfansyndrome,thegenes.TrendsBiochemSci1998;23:59.mucopolysaccharidoses,achondroplasia,Alportsyndrome,CompstonJE:Sexsteroidsandbone.PhysiolRev2001;81:419.andcraniosynostosissyndromes.)FullerGM,ShieldsD:MolecularBasisofMedicalCellBiology.Ap-SelleckSB:Geneticdissectionofproteoglycanfunctioninpleton&Lange,1998.DrosophilaandC.elegans.SeminCellDevBiol2001;12:127.HermanT,HorvitzHR:ThreeproteinsinvolvedinCaenorhabditiselegansvulvalinvaginationaresimilartocomponentsofagly-cosylationpathway.ProcNatlAcadSciUSA1999;96:974.
554Muscle&theCytoskeleton49RobertK.Murray,MD,PhDBIOMEDICALIMPORTANCEshalldiscussaspectsofthethreetypesofmusclefoundinvertebrates:skeletal,cardiac,andsmooth.BothProteinsplayanimportantroleinmovementatbothskeletalandcardiacmuscleappearstriateduponmicro-theorgan(eg,skeletalmuscle,heart,andgut)andcellu-scopicobservation;smoothmuscleisnonstriated.Al-larlevels.Inthischapter,therolesofspecificproteinsandcertainotherkeymolecules(eg,Ca2+)inmuscularthoughskeletalmuscleisundervoluntarynervouscon-trol,thecontrolofbothcardiacandsmoothmuscleiscontractionaredescribed.Abriefcoverageofcyto-involuntary.skeletalproteinsisalsopresented.Knowledgeofthemolecularbasesofanumberofconditionsthataffectmusclehasadvancedgreatlyinre-TheSarcoplasmofMuscleCellscentyears.UnderstandingofthemolecularbasisofContainsATP,Phosphocreatine,Duchenne-typemusculardystrophywasgreatlyen-&GlycolyticEnzymeshancedwhenitwasfoundthatitwasduetomutationsStriatedmuscleiscomposedofmultinucleatedmuscleinthegeneencodingdystrophin.Significantprogressfibercellssurroundedbyanelectricallyexcitableplasmahasalsobeenmadeinunderstandingthemolecularmembrane,thesarcolemma.Anindividualmusclebasisofmalignanthyperthermia,aseriouscomplica-fibercell,whichmayextendtheentirelengthofthetionforsomepatientsundergoingcertaintypesofanes-muscle,containsabundleofmanymyofibrilsarrangedthesia.Heartfailureisaverycommonmedicalcondi-inparallel,embeddedinintracellularfluidtermedsar-tion,withavarietyofcauses;itsrationaltherapycoplasm.Withinthisfluidiscontainedglycogen,therequiresunderstandingofthebiochemistryofhearthigh-energycompoundsATPandphosphocreatine,muscle.Onegroupofconditionsthatcauseheartfail-andtheenzymesofglycolysis.urearethecardiomyopathies,someofwhicharege-neticallydetermined.Nitricoxide(NO)hasbeenfoundtobeamajorregulatorofsmoothmuscletone.TheSarcomereIstheFunctionalManywidelyusedvasodilators—suchasnitroglycerin,UnitofMuscleusedinthetreatmentofanginapectoris—actbyin-AnoverallviewofvoluntarymuscleatseverallevelsofcreasingtheformationofNO.Muscle,partlybecauseorganizationispresentedinFigure49–1.ofitsmass,playsmajorrolesintheoverallmetabolismWhenthemyofibrilisexaminedbyelectronmi-ofthebody.croscopy,alternatingdarkandlightbands(anisotropicbands,meaningbirefringentinpolarizedlight;andisotropicbands,meaningnotalteredbypolarizedlight)MUSCLETRANSDUCESCHEMICALcanbeobserved.ThesebandsarethusreferredtoasAENERGYINTOMECHANICALENERGYandIbands,respectively.ThecentralregionoftheAband(theHband)appearslessdensethantherestofMuscleisthemajorbiochemicaltransducer(machine)theband.TheIbandisbisectedbyaverydenseandthatconvertspotential(chemical)energyintokineticnarrowZline(Figure49–2).(mechanical)energy.Muscle,thelargestsingletissueinThesarcomereisdefinedastheregionbetweentwothehumanbody,makesupsomewhatlessthan25%ofZlines(Figures49–1and49–2)andisrepeatedalongbodymassatbirth,morethan40%intheyoungadult,theaxisofafibrilatdistancesof1500–2300nmde-andsomewhatlessthan30%intheagedadult.Wependinguponthestateofcontraction.556
555MUSCLE&THECYTOSKELETON/557AMuscleBMusclefasciculusC20–100μmMusclefiberHZAIbandlinebandbandD1–2μmZ–Sarcomere–ZMyofibrilFigure49–1.Thestructureofvoluntarymuscle.ThesarcomereistheregionbetweentheZlines.(DrawingbySylviaColardKeene.Reproduced,withpermission,fromBloomW,FawcettDW:ATextbookofHistology,10thed.Saunders,1975.)Thestriatedappearanceofvoluntaryandcardiacsection),andeachthickfilamentissurroundedsym-muscleinlightmicroscopicstudiesresultsfromtheirmetricallybysixthinfilaments.highdegreeoforganization,inwhichmostmusclefiberThethickandthinfilamentsinteractviacross-cellsarealignedsothattheirsarcomeresareinparallelbridgesthatemergeatintervalsof14nmalongtheregister(Figure49–1).thickfilaments.AsdepictedinFigure49–2,thecross-bridges(drawnasarrowheadsateachendofthemyosinThickFilamentsContainMyosin;filaments,butnotshownextendingfullyacrosstotheThinFilamentsContainActin,thinfilaments)haveoppositepolaritiesatthetwoendsofthethickfilaments.Thetwopolesofthethickfila-Tropomyosin,&Troponinmentsareseparatedbya150-nmsegment(theMband,Whenmyofibrilsareexaminedbyelectronmicroscopy,notlabeledinthefigure)thatisfreeofprojections.itappearsthateachoneisconstructedoftwotypesoflongitudinalfilaments.Onetype,thethickfilament,confinedtotheAband,containschieflytheproteinTheSlidingFilamentCross-Bridgemyosin.Thesefilamentsareabout16nmindiameterModelIstheFoundationonWhichandarrangedincross-sectionasahexagonalarray(Fig-CurrentThinkingAboutMuscleure49–2,center;right-handcross-section).ContractionIsBuiltThethinfilament(about7nmindiameter)liesintheIbandandextendsintotheAbandbutnotintoitsThismodelwasproposedindependentlyinthe1950sHzone(Figure49–2).Thinfilamentscontainthepro-byHenryHuxleyandAndrewHuxleyandtheircol-teinsactin,tropomyosin,andtroponin(Figure49–3).leagues.Itwaslargelybasedoncarefulmorphologicob-IntheAband,thethinfilamentsarearrangedaroundservationsonresting,extended,andcontractingmus-thethick(myosin)filamentasasecondaryhexagonalcle.Basically,whenmusclecontracts,thereisnochangearray.Eachthinfilamentliessymmetricallybetweeninthelengthsofthethickandthinfilaments,butthethreethickfilaments(Figure49–2,center;midcross-HzonesandtheIbandsshorten(seelegendtoFig-
556558/CHAPTER49HbandA.ExtendedIbandAbandZline2300nmα-ActininActinfilaments6-nmdiameterMyosinfilaments16-nmdiameterCrosssection:B.ContractedThin6-nmdiameterfilamentThick16-nmdiameterfilament1500nmFigure49–2.Arrangementoffilamentsinstriatedmuscle.A:Extended.ThepositionsoftheI,A,andHbandsintheextendedstateareshown.Thethinfilamentspartlyoverlaptheendsofthethickfilaments,andthethinfilamentsareshownanchoredintheZlines(oftencalledZdisks).InthelowerpartofFigure49–2A,“arrowheads,”pointinginoppositedirections,areshownemanat-ingfromthemyosin(thick)filaments.Fouractin(thin)filamentsareshownattachedtotwoZlinesviaα-actinin.Thecentralregionofthethreemyosinfilaments,freeofarrowheads,iscalledtheMband(notlabeled).Cross-sectionsthroughtheMbands,throughanareawheremyosinandactinfilamentsoverlapandthroughanareainwhichsolelyactinfilamentsarepresent,areshown.B:Contracted.Theactinfilamentsareseentohaveslippedalongthesidesofthemyosinfibersto-wardeachother.Thelengthsofthethickfilaments(indicatedbytheAbands)andthethinfila-ments(distancebetweenZlinesandtheadjacentedgesoftheHbands)havenotchanged.How-ever,thelengthsofthesarcomereshavebeenreduced(from2300nmto1500nm),andthelengthsoftheHandIbandsarealsoreducedbecauseoftheoverlapbetweenthethickandthinfilaments.Thesemorphologicobservationsprovidedpartofthebasisfortheslidingfilamentmodelofmusclecontraction.
557MUSCLE&THECYTOSKELETON/559G-actinF-actin6–7nmTropomyosinTroponinTpC38.5nmTpITpT35.5nmTheassembledthinfilamentFigure49–3.Schematicrepresentationofthethinfilament,showingthespatialconfigurationofitsthreemajorproteincomponents:actin,myosin,andtropomyosin.TheupperpanelshowsindividualmoleculesofG-actin.ThemiddlepanelshowsactinmonomersassembledintoF-actin.Individualmoleculesoftropomyosin(twostrandswoundaroundoneanother)andoftroponin(madeupofitsthreesubunits)arealsoshown.Thelowerpanelshowstheassembledthinfilament,consistingofF-actin,tropomyosin,andthethreesubunitsoftroponin(TpC,TpI,andTpT).ure49–2).Thus,thearraysofinterdigitatingfilamentsACTIN&MYOSINARETHEMAJORmustslidepastoneanotherduringcontraction.Cross-PROTEINSOFMUSCLEbridgesthatlinkthickandthinfilamentsatcertainstagesinthecontractioncyclegenerateandsustaintheThemassofamuscleismadeupof75%waterandtension.Thetensiondevelopedduringmusclecontrac-morethan20%protein.Thetwomajorproteinsaretionisproportionatetothefilamentoverlapandtotheactinandmyosin.numberofcross-bridges.Eachcross-bridgeheadiscon-MonomericG-actin(43kDa;G,globular)makesnectedtothethickfilamentviaaflexiblefibrousseg-up25%ofmuscleproteinbyweight.Atphysiologicmentthatcanbendoutwardfromthethickfilament.ionicstrengthandinthepresenceofMg2+,G-actinThisflexiblesegmentfacilitatescontactoftheheadpolymerizesnoncovalentlytoformaninsolubledoublewiththethinfilamentwhennecessarybutisalsosuffi-helicalfilamentcalledF-actin(Figure49–3).ThecientlyplianttobeaccommodatedintheinterfilamentF-actinfiberis6–7nmthickandhasapitchorrepeat-spacing.ingstructureevery35.5nm.
558560/CHAPTER49Myosinsconstituteafamilyofproteins,withatlightchain.Skeletalmusclemyosinbindsactintoformleast15membershavingbeenidentified.Themyosinactomyosin(actin-myosin),anditsintrinsicATPaseac-discussedinthischapterismyosin-II,andwhenmyosintivityismarkedlyenhancedinthiscomplex.Isoformsisreferredtointhistext,itisthisspeciesthatismeantofmyosinexistwhoseamountscanvaryindifferentunlessotherwiseindicated.Myosin-Iisamonomericanatomic,physiologic,andpathologicsituations.speciesthatbindstocellmembranes.ItmayserveasaThestructuresofactinandoftheheadofmyosinlinkagebetweenmicrofilamentsandthecellmembranehavebeendeterminedbyx-raycrystallography;theseincertainlocations.studieshaveconfirmedanumberofearlierfindingsMyosincontributes55%ofmuscleproteinbyconcerningtheirstructuresandhavealsogivenrisetoweightandformsthethickfilaments.Itisanasymmet-muchnewinformation.richexamerwithamolecularmassofapproximately460kDa.Myosinhasafibroustailconsistingoftwoin-LimitedDigestionofMyosinWithtertwinedhelices.Eachhelixhasaglobularheadpor-ProteasesHasHelpedtoElucidatetionattachedatoneend(Figure49–4).ThehexamerItsStructure&Functionconsistsofonepairofheavy(H)chainseachofap-proximately200kDAmolecularmass,andtwopairsofWhenmyosinisdigestedwithtrypsin,twomyosinlight(L)chainseachwithamolecularmassofapproxi-fragments(meromyosins)aregenerated.Lightmero-mately20kDa.TheLchainsdiffer,onebeingcalledmyosin(LMM)consistsofaggregated,insolubleα-he-theessentiallightchainandtheothertheregulatorylicalfibersfromthetailofmyosin(Figure49–4).LMMLLLLLLGGGGHMMS-1GG9nmLLLLLLPAPAINHMMTRYPSINHMMS-2134nmLMM85nmFigure49–4.Diagramofamyosinmoleculeshowingthetwointertwinedα-helices(fibrousportion),theglobularregionorhead(G),thelightchains(L),andtheeffectsofproteolyticcleavagebytrypsinandpapain.Theglobularregion(myosinhead)containsanactin-bindingsiteandanLchain-bindingsiteandalsoattachestotheremainderofthemyosinmolecule.
559MUSCLE&THECYTOSKELETON/561exhibitsnoATPaseactivityanddoesnotbindtointhepowerstroke,whichdrivesmovementofactinF-actin.filamentspastmyosinfilaments.TheenergyfortheHeavymeromyosin(HMM;molecularmassaboutpowerstrokeisultimatelysuppliedbyATP,whichis340kDa)isasolubleproteinthathasbothafibroushydrolyzedtoADPandPi.However,thepowerstrokeportionandaglobularportion(Figure49–4).Itex-itselfoccursasaresultofconformationalchangesinhibitsATPaseactivityandbindstoF-actin.DigestionthemyosinheadwhenADPleavesit.ofHMMwithpapaingeneratestwosubfragments,S-1ThemajorbiochemicaleventsoccurringduringoneandS-2.TheS-2fragmentisfibrousincharacter,hascycleofmusclecontractionandrelaxationcanberepre-noATPaseactivity,anddoesnotbindtoF-actin.sentedinthefivestepsshowninFigure49–6:S-1(molecularmassapproximately115kDa)doesexhibitATPaseactivity,bindsLchains,andintheab-(1)Intherelaxationphaseofmusclecontraction,senceofATPwillbindtoanddecorateactinwith“ar-theS-1headofmyosinhydrolyzesATPtoADPandPi,rowheads”(Figure49–5).BothS-1andHMMexhibitbuttheseproductsremainbound.TheresultantADP-ATPaseactivity,whichisaccelerated100-to200-foldbyPi-myosincomplexhasbeenenergizedandisinaso-complexingwithF-actin.Asdiscussedbelow,F-actincalledhigh-energyconformation.greatlyenhancestherateatwhichmyosinATPasere-(2)Whencontractionofmuscleisstimulated(vialeasesitsproducts,ADPandP.Thus,althoughF-actin2+ieventsinvolvingCa,troponin,tropomyosin,anddoesnotaffectthehydrolysisstepperse,itsabilitytoactin,whicharedescribedbelow),actinbecomesacces-promotereleaseoftheproductsproducedbytheATPasesibleandtheS-1headofmyosinfindsit,bindsit,andactivitygreatlyacceleratestheoverallrateofcatalysis.formstheactin-myosin-ADP-Picomplexindicated.(3)Formationofthiscomplexpromotesthere-CHANGESINTHECONFORMATIONleaseofPi,whichinitiatesthepowerstroke.Thisisfol-OFTHEHEADOFMYOSINDRIVElowedbyreleaseofADPandisaccompaniedbyalargeMUSCLECONTRACTIONconformationalchangeintheheadofmyosininrela-tiontoitstail(Figure49–7),pullingactinabout10nmHowcanhydrolysisofATPproducemacroscopictowardthecenterofthesarcomere.Thisisthepowermovement?Musclecontractionessentiallyconsistsofstroke.Themyosinisnowinaso-calledlow-energythecyclicattachmentanddetachmentoftheS-1headofstate,indicatedasactin-myosin.myosintotheF-actinfilaments.Thisprocesscanalsobe(4)AnothermoleculeofATPbindstotheS-1head,referredtoasthemakingandbreakingofcross-bridges.forminganactin-myosin-ATPcomplex.Theattachmentofactintomyosinisfollowedbycon-(5)Myosin-ATPhasalowaffinityforactin,andformationalchangeswhichareofparticularimportanceactinisthusreleased.Thislaststepisakeycompo-intheS-1headandaredependentuponwhichnu-nentofrelaxationandisdependentuponthebindingcleotideispresent(ADPorATP).ThesechangesresultofATPtotheactin-myosincomplex.ActinATP-MyosinHO251Actin-MyosinATPATP4ADP-Pi-MyosinActin-MyosinActin23ADP+Actin-MyosinPiADP-PiFigure49–6.ThehydrolysisofATPdrivesthecyclicFigure49–5.ThedecorationofactinfilamentswithassociationanddissociationofactinandmyosininfivetheS-1fragmentsofmyosintoform“arrowheads.”reactionsdescribedinthetext.(ModifiedfromStryerL:(CourtesyofJASpudich.)Biochemistry,2nded.Freeman,1981.)
560562/CHAPTER49ADP.Thehingeregionsofmyosin(referredtoasflexi-blepointsateachendofS-2inthelegendtoFigure149–7)permitthelargerangeofmovementofS-1andalsoallowS-1tofindactinfilaments.IfintracellularlevelsofATPdrop(eg,afterdeath),ThickfilamentATPisnotavailabletobindtheS-1head(step4LMMabove),actindoesnotdissociate,andrelaxation(step5)2S-2S-1doesnotoccur.Thisistheexplanationforrigormor-tis,thestiffeningofthebodythatoccursafterdeath.CalculationshaveindicatedthattheefficiencyofThinfilamentcontractionisabout50%;thatoftheinternalcombus-tionengineislessthan20%.3Tropomyosin&theTroponinComplexPresentinThinFilamentsPerformKeyFigure49–7.Representationoftheactivecross-FunctionsinStriatedMusclebridgesbetweenthickandthinfilaments.ThisdiagramwasadaptedbyAFHuxleyfromHEHuxley:TheInstriatedmuscle,therearetwootherproteinsthatareminorintermsoftheirmassbutimportantintermsofmechanismofmuscularcontraction.Sciencetheirfunction.Tropomyosinisafibrousmoleculethat1969;164:1356.Thelatterproposedthattheforcein-consistsoftwochains,alphaandbeta,thatattachtovolvedinmuscularcontractionoriginatesinatendencyF-actininthegroovebetweenitsfilaments(Figure49–3).forthemyosinhead(S-1)torotaterelativetothethinTropomyosinispresentinallmuscularandmuscle-likefilamentandistransmittedtothethickfilamentbythestructures.ThetroponincomplexisuniquetostriatedS-2portionofthemyosinmoleculeactingasaninex-muscleandconsistsofthreepolypeptides.TroponinTtensiblelink.FlexiblepointsateachendofS-2permit(TpT)bindstotropomyosinaswellastotheothertwoS-1torotateandallowforvariationsintheseparationtroponincomponents.TroponinI(TpI)inhibitsthebetweenfilaments.ThepresentfigureisbasedonHEF-actin-myosininteractionandalsobindstotheotherHuxley’sproposalbutalsoincorporateselastic(thecoilscomponentsoftroponin.TroponinC(TpC)isacal-intheS-2portion)andstepwise-shorteningelementscium-bindingpolypeptidethatisstructurallyandfunc-(depictedhereasfoursitesofinteractionbetweenthetionallyanalogoustocalmodulin,animportantcal-S-1portionandthethinfilament).(SeeHuxleyAF,Sim-cium-bindingproteinwidelydistributedinnature.monsRM:ProposedmechanismofforcegenerationinFourmoleculesofcalciumionareboundpermoleculestriatedmuscle.Nature[Lond]1971;233:533.)TheoftroponinCorcalmodulin,andbothmoleculeshavestrengthsofbindingoftheattachedsitesarehigherinamolecularmassof17kDa.position2thaninposition1andhigherinposition3thanposition2.ThemyosinheadcanbedetachedfromCa2+PlaysaCentralRoleinRegulationposition3withtheutilizationofamoleculeofATP;thisofMuscleContractionisthepredominantprocessduringshortening.TheThecontractionofmusclesfromallsourcesoccursbymyosinheadisseentovaryinitspositionfromaboutthegeneralmechanismdescribedabove.Musclesfrom90°toabout45°,asindicatedinthetext.(S-1,myosindifferentorganismsandfromdifferentcellsandtissueshead;S-2,portionofthemyosinmolecule;LMM,lightwithinthesameorganismmayhavedifferentmolecularmeromyosin)(seelegendtoFigure49–4).(Reproducedmechanismsresponsiblefortheregulationoftheircon-fromHuxleyAF:Muscularcontraction.JPhysiol1974;2+tractionandrelaxation.Inallsystems,Caplaysakey243:1.BykindpermissionoftheauthorandtheJournalofregulatoryrole.TherearetwogeneralmechanismsofPhysiology.)regulationofmusclecontraction:actin-basedandmyosin-based.Theformeroperatesinskeletalandcar-diacmuscle,thelatterinsmoothmuscle.AnothercyclethencommenceswiththehydrolysisofATP(step1ofFigure49–6),re-formingthehigh-Actin-BasedRegulationOccursenergyconformation.inStriatedMuscleThus,hydrolysisofATPisusedtodrivethecycle,withtheactualpowerstrokebeingtheconformationalActin-basedregulationofmuscleoccursinvertebratechangeintheS-1headthatoccursuponthereleaseofskeletalandcardiacmuscles,bothstriated.Inthegen-
561MUSCLE&THECYTOSKELETON/563eralmechanismdescribedabove(Figure49–6),theTtubuleonlypotentiallylimitingfactorinthecycleofmuscleSarcolemmacontractionmightbeATP.TheskeletalmusclesystemDihydropyridineisinhibitedatrest;thisinhibitionisrelievedtoactivatereceptorcontraction.Theinhibitorofstriatedmuscleisthetro-Ca2+releaseponinsystem,whichisboundtotropomyosinandCalsequestrinchannelF-actininthethinfilament(Figure49–3).Instriatedmuscle,thereisnocontrolofcontractionunlesstheCa2+tropomyosin-troponinsystemsarepresentalongwith2+Catheactinandmyosinfilaments.Asdescribedabove,CisternaCa2+tropomyosinliesalongthegrooveofF-actin,andCa2+Cisternathethreecomponentsoftroponin—TpT,TpI,andTpC—areboundtotheF-actin–tropomyosincomplex.Ca2+ATPaseTpIpreventsbindingofthemyosinheadtoitsF-actinCalsequestrin2+attachmentsiteeitherbyalteringtheconformationofCaF-actinviathetropomyosinmoleculesorbysimply2+CarollingtropomyosinintoapositionthatdirectlyblocksthesitesonF-actintowhichthemyosinheadsattach.EitherwaypreventsactivationofthemyosinATPasethatismediatedbybindingofthemyosinheadtoSarcomereF-actin.Hence,theTpIsystemblocksthecontractionFigure49–8.Diagramoftherelationshipsamongcycleatstep2ofFigure49–6.Thisaccountsforthein-thesarcolemma(plasmamembrane),aTtubule,andhibitedstateofrelaxedstriatedmuscle.twocisternaeofthesarcoplasmicreticulumofskeletalmuscle(nottoscale).TheTtubuleextendsinwardfromTheSarcoplasmicReticulumthesarcolemma.Awaveofdepolarization,initiatedbyRegulatesIntracellularLevelsanerveimpulse,istransmittedfromthesarcolemma2+ofCainSkeletalMuscledowntheTtubule.ItisthenconveyedtotheCa2+re-Inthesarcoplasmofrestingmuscle,theconcentrationleasechannel(ryanodinereceptor),perhapsbyinterac-ofCa2+is10−8to10−7mol/L.Therestingstateistionbetweenitandthedihydropyridinereceptor(slow2+Ca2+voltagechannel),whichareshownincloseprox-achievedbecauseCaispumpedintothesarcoplasmic2+2+reticulumthroughtheactionofanactivetransportsys-imity.ReleaseofCafromtheCareleasechannelinto2+thecytosolinitiatescontraction.Subsequently,Ca2+istem,calledtheCaATPase(Figure49–8),initiatingrelaxation.Thesarcoplasmicreticulumisanetworkofpumpedbackintothecisternaeofthesarcoplasmicfinemembranoussacs.Insidethesarcoplasmicreticu-2+2+reticulumbytheCaATPase(Capump)andstored2+2+lum,CaisboundtoaspecificCa-bindingproteinthere,inpartboundtocalsequestrin.designatedcalsequestrin.Thesarcomereissurroundedbyanexcitablemembrane(theTtubulesystem)com-posedoftransverse(T)channelscloselyassociatedwiththesarcoplasmicreticulum.receptor,RYR1andRYR2,theformerbeingpresentinWhenthesarcolemmaisexcitedbyanerveimpulse,skeletalmuscleandthelatterinheartmuscleandbrain.thesignalistransmittedintotheTtubulesystemandaRyanodineisaplantalkaloidthatbindstoRYR1and2+Careleasechannelinthenearbysarcoplasmicreticu-RYR2specificallyandmodulatestheiractivities.The2+2+lumopens,releasingCafromthesarcoplasmicreticu-Careleasechannelisahomotetramermadeupoffour2+lumintothesarcoplasm.TheconcentrationofCainsubunitsofkDa565.Ithastransmembranesequences−52+thesarcoplasmrisesrapidlyto10mol/L.TheCa-atitscarboxylterminal,andtheseprobablyformthe2+bindingsitesonTpCinthethinfilamentarequicklyCachannel.Theremainderoftheproteinprotrudes2+2+occupiedbyCa.TheTpC-4CainteractswithTpIintothecytosol,bridgingthegapbetweenthesar-andTpTtoaltertheirinteractionwithtropomyosin.coplasmicreticulumandthetransversetubularmem-2+Accordingly,tropomyosinmovesoutofthewayoral-brane.Thechannelisligand-gated,CaandATPterstheconformationofF-actinsothatthemyosinworkingsynergisticallyinvitro,althoughhowitoper-head-ADP-Pi(Figure49–6)caninteractwithF-actintoatesinvivoisnotclear.Apossiblesequenceofeventsstartthecontractioncycle.leadingtoopeningofthechannelisshowninFigure2+TheCareleasechannelisalsoknownastheryan-49–9.Thechannelliesveryclosetothedihydropyri-odinereceptor(RYR).Therearetwoisoformsofthisdinereceptor(DHPR;avoltage-gatedslowKtype
562564/CHAPTER49DepolarizationofnerveTable49–1summarizestheoveralleventsincon-tractionandrelaxationofskeletalmuscle.Depolarizationofskeletalmuscle2+MutationsintheGeneEncodingtheCaReleaseChannelAreOneCauseofHumanDepolarizationofthetransversetubularmembraneMalignantHyperthermiaSomegeneticallypredisposedpatientsexperiencease-ChargemovementoftheslowCa2+voltageverereaction,designatedmalignanthyperthermia,onchannel(DHPR)ofthetransversetubularmembraneexposuretocertainanesthetics(eg,halothane)andde-polarizingskeletalmusclerelaxants(eg,succinyl-OpeningoftheCa2+releasechannel(RYR1)choline).Thereactionconsistsprimarilyofrigidityofskeletalmuscles,hypermetabolism,andhighfever.AFigure49–9.PossiblechainofeventsleadingtohighcytosolicconcentrationofCa2+inskeletalmus-2+openingoftheCareleasechannel.Asindicatedinthecleisamajorfactorinitscausation.Unlessmalignant2+2+text,theCavoltagechannelandtheCareleasehyperthermiaisrecognizedandtreatedimmediately,channelhavebeenshowntointeractwitheachotherinpatientsmaydieacutelyofventricularfibrillationorvitroviaspecificregionsintheirpolypeptidechains.survivetosuccumbsubsequentlyfromotherserious(DHPR,dihydropyridinereceptor;RYR1,ryanodinere-complications.Appropriatetreatmentistostoptheceptor1.)anestheticandadministerthedrugdantroleneintra-venously.Dantroleneisaskeletalmusclerelaxantthat2+actstoinhibitreleaseofCafromthesarcoplasmicreticulumintothecytosol,thuspreventingtheincreaseCa2+channel)ofthetransversetubulesystem(FigureofcytosolicCa2+foundinmalignanthyperthermia.49–8).Experimentsinvitroemployinganaffinitycol-umnchromatographyapproachhaveindicatedthata37-amino-acidstretchinRYR1interactswithonespe-cificloopofDHPR.Table49–1.Sequenceofeventsincontraction2+RelaxationoccurswhensarcoplasmicCafallsandrelaxationofskeletalmuscle.1−7below10mol/Lowingtoitsresequestrationintothe2+2+sarcoplasmicreticulumbyCaATPase.TpC.4Ca2+StepsincontractionthuslosesitsCa.Consequently,troponin,viainterac-(1)Dischargeofmotorneurontionwithtropomyosin,inhibitsfurthermyosinhead(2)Releaseoftransmitter(acetylcholine)atmotorend-andF-actininteraction,andinthepresenceofATPtheplatemyosinheaddetachesfromtheF-actin.(3)Bindingofacetylcholinetonicotinicacetylcholinere-2+Thus,Cacontrolsskeletalmusclecontractionandceptorsrelaxationbyanallostericmechanismmediatedby(4)IncreasedNa+andK+conductanceinendplatemem-TpC,TpI,TpT,tropomyosin,andF-actin.braneAdecreaseintheconcentrationofATPinthesar-(5)Generationofendplatepotentialcoplasm(eg,byexcessiveusageduringthecycleofcon-(6)Generationofactionpotentialinmusclefiberstraction-relaxationorbydiminishedformation,suchas(7)InwardspreadofdepolarizationalongTtubulesmightoccurinischemia)hastwomajoreffects:(1)The2+(8)ReleaseofCafromterminalcisternsofsarcoplasmicCa2+ATPase(Ca2+pump)inthesarcoplasmicreticu-reticulumanddiffusiontothickandthinfilaments2+(9)BindingofCa2+totroponinC,uncoveringmyosinlumceasestomaintainthelowconcentrationofCainthesarcoplasm.Thus,theinteractionofthemyosinbindingsitesofactinheadswithF-actinispromoted.(2)TheATP-depen-(10)Formationofcross-linkagesbetweenactinanddentdetachmentofmyosinheadsfromF-actincannotmyosinandslidingofthinonthickfilaments,produc-occur,andrigidity(contracture)setsin.Theconditioningshorteningofrigormortis,followingdeath,isanextensionofStepsinrelaxation2+theseevents.(1)Capumpedbackintosarcoplasmicreticulum2+Musclecontractionisadelicatedynamicbalanceof(2)ReleaseofCafromtroponin(3)Cessationofinteractionbetweenactinandmyosintheattachmentanddetachmentofmyosinheadsto1F-actin,subjecttofineregulationviathenervousReproduced,withpermission,fromGanongWF:ReviewofMed-system.icalPhysiology,21sted.McGraw-Hill,2003.
563MUSCLE&THECYTOSKELETON/565Malignanthyperthermiaalsooccursinswine.Sus-MutationsintheRYR1geneceptibleanimalshomozygousformalignanthyperther-miarespondtostresswithafatalreaction(porcineAlteredCa2+releasechannelprotein(RYR1)stresssyndrome)similartothatexhibitedbyhumans.(eg,substitutionofCysforArg615)Ifthereactionoccurspriortoslaughter,itaffectsthequalityoftheporkadversely,resultinginaninferiorproduct.Botheventscanresultinconsiderableeco-Mutatedchannelopensmoreeasilyandstaysopennomiclossesfortheswineindustry.longer,thusfloodingthecytosolwithCa2+2+ThefindingofahighlevelofcytosolicCainmus-cleinmalignanthyperthermiasuggestedthatthecon-2+2+HighintracellularlevelsofCastimulatesustainedditionmightbecausedbyabnormalitiesoftheCa2+musclecontraction(rigidity);highCaalsostimulates2+ATPaseoroftheCareleasechannel.Noabnormali-breakdownofglycogen,glycolysis,andaerobictiesweredetectedintheformer,butsequencingofmetabolism(resultinginexcessiveproductionofheat)cDNAsforthelatterproteinprovedinsightful,particu-larlyinswine.AllcDNAsfromswinewithmalignantFigure49–10.Simplifiedschemeofthecausationofhyperthermiasofarexaminedhaveshownasubstitu-malignanthyperthermia(MIM145600).Atleast17dif-tionofTforC1843,resultinginthesubstitutionofferentpointmutationshavebeendetectedintheRYR16152+CysforArgintheCareleasechannel.Themuta-gene,someofwhichareassociatedwithcentralcoretionaffectsthefunctionofthechannelinthatitopensdisease(MIM117000).Itisestimatedthatatleast50%moreeasilyandremainsopenlonger;thenetresultis2+offamilieswithmemberswhohavemalignanthyper-massivereleaseofCaintothecytosol,ultimatelycaus-thermiaarelinkedtotheRYR1gene.Someindividualsingsustainedmusclecontraction.withmutationsinthegeneencodingDHPRhavealsoThepictureismorecomplexinhumans,sincema-beendetected;itispossiblethatmutationsinotherlignanthyperthermiaexhibitsgeneticheterogeneity.Membersofanumberoffamilieswhosufferfromma-genesforproteinsinvolvedincertainaspectsofmusclelignanthyperthermiahavenotshowngeneticlinkagemetabolismwillalsobefound.totheRYR1gene.Somehumanssusceptibletomalig-nanthyperthermiahavebeenfoundtoexhibitthesamemutationfoundinswine,andothershaveavari-etyofpointmutationsatdifferentlociintheRYR1MUTATIONSINTHEGENEENCODINGgene.CertainfamilieswithmalignanthypertensionDYSTROPHINCAUSEDUCHENNEhavebeenfoundtohavemutationsaffectingtheMUSCULARDYSTROPHYDHPR.Figure49–10summarizestheprobablechainofeventsinmalignanthyperthermia.ThemajorAnumberofadditionalproteinsplayvariousrolesinpromiseofthesefindingsisthat,onceadditionalmu-thestructureandfunctionofmuscle.Theyincludetitintationsaredetected,itwillbepossibletoscreen,using(thelargestproteinknown),nebulin,α-actinin,desmin,suitableDNAprobes,forindividualsatriskofdevel-dystrophin,andcalcineurin.Somepropertiesoftheseopingmalignanthyperthermiaduringanesthesia.Cur-proteinsaresummarizedinTable49–2.rentscreeningtests(eg,theinvitrocaffeine-halothaneDystrophinisofspecialinterest.Mutationsinthetest)arerelativelyunreliable.Affectedindividualsgeneencodingthisproteinhavebeenshowntobethecouldthenbegivenalternativeanesthetics,whichcauseofDuchennemusculardystrophyandthemilderwouldnotendangertheirlives.Itshouldalsobepossi-Beckermusculardystrophy(seeFigure49–11).Theyble,ifdesired,toeliminatemalignanthyperthermiaarealsoimplicatedinsomecasesofdilatedcardiomy-fromswinepopulationsusingsuitablebreedingprac-opathy(seebelow).Thegeneencodingdystrophinistices.thelargestgeneknown(≈2300kb)andissituatedonAnotherconditionduetomutationsintheRYR1theXchromosome,accountingforthematernalinheri-geneiscentralcoredisease.ThisisararemyopathytancepatternofDuchenneandBeckermusculardys-presentingininfancywithhypotoniaandproximaltrophies.AsshowninFigure49–12,dystrophinformsmuscleweakness.Electronmicroscopyrevealsanab-partofalargecomplexofproteinsthatattachtoorin-senceofmitochondriainthecenterofmanytypeI(seeteractwiththeplasmalemma.Dystrophinlinksthebelow)musclefibers.DamagetomitochondriainducedactincytoskeletontotheECMandappearstobe2+neededforassemblyofthesynapticjunction.Impair-byhighintracellularlevelsofCasecondarytoabnor-malfunctioningofRYR1appearstoberesponsibleformentoftheseprocessesbyformationofdefectivedys-themorphologicfindings.trophinispresumablycriticalinthecausationof
564566/CHAPTER49Table49–2.SomeotherimportantproteinsDuchennemusculardystrophy.Mutationsinthegenesofmuscle.encodingsomeofthecomponentsofthesarcoglycancomplexshowninFigure49–12areresponsibleforlimb-girdleandcertainothercongenitalformsofmus-ProteinLocationCommentorFunctionculardystrophy.TitinReachesfromtheZLargestproteininbody.linetotheMlineRoleinrelaxationofmuscle.CARDIACMUSCLERESEMBLESSKELETALNebulinFromZlinealongMayregulateassemblyMUSCLEINMANYRESPECTSlengthofactinandlengthofactinThegeneralpictureofmusclecontractionintheheartfilamentsfilaments.resemblesthatofskeletalmuscle.Cardiacmuscle,likeα-ActininAnchorsactintoZStabilizesactinskeletalmuscle,isstriatedandusestheactin-myosin-linesfilaments.tropomyosin-troponinsystemdescribedabove.Unlikeskeletalmuscle,cardiacmuscleexhibitsintrinsicrhyth-DesminLiesalongsideactinAttachestoplasmamicity,andindividualmyocytescommunicatewithfilamentsmembrane(plasma-eachotherbecauseofitssyncytialnature.TheTtubu-lemma).larsystemismoredevelopedincardiacmuscle,DystrophinAttachedtoplasma-DeficientinDuchennewhereasthesarcoplasmicreticulumislessextensivelemmamusculardystrophy.andconsequentlytheintracellularsupplyofCa2+forMutationsofitsgenecontractionisless.Cardiacmusclethusreliesonextra-canalsocausedilatedcellularCa2+forcontraction;ifisolatedcardiacmusclecardiomyopathy.isdeprivedofCa2+,itceasestobeatwithinapproxi-CalcineurinCytosolAcalmodulin-regulatedmately1minute,whereasskeletalmusclecancontinueproteinphosphatase.2+tocontractwithoutanextracellularsourceofCa.MayplayimportantCyclicAMPplaysamoreprominentroleincardiacrolesincardiachyper-thaninskeletalmuscle.ItmodulatesintracellularlevelstrophyandinregulatingofCa2+throughtheactivationofproteinkinases;theseamountsofslowandenzymesphosphorylatevarioustransportproteinsinfasttwitchmuscles.thesarcolemmaandsarcoplasmicreticulumandalsoinMyosin-Arrangedtrans-Bindsmyosinandtitin.thetroponin-tropomyosinregulatorycomplex,affect-2+bindingverselyinsarcomerePlaysaroleinmain-ingintracellularlevelsofCaorresponsestoit.ThereproteinCA-bandstainingthestructuralisaroughcorrelationbetweenthephosphorylationofintegrityofthesarco-TpIandtheincreasedcontractionofcardiacmusclein-mere.ducedbycatecholamines.Thismayaccountforthein-otropiceffects(increasedcontractility)ofβ-adrenergiccompoundsontheheart.Somedifferencesamongskeletal,cardiac,andsmoothmusclearesummarizedinTable49–3.Deletionofpartofthestructuralgenefordystrophin,locatedontheXchromosome2+2+CaEntersMyocytesviaCaChannels&LeavesviatheNa+-Ca2+ExchangerDiminishedsynthesisofthemRNAfordystrophin&theCa2+ATPase2+Asstatedabove,extracellularCaplaysanimportantLowlevelsorabsenceofdystrophinroleincontractionofcardiacmusclebutnotinskeletal2+muscle.ThismeansthatCabothentersandleavesMusclecontraction/relaxationaffected;myocytesinaregulatedmanner.Weshallbrieflycon-precisemechanismsnotelucidatedsiderthreetransmembraneproteinsthatplayrolesinthisprocess.Progressive,usuallyfatalmuscularweakness2+A.CaCHANNELS2+Figure49–11.SummaryofthecausationofCaentersmyocytesviathesechannels,whichallow2+Duchennemusculardystrophy(MIM310200).entryonlyofCaions.Themajorportalofentryisthe
565MUSCLE&THECYTOSKELETON/567Figure49–12.Organizationofdystrophinandotherproteinsinrelationtotheplasmamembraneofmusclecells.Dystrophinispartofalargeoligomericcomplexassociatedwithseveralotherproteincomplexes.Thedystroglycancomplexconsistsofα-dystroglycan,whichassociateswiththebasallaminaproteinmerosin,andβ-dystroglycan,whichbindsα-dystroglycananddystrophin.Syntrophinbindstothecarboxylterminalofdystrophin.Thesarcogly-cancomplexconsistsoffourtransmembraneproteins:α-,β-,γ-,andδ-sarcoglycan.Thefunctionofthesarcoglycancomplexandthenatureoftheinteractionswithinthecomplexandbetweenitandtheothercomplexesarenotclear.Thesarcoglycancomplexisformedonlyinstriatedmuscle,anditssubunitspreferentiallyassociatewitheachother,suggestingthatthecomplexmayfunctionasasingleunit.MutationsinthegeneencodingdystrophincauseDuchenneandBeckermusculardystrophy;mutationsinthegenesencodingthevarioussarcoglycanshavebeenshowntoberesponsibleforlimb-girdledystrophies(eg,MIM601173).(Reproduced,withpermission,fromDugganDJetal:Mutationsinthesarcoglycangenesinpatientswithmyopathy.NEnglJMed1997;336:618.)L-type(long-durationcurrent,largeconductance)or(CICR).Itisestimatedthatapproximately10%ofthe2+2+slowCachannel,whichisvoltage-gated,openingCainvolvedincontractionentersthecytosolfromduringdepolarizationinducedbyspreadofthecardiactheextracellularfluidand90%fromthesarcoplasmicactionpotentialandclosingwhentheactionpotentialreticulum.However,theformer10%isimportant,as2+declines.Thesechannelsareequivalenttothedihy-therateofincreaseofCainthemyoplasmisimpor-2+dropyridinereceptorsofskeletalmuscle(Figure49–8).tant,andentryviatheCachannelscontributesappre-2+SlowCachannelsareregulatedbycAMP-dependentciablytothis.proteinkinases(stimulatory)andcGMP-proteinki-nases(inhibitory)andareblockedbyso-calledcalcium2++channelblockers(eg,verapamil).Fast(orT,transient)B.Ca-NaEXCHANGERCa2+channelsarealsopresentintheplasmalemma,ThisistheprincipalrouteofexitofCa2+frommyo-thoughinmuchlowernumbers;theyprobablycon-cytes.Inrestingmyocytes,ithelpstomaintainalowtributetotheearlyphaseofincreaseofmyoplasmicleveloffreeintracellularCa2+byexchangingoneCa2+Ca2+.forthreeNa+.TheenergyfortheuphillmovementofTheresultantincreaseofCa2+inthemyoplasmactsCa2+outofthecellcomesfromthedownhillmove-ontheCa2+releasechannelofthesarcoplasmicreticu-+mentofNaintothecellfromtheplasma.Thisex-2+2+lumtoopenit.ThisiscalledCa-inducedCareleasechangecontributestorelaxationbutmayruninthere-
566568/CHAPTER49Table49–3.Somedifferencesbetweenskeletal,cardiac,andsmoothmuscle.SkeletalMuscleCardiacMuscleSmoothMuscle1.Striated.1.Striated.1.Nonstriated.2.Nosyncytium.2.Syncytial.2.Syncytial.3.SmallTtubules.3.LargeTtubules.3.GenerallyrudimentaryTtubules.4.Sarcoplasmicreticulumwell-4.Sarcoplasmicreticulumpresentand4.Sarcoplasmicreticulumoftenrudimen-2+2+2+developedandCapumpactsCapumpactsrelativelyrapidly.taryandCapumpactsslowly.rapidly.5.Plasmalemmalacksmanyhormone5.Plasmalemmacontainsavarietyof5.Plasmalemmacontainsavarietyofreceptors.receptors(eg,α-andβ-adrenergic).receptors(eg,α-andβ-adrenergic).6.Nerveimpulseinitiatescontraction.6.Hasintrinsicrhythmicity.6.Contractioninitiatedbynerveimpulses,hormones,etc.2+2+2+7.ExtracellularfluidCanotimportant7.ExtracellularfluidCaimportant7.ExtracellularfluidCaimportantforforcontraction.forcontraction.contraction.8.Troponinsystempresent.8.Troponinsystempresent.8.Lackstroponinsystem;usesregulatoryheadofmyosin.9.Caldesmonnotinvolved.9.Caldesmonnotinvolved.9.Caldesmonisimportantregulatoryprotein.10.Veryrapidcyclingofthe10.Relativelyrapidcyclingofthecross-10.Slowcyclingofthecross-bridgesper-cross-bridges.bridges.mitsslowprolongedcontractionandlessutilizationofATP.versedirectionduringexcitation.BecauseoftheCa2+-importantinskeletalmuscle.Mutationsingenesen-Na+exchanger,anythingthatcausesintracellularNa+codingionchannelshavebeenshowntoberesponsible(Na+)torisewillsecondarilycauseCa2+torise,caus-foranumberofrelativelyrareconditionsaffectingmus-iiingmoreforcefulcontraction.Thisisreferredtoasacle.Theseandotherdiseasesduetomutationsofionpositiveinotropiceffect.Oneexampleiswhenthedrugchannelshavebeentermedchannelopathies;somearedigitalisisusedtotreatheartfailure.DigitalisinhibitslistedinTable49–5.thesarcolemmalNa+-K+ATPase,diminishingexitofNa+andthusincreasingNa+.ThisinturncausesCa2+itoincrease,viatheCa2+-Na+exchanger.Theincreased2+Table49–4.MajortypesofionchannelsfoundCairesultsinincreasedforceofcardiaccontraction,ofincells.benefitinheartfailure.2+C.CaATPASETypeComment2+ThisCapump,situatedinthesarcolemma,alsocon-2+ExternalOpeninresponsetoaspecificextracellulartributestoCaexitbutisbelievedtoplayarelativelyligand-gatedmolecule,eg,acetylcholine.minorroleascomparedwiththeCa2+-Na+exchanger.ItshouldbenotedthatthereareavarietyofionInternalOpenorcloseinresponsetoaspecificintra-channels(Chapter41)inmostcells,forNa+,K+,Ca2+,ligand-gatedcellularmolecule,eg,acyclicnucleotide.etc.ManyofthemhavebeenclonedinrecentyearsandVoltage-gatedOpeninresponsetoachangeinmembranetheirdispositionsintheirrespectivemembranesworkedpotential,eg,Na+,K+,andCa2+channelsinout(numberoftimeseachonecrossesitsmembrane,heart.locationoftheactualiontransportsiteintheprotein,MechanicallyOpeninresponsetochangeinmechanicaletc).TheycanbeclassifiedasindicatedinTable49–4.gatedpressure.Cardiacmuscleisrichinionchannels,andtheyarealso
567MUSCLE&THECYTOSKELETON/569Table49–5.Somedisorders(channelopathies)Table49–6.Biochemicalcausesofinherited1,2duetomutationsingenesencodingpolypeptidecardiomyopathies.1constituentsofionchannels.ProteinsorProcessIonChannelandMajorCauseAffected2DisorderOrgansInvolvedInbornerrorsoffattyacidCarnitineentryintocellsandCentralcorediseaseCa2+releasechannel(RYR1)oxidationmitochondria(MIM117000)SkeletalmuscleCertainenzymesoffattyacidoxidation−CysticfibrosisCFTR(Clchannel)(MIM219700)Lungs,pancreasDisordersofmitochondrialProteinsencodedbymito-oxidativephosphorylationchondrialgenesHyperkalemicperiodicSodiumchannelProteinsencodedbynuclearparalysis(MIM170500)Skeletalmusclegenes2+HypokalemicperiodicSlowCavoltagechannel(DHPR)Abnormalitiesofmyocardialβ-Myosinheavychains,tropo-paralysis(MIM114208)Skeletalmusclecontractileandstructuralnin,tropomyosin,dys-MalignanthyperthermiaCa2+releasechannel(RYR1)proteinstrophin(MIM180901)Skeletalmuscle1BasedonKellyDP,StraussAW:Inheritedcardiomyopathies.MyotoniacongenitaChloridechannelNEnglJMed1994;330:913.2Mutations(eg,pointmutations,orinsomecasesdeletions)in(MIM160800)Skeletalmusclethegenes(nuclearormitochondrial)encodingvariousproteins,1DatainpartfromAckermanNJ,ClaphamDE:Ionchannels—enzymes,ortRNAmoleculesarethefundamentalcausesofthebasicscienceandclinicaldisease.NEnglJMed1997;336:1575.inheritedcardiomyopathies.Someconditionsaremild,whereas2OtherchannelopathiesincludethelongQTsyndrome(MIMothersaresevereandmaybepartofasyndromeaffectingother192500);pseudoaldosteronism(Liddlesyndrome,MIM177200);tissues.persistenthyperinsulinemichypoglycemiaofinfancy(MIM601820);hereditaryX-linkedrecessivetypeIInephrolithiasisofin-fancy(Dentsyndrome,MIM300009);andgeneralizedmyotonia,recessive(Beckerdisease,MIM255700).Theterm“myotonia”sig-nifiesanyconditioninwhichmusclesdonotrelaxaftercontrac-MutationsintheCardiac-MyosinHeavytion.ChainGeneAreOneCauseofFamilialHypertrophicCardiomyopathyFamilialhypertrophiccardiomyopathyisoneoftheInheritedCardiomyopathiesAreDuemostfrequenthereditarycardiacdiseases.Patientsex-toDisordersofCardiacEnergyhibithypertrophy—oftenmassive—ofoneorbothven-MetabolismortoAbnormaltricles,startingearlyinlife,andnotrelatedtoanyex-trinsiccausesuchashypertension.MostcasesareMyocardialProteinstransmittedinanautosomaldominantmanner;therestAninheritedcardiomyopathyisanystructuralorfunc-aresporadic.Untilrecently,itscausewasobscure.How-tionalabnormalityoftheventricularmyocardiumdueever,thissituationchangedwhenstudiesofoneaffectedtoaninheritedcause.Therearenonheritabletypesoffamilyshowedthatamissensemutation(ie,substitu-cardiomyopathy,butthesewillnotbedescribedhere.tionofoneaminoacidbyanother)intheβ-myosinAsshowninTable49–6,thecausesofinheritedcar-heavychaingenewasresponsibleforthecondition.diomyopathiesfallintotwobroadclasses:(1)disordersSubsequentstudieshaveshownanumberofmissenseofcardiacenergymetabolism,mainlyreflectingmuta-mutationsinthisgene,allcodingforhighlyconservedtionsingenesencodingenzymesorproteinsinvolvedinresidues.Someindividualshaveshownothermutations,fattyacidoxidation(amajorsourceofenergyforthesuchasformationofanα/β-myosinheavychainhybridmyocardium)andoxidativephosphorylation;andgene.Patientswithfamilialhypertrophiccardiomyopa-(2)mutationsingenesencodingproteinsinvolvedinorthycanshowgreatvariationinclinicalpicture.Thisinaffectingmyocardialcontraction,suchasmyosin,partreflectsgeneticheterogeneity;ie,mutationinatropomyosin,thetroponins,andcardiacmyosin-numberofothergenes(eg,thoseencodingcardiacbindingproteinC.Mutationsinthegenesencodingactin,tropomyosin,cardiactroponinsIandT,essentialtheselatterproteinscausefamilialhypertrophiccar-andregulatorymyosinlightchains,andcardiacmyosin-diomyopathy,whichwillnowbediscussed.bindingproteinC)mayalsocausefamilialhypertrophic
568570/CHAPTER49cardiomyopathy.Inaddition,mutationsatdifferentintheregulationofanumberofgenesinthesecells.sitesinthegeneforβ-myosinheavychainmayaffecttheCurrentresearchisnotonlyelucidatingthemolecularfunctionoftheproteintoagreaterorlesserextent.Thecausesofthecardiomyopathiesbutisalsodisclosingmissensemutationsareclusteredintheheadandhead-mutationsthatcausecardiacdevelopmentaldisordersrodregionsofmyosinheavychain.Onehypothesisis(eg,septaldefects)andarrhythmias(eg,duetomuta-thatthemutantpolypeptides(“poisonpolypeptides”)tionsaffectingionchannels).causeformationofabnormalmyofibrils,eventuallyre-sultingincompensatoryhypertrophy.SomemutationsCa2+AlsoRegulatesContractionalterthechargeoftheaminoacid(eg,substitutionofofSmoothMusclearginineforglutamine),presumablyaffectingthecon-formationoftheproteinmoremarkedlyandthusaffect-Whileallmusclescontainactin,myosin,andtropo-ingitsfunction.Patientswiththesemutationshaveamyosin,onlyvertebratestriatedmusclescontainthesignificantlyshorterlifeexpectancythanpatientsintroponinsystem.Thus,themechanismsthatregulatewhomthemutationproducednoalterationincharge.contractionmustdifferinvariouscontractilesystems.Thus,definitionoftheprecisemutationsinvolvedintheSmoothmuscleshavemolecularstructuressimilartogenesisofFHCmayprovetobeofimportantprognos-thoseinstriatedmuscle,butthesarcomeresarenotticvalue;itcanbeaccomplishedbyappropriateuseofalignedsoastogeneratethestriatedappearance.thepolymerasechainreactionongenomicDNAob-Smoothmusclescontainα-actininandtropomyosintainedfromonesampleofbloodlymphocytes.Figuremolecules,asdoskeletalmuscles.Theydonothavethe49–13isasimplifiedschemeoftheeventscausingfa-troponinsystem,andthelightchainsofsmoothmusclemilialhypertrophiccardiomyopathy.myosinmoleculesdifferfromthoseofstriatedmuscleAnothertypeofcardiomyopathyistermeddilatedmyosin.Regulationofsmoothmusclecontractioniscardiomyopathy.Mutationsinthegenesencodingdys-myosin-based,unlikestriatedmuscle,whichisactin-trophin,muscleLIMprotein(socalledbecauseitwasbased.However,likestriatedmuscle,smoothmuscle2+foundtocontainacysteine-richdomainoriginallyde-contractionisregulatedbyCa.tectedinthreeproteins:Lin-II,Isl-1,andMec-3),andthecyclicresponse-elementbindingprotein(CREB)PhosphorylationofMyosinLightChainshavebeenimplicatedinthecausationofthiscondition.InitiatesContractionofSmoothMuscleThefirsttwoproteinshelporganizethecontractileap-paratusofcardiacmusclecells,andCREBisinvolvedWhensmoothmusclemyosinisboundtoF-actinintheabsenceofothermuscleproteinssuchastropomyosin,thereisnodetectableATPaseactivity.Thisabsenceofactivityisquiteunlikethesituationdescribedforstri-Predominantlymissensemutationsintheβ-myosinatedmusclemyosinandF-actin,whichhasabundantheavychaingeneonchromosome14ATPaseactivity.SmoothmusclemyosincontainslightchainsthatpreventthebindingofthemyosinheadtoMutantpolypeptidechains(“poisonpolypeptides”)F-actin;theymustbephosphorylatedbeforetheyallowthatleadtoformationofdefectivemyofibrilsF-actintoactivatemyosinATPase.TheATPaseactivitythenattainedhydrolyzesATPabouttenfoldmoreslowlythanthecorrespondingactivityinskeletalmus-Compensatoryhypertrophyofonecle.Thephosphateonthemyosinlightchainsmayformorbothcardiacventricles2+achelatewiththeCaboundtothetropomyosin-TpC-actincomplex,leadingtoanincreasedrateofformationCardiomegalyandvariouscardiacsignsandofcross-bridgesbetweenthemyosinheadsandactin.symptoms,includingsuddendeathThephosphorylationoflightchainsinitiatestheattach-ment-detachmentcontractioncycleofsmoothmuscle.Figure49–13.Simplifiedschemeofthecausationoffamilialhypertrophiccardiomyopathy(MIM192600)MyosinLightChainKinaseIsActivatedduetomutationsinthegeneencodingβ-myosinheavy2+byCalmodulin-4Ca&Thenchain.Mutationsingenesencodingotherproteins,PhosphorylatestheLightChainssuchasthetroponins,tropomyosin,andcardiacmyosin-bindingproteinCcanalsocausethiscondition.SmoothmusclesarcoplasmcontainsamyosinlightMutationsingenesencodingyetotherproteins(eg,chainkinasethatiscalcium-dependent.TheCa2+acti-dystrophin)areinvolvedinthecausationofdilatedvationofmyosinlightchainkinaserequiresbindingofcardiomyopathy.2+calmodulin-4Catoitskinasesubunit(Figure49–14).
569MUSCLE&THECYTOSKELETON/571CalmodulinTable49–7summarizesandcomparestheregula-Myosinkinasetionofactin-myosininteractions(activationofmyosin(inactive)10–5mol/LCa2+10–7mol/LCa2+ATPase)instriatedandsmoothmuscles.Themyosinlightchainkinaseisnotdirectlyaf-Ca2+•calmodulinfectedoractivatedbycAMP.However,cAMP-acti-vatedproteinkinasecanphosphorylatethemyosinlightchainkinase(notthelightchainsthemselves).Thephosphorylatedmyosinlightchainkinaseexhibitsasig-2+nificantlyloweraffinityforcalmodulin-CaandthusisATPCa2+•CALMODULIN–MYOSINlesssensitivetoactivation.Accordingly,anincreaseinKINASE(ACTIVE)cAMPdampensthecontractionresponseofsmooth2+muscletoagivenelevationofsarcoplasmicCa.ThisL-myosin(inhibitsADPmolecularmechanismcanexplaintherelaxingeffectofmyosin-actininteraction)β-adrenergicstimulationonsmoothmuscle.2+AnotherproteinthatappearstoplayaCa-depen-dentroleintheregulationofsmoothmusclecontrac-pL-myosin(doesnottioniscaldesmon(87kDa).Thisproteinisubiquitousinhibitmyosin-actininteraction)insmoothmuscleandisalsofoundinnonmuscletis-2+sue.AtlowconcentrationsofCa,itbindstotro-HPO–pomyosinandactin.Thispreventsinteractionofactin24withmyosin,keepingmuscleinarelaxedstate.At2+2+higherconcentrationsofCa,Ca-calmodulinbindsPHOSPHATASEcaldesmon,releasingitfromactin.ThelatteristhenFigure49–14.Regulationofsmoothmusclecon-freetobindtomyosin,andcontractioncanoccur.tractionbyCa2+.pL-myosinisthephosphorylatedlightCaldesmonisalsosubjecttophosphorylation-dephos-phorylation;whenphosphorylated,itcannotbindchainofmyosin;L-myosinisthedephosphorylatedactin,againfreeingthelattertointeractwithmyosin.lightchain.(AdaptedfromAdelsteinRS,EisenbergR:Reg-Caldesmonmayalsoparticipateinorganizingthestruc-ulationandkineticsofactin-myosinATPinteraction.Annutureofthecontractileapparatusinsmoothmuscle.RevBiochem1980;49:921.)Manyofitseffectshavebeendemonstratedinvitro,anditsphysiologicsignificanceisstillunderinvestiga-tion.AsnotedinTable49–3,slowcyclingofthecross-Thecalmodulin-4Ca2+-activatedlightchainkinasebridgespermitsslowprolongedcontractionofsmoothphosphorylatesthelightchains,whichthenceasestoin-muscle(eg,invisceraandbloodvessels)withlessuti-hibitthemyosin–F-actininteraction.ThecontractionlizationofATPcomparedwithstriatedmuscle.Thecyclethenbegins.abilityofsmoothmuscletomaintainforceatreducedvelocitiesofcontractionisreferredtoasthelatchstate;SmoothMuscleRelaxesWhenthisisanimportantfeatureofsmoothmuscle,andits2+precisemolecularbasesareunderstudy.theConcentrationofCaFalls−7Below10MolarNitricOxideRelaxestheSmoothMuscleRelaxa2+tionofsmoot−h7muscleoccursw2+hensarcoplasmicofBloodVessels&AlsoHasManyOtherCafallsbelow10mol/L.TheCadissociatesfromImportantBiologicFunctionscalmodulin,whichinturndissociatesfromthemyosinlightchainkinase,inactivatingthekinase.NonewAcetylcholineisavasodilatorthatactsbycausingrelax-phosphatesareattachedtothep-lightchain,andlightationofthesmoothmuscleofbloodvessels.However,chainproteinphosphatase,whichiscontinuallyactiveitdoesnotactdirectlyonsmoothmuscle.Akeyobser-andcalcium-independent,removestheexistingphos-vationwasthatifendothelialcellswerestrippedawayphatesfromthelightchains.Dephosphorylatedmyosinfromunderlyingsmoothmusclecells,acetylcholinenop-lightchaintheninhibitsthebindingofmyosinheadslongerexerteditsvasodilatoreffect.Thisfindingindi-toF-actinandtheATPaseactivity.Themyosinheadcatedthatvasodilatorssuchasacetylcholineinitiallyin-detachesfromtheF-actininthepresenceofATP,butteractwiththeendothelialcellsofsmallbloodvesselsitcannotreattachbecauseofthepresenceofdephos-viareceptors.Thereceptorsarecoupledtothephos-phorylatedp-lightchain;hence,relaxationoccurs.phoinositidecycle,leadingtotheintracellularreleaseof
570572/CHAPTER49Table49–7.Actin-myosininteractionsinstriatedandsmoothmuscle.SmoothMuscleStriatedMuscle(andNonmuscleCells)ProteinsofmusclefilamentsActinActin1MyosinMyosinTropomyosinTropomyosinTroponin(Tpl,TpT,TpC)SpontaneousinteractionofF-actinandYesNomyosinalone(spontaneousactivationofmyosinATPasebyF-actinInhibitorofF-actin–myosininteraction(in-Troponinsystem(Tpl)UnphosphorylatedmyosinlightchainhibitorofF-actin–dependentactivationofATPase)2+2+ContractionactivatedbyCaCa2+2+2+DirecteffectofCa4CabindtoTpC4Cabindtocalmodulin2+2+2+Effectofprotein-boundCaTpC⋅4CaantagonizesTplinhibitionCalmodulin⋅4CaactivatesmyosinlightofF-actin–myosininteraction(allowschainkinasethatphosphorylatesmyosinF-actinactivationofATPase)p-lightchain.Thephosphorylatedp-lightchainnolongerinhibitsF-actin–myosininteraction(allowsF-actinactivationofATPase).1Lightchainsofmyosinaredifferentinstriatedandsmoothmuscles.Ca2+throughtheactionofinositoltrisphosphate.Inzyme.NOsynthaseisaverycomplexenzyme,employ-turn,theelevationofCa2+leadstotheliberationofen-ingfiveredoxcofactors:NADPH,FAD,FMN,heme,dothelium-derivedrelaxingfactor(EDRF),whichandtetrahydrobiopterin.NOcanalsobeformedfromdiffusesintotheadjacentsmoothmuscle.There,itre-nitrite,derivedfromvasodilatorssuchasglyceryltrini-actswiththehememoietyofasolubleguanylylcyclase,trateduringtheirmetabolism.NOhasaveryshortresultinginactivationofthelatter,withaconsequenthalf-life(approximately3–4seconds)intissuesbecauseelevationofintracellularlevelsofcGMP(Figureitreactswithoxygenandsuperoxide.Theproductof−49–15).Thisinturnstimulatestheactivitiesofcertainthereactionwithsuperoxideisperoxynitrite(ONOO),•cGMP-dependentproteinkinases,whichprobablywhichdecomposestoformthehighlyreactiveOHphosphorylatespecificmuscleproteins,causingrelax-radical.NOisinhibitedbyhemoglobinandotheration;however,thedetailsarestillbeingclarified.Thehemeproteins,whichbindittightly.Chemicalin-importantcoronaryarteryvasodilatornitroglycerin,hibitorsofNOsynthasearenowavailablethatcanwidelyusedtorelieveanginapectoris,actstoincreasemarkedlydecreaseformationofNO.AdministrationofintracellularreleaseofEDRFandthusofcGMP.suchinhibitorstoanimalsandhumansleadstovaso-Quiteunexpectedly,EDRFwasfoundtobethegasconstrictionandamarkedelevationofbloodpressure,nitricoxide(NO).NOisformedbytheactionoftheindicatingthatNOisofmajorimportanceinthemain-enzymeNOsynthase,whichiscytosolic.Theendothe-tenanceofbloodpressureinvivo.AnotherimportantlialandneuronalformsofNOsynthaseareactivatedbycardiovasculareffectisthatbyincreasingsynthesisofCa2+(Table49–8).Thesubstrateisarginine,andthecGMP,itactsasaninhibitorofplateletaggregationproductsarecitrullineandNO:(Chapter51).SincethediscoveryoftheroleofNOasavasodila-NOSYNTHASEtor,therehasbeenintenseexperimentalinterestinthisArginineCitrulline+NOsubstance.Ithasturnedouttohaveavarietyofphysio-logicroles,involvingvirtuallyeverytissueofthebodyNOsynthasecatalyzesafive-electronoxidationof(Table49–9).ThreemajorisoformsofNOsynthaseanamidinenitrogenofarginine.L-Hydroxyarginineishavebeenidentified,eachofwhichhasbeencloned,anintermediatethatremainstightlyboundtotheen-andthechromosomallocationsoftheirgenesinhu-
571MUSCLE&THECYTOSKELETON/573GlycerylAcetylcholinephosphate,and(4)fromtwomoleculesofADPinare-trinitrateactioncatalyzedbyadenylylkinase(Figure49–16).TheamountofATPinskeletalmuscleisonlysufficienttoRENDOTHELIALprovideenergyforcontractionforafewseconds,soCELLthatATPmustbeconstantlyrenewedfromoneorArgininemoreoftheabovesources,dependinguponmetabolicconditions.Asdiscussedbelow,thereareatleasttwo↑Ca2++NOsynthasedistincttypesoffibersinskeletalmuscle,onepredomi-nantlyactiveinaerobicconditionsandtheotherinNO+Citrullineanaerobicconditions;notunexpectedly,theyuseeachoftheabovesourcesofenergytodifferentextents.SkeletalMuscleContainsLargeGTPSuppliesofGlycogenGuanylylThesarcoplasmofskeletalmusclecontainslargestoresNitrateNitriteNO+cyclaseofglycogen,locatedingranulesclosetotheIbands.ThereleaseofglucosefromglycogenisdependentonacGMPcGMPproteinspecificmuscleglycogenphosphorylase(Chapter18),kinaseswhichcanbeactivatedbyCa2+,epinephrine,andAMP.Togenerateglucose6-phosphateforglycolysisinskele-+talmuscle,glycogenphosphorylasebmustbeactivatedtophosphorylaseaviaphosphorylationbyphosphory-RelaxationSMOOTHMUSCLECELLlasebkinase(Chapter18).Ca2+promotestheactiva-tionofphosphorylasebkinase,alsobyphosphoryla-Figure49–15.Diagramshowingformationinanen-tion.Thus,Ca2+bothinitiatesmusclecontractionanddothelialcellofnitricoxide(NO)fromarginineinare-activatesapathwaytoprovidenecessaryenergy.TheactioncatalyzedbyNOsynthase.Interactionofanago-hormoneepinephrinealsoactivatesglycogenolysisinnist(eg,acetylcholine)withareceptor(R)probablymuscle.AMP,producedbybreakdownofADPduring2+leadstointracellularreleaseofCaviainositoltrisphos-muscularexercise,canalsoactivatephosphorylasebphategeneratedbythephosphoinositidepathway,re-withoutcausingphosphorylation.MuscleglycogensultinginactivationofNOsynthase.TheNOsubse-phosphorylasebisinactiveinMcArdledisease,oneofquentlydiffusesintoadjacentsmoothmuscle,whereittheglycogenstoragediseases(Chapter18).leadstoactivationofguanylylcyclase,formationofcGMP,stimulationofcGMP-proteinkinases,andsubse-UnderAerobicConditions,Musclequentrelaxation.ThevasodilatornitroglycerinisshownGeneratesATPMainlybyOxidativeenteringthesmoothmusclecell,whereitsmetabolismPhosphorylationalsoleadstoformationofNO.SynthesisofATPviaoxidativephosphorylationre-quiresasupplyofoxygen.Musclesthathaveahighde-mandforoxygenasaresultofsustainedcontractionmanshavebeendetermined.Geneknockoutexperi-(eg,tomaintainposture)storeitattachedtothehemementshavebeenperformedoneachofthethreeiso-moietyofmyoglobin.Becauseofthehememoiety,formsandhavehelpedestablishsomeofthepostulatedmusclescontainingmyoglobinarered,whereasmusclesfunctionsofNO.withlittleornomyoglobinarewhite.Glucose,derivedTosummarize,researchinthepastdecadehasfromthebloodglucoseorfromendogenousglycogen,shownthatNOplaysanimportantroleinmanyphysi-andfattyacidsderivedfromthetriacylglycerolsofadi-ologicandpathologicprocesses.posetissuearetheprincipalsubstratesusedforaerobicmetabolisminmuscle.SEVERALMECHANISMSREPLENISHSTORESOFATPINMUSCLECreatinePhosphateConstitutesaMajorEnergyReserveinMuscleTheATPrequiredastheconstantenergysourceforthecontraction-relaxationcycleofmusclecanbegeneratedCreatinephosphatepreventstherapiddepletionof(1)byglycolysis,usingbloodglucoseormuscleglyco-ATPbyprovidingareadilyavailablehigh-energyphos-gen,(2)byoxidativephosphorylation,(3)fromcreatinephatethatcanbeusedtoregenerateATPfromADP.
572574/CHAPTER49Table49–8.SummaryofthenomenclatureoftheNOsynthasesandoftheeffectsofknockoutoftheir1genesinmice.ResultofGene23SubtypeNameCommentsKnockoutinMice2+1nNOSActivitydependsonelevatedCa.FirstPyloricstenosis,resistanttovascularstroke,aggressiveidentifiedinneurons.Calmodulin-activated.sexualbehavior(males).42+2iNOSIndependentofelevatedCa.Moresusceptibletocertaintypesofinfection.Prominentinmacrophages.2+3eNOSActivitydependsonelevatedCa.Elevatedmeanbloodpressure.Firstidentifiedinendothelialcells.1AdaptedfromSnyderSH:NoendothelialNO.Nature1995;377:196.2n,neuronal;i,inducible;e,endothelial.3Geneknockoutswereperformedbyhomologousrecombinationinmice.Theenzymesarecharacterizedasneuronal,inducible(macrophage),andendothelialbecausethesewerethesitesinwhichtheywerefirstidentified.However,allthreeenzymeshavebeenfoundinothersites,andtheneuronalenzymeisalsoinducible.Eachgenehasbeencloned,anditschromosomallocationinhumanshasbeendetermined.42+iNOSisCa-independentbutbindscalmodulinverytightly.CreatinephosphateisformedfromATPandcreatineidative)andtypeII(fasttwitch,glycolytic)(Table(Figure49–16)attimeswhenthemuscleisrelaxedand49–10).ThetypeIfibersareredbecausetheycontaindemandsforATParenotsogreat.Theenzymecatalyz-myoglobinandmitochondria;theirmetabolismisaero-ingthephosphorylationofcreatineiscreatinekinasebic,andtheymaintainrelativelysustainedcontractions.(CK),amuscle-specificenzymewithclinicalutilityinThetypeIIfibers,lackingmyoglobinandcontainingthedetectionofacuteorchronicdiseasesofmuscle.fewmitochondria,arewhite:theyderivetheirenergyfromanaerobicglycolysisandexhibitrelativelyshortdu-SKELETALMUSCLECONTAINSSLOWrationsofcontraction.Theproportionofthesetwo(RED)&FAST(WHITE)TWITCHFIBERStypesoffibersvariesamongthemusclesofthebody,de-pendingonfunction(eg,whetherornotamuscleisin-Differenttypesoffibershavebeendetectedinskeletalvolvedinsustainedcontraction,suchasmaintainingmuscle.OneclassificationsubdividesthemintotypeIposture).Theproportionalsovarieswithtraining;for(slowtwitch),typeIIA(fasttwitch-oxidative),andtypeexample,thenumberoftypeIfibersincertainlegmus-IIB(fasttwitch-glycolytic).Forthesakeofsimplicity,clesincreasesinathletestrainingformarathons,whereasweshallconsideronlytwotypes:typeI(slowtwitch,ox-thenumberoftypeIIfibersincreasesinsprinters.ASprinterUsesCreatinePhosphate&AnaerobicGlycolysistoMakeATP,Table49–9.SomephysiologicfunctionsandWhereasaMarathonRunnerUsespathologicinvolvementsofnitricoxide(NO).OxidativePhosphorylation•Vasodilator,importantinregulationofbloodpressureInviewofthetwotypesoffibersinskeletalmuscleand•Involvedinpenileerection;sildenafilcitrate(Viagra)affectsofthevariousenergysourcesdescribedabove,itisofthisprocessbyinhibitingacGMPphosphodiesteraseinteresttocomparetheirinvolvementinasprint(eg,•Neurotransmitterinthebrainandperipheralautonomic100meters)andinthemarathon(42.2km;justovernervoussystem26miles)(Table49–11).•Roleinlong-termpotentiationThemajorsourcesofenergyinthe100-msprint•Roleinneurotoxicityarecreatinephosphate(first4–5seconds)andthen•LowlevelofNOinvolvedincausationofpylorospasminin-anaerobicglycolysis,usingmuscleglycogenasthefantilehypertrophicpyloricstenosissourceofglucose.Thetwomainsitesofmetaboliccon-•MayhaveroleinrelaxationofskeletalmuscletrolareatglycogenphosphorylaseandatPFK-1.The•MayconstitutepartofaprimitiveimmunesystemformerisactivatedbyCa2+(releasedfromthesarcoplas-•Inhibitsadhesion,activation,andaggregationofplateletsmicreticulumduringcontraction),epinephrine,and
573MUSCLE&THECYTOSKELETON/575CreatinephosphateMuscleglycogenCREATINEPHOSPHOKINASEADPMUSCLEPHOSPHORYLASECreatineGlucose6-PGLYCOLYSISMuscleATPcontractionMYOSINOXIDATIVEATPasePHOSPHORYLATIONADP+PiAMPADPADENYLYLKINASEFigure49–16.ThemultiplesourcesofATPinmuscle.AMP.PFK-1isactivatedbyAMP,Pi,andNH3.Attest-ergyduringamarathonfor4minutes,18minutes,70ingtotheefficiencyoftheseprocesses,thefluxthroughminutes,andapproximately4000minutes,respec-glycolysiscanincreaseasmuchas1000-foldduringatively.However,therateofoxidationoffattyacidsbysprint.muscleisslowerthanthatofglucose,sothatoxidationIncontrast,inthemarathon,aerobicmetabolismisofglucoseandoffattyacidsarebothmajorsourcesoftheprincipalsourceofATP.Themajorfuelsourcesareenergyinthemarathon.bloodglucoseandfreefattyacids,largelyderivedfromAnumberofprocedureshavebeenusedbyathletesthebreakdownoftriacylglycerolsinadiposetissue,tocounteractmusclefatigueandinadequatestrength.stimulatedbyepinephrine.Hepaticglycogenisde-Theseincludecarbohydrateloading,soda(sodiumbi-gradedtomaintainthelevelofbloodglucose.Muscleglycogenisalsoafuelsource,butitisdegradedmuchmoregraduallythaninasprint.Ithasbeencalculatedthattheamountsofglucoseintheblood,ofglycogeninTable49–11.Typesofmusclefibersandmajortheliver,ofglycogeninmuscle,andoftriacylglycerolinfuelsourcesusedbyasprinterandbyamarathonadiposetissuearesufficienttosupplymusclewithen-runner.Sprinter(100m)MarathonRunnerTable49–10.CharacteristicsoftypeIandtypeIITypeII(glycolytic)fibersareTypeI(oxidative)fibersarefibersofskeletalmuscle.usedpredominantly.usedpredominantly.CreatinephosphateistheATPisthemajorenergyTypeITypeIImajorenergysourcedur-sourcethroughout.SlowTwitchFastTwitchingthefirst4–5seconds.MyosinATPaseLowHighGlucosederivedfrommuscleBloodglucoseandfreefattyEnergyutilizationLowHighglycogenandmetabolizedacidsarethemajorfuelMitochondriaManyFewbyanaerobicglycolysisissources.ColorRedWhitethemajorfuelsource.MyoglobinYesNoContractionrateSlowFastMuscleglycogenisrapidlyMuscleglycogenisslowlyDurationProlongedShortdepleted.depleted.
574576/CHAPTER49carbonate)loading,blooddoping(administrationofTable49–12.Summaryofmajorfeaturesofredbloodcells),andingestionofcreatineandan-thebiochemistryofskeletalmusclerelatedtodrostenedione.Theirrationalesandefficacieswillnotitsmetabolism.1bediscussedhere.•Skeletalmusclefunctionsunderbothaerobic(resting)andSKELETALMUSCLECONSTITUTESanaerobic(eg,sprinting)conditions,sobothaerobicandTHEMAJORRESERVEOFanaerobicglycolysisoperate,dependingonconditions.PROTEININTHEBODY•Skeletalmusclecontainsmyoglobinasareservoirofoxy-gen.Inhumans,skeletalmuscleproteinisthemajornonfat•Skeletalmusclecontainsdifferenttypesoffibersprimarilysourceofstoredenergy.Thisexplainstheverylargesuitedtoanaerobic(fasttwitchfibers)oraerobic(slowlossesofmusclemass,particularlyinadults,resultingtwitchfibers)conditions.fromprolongedcaloricundernutrition.•Actin,myosin,tropomyosin,troponincomplex(TpT,Tpl,2+Thestudyoftissueproteinbreakdowninvivoisdif-andTpC),ATP,andCaarekeyconstituentsinrelationtoficult,becauseaminoacidsreleasedduringintracellularcontraction.2+2+breakdownofproteinscanbeextensivelyreutilizedfor•TheCaATPase,theCareleasechannel,andcalse-2+proteinsynthesiswithinthecell,ortheaminoacidsquestrinareproteinsinvolvedinvariousaspectsofCame-maybetransportedtootherorganswheretheyentertabolisminmuscle.anabolicpathways.However,actinandmyosinare•Insulinactsonskeletalmuscletoincreaseuptakeofglu-methylatedbyaposttranslationalreaction,formingcose.3-methylhistidine.Duringintracellularbreakdownof•Inthefedstate,mostglucoseisusedtosynthesizeglyco-gen,whichactsasastoreofglucoseforuseinexercise;actinandmyosin,3-methylhistidineisreleasedandex-“preloading”withglucoseisusedbysomelong-distancecretedintotheurine.Theurinaryoutputofthemethy-athletestobuildupstoresofglycogen.latedaminoacidprovidesareliableindexoftherateof•Epinephrinestimulatesglycogenolysisinskeletalmuscle,myofibrillarproteinbreakdowninthemusculatureofwhereasglucagondoesnotbecauseofabsenceofitsre-humansubjects.ceptors.Variousfeaturesofmusclemetabolism,mostof•Skeletalmusclecannotcontributedirectlytobloodglucosewhicharedealtwithinotherchaptersofthistext,arebecauseitdoesnotcontainglucose-6-phosphatase.summarizedinTable49–12.•Lactateproducedbyanaerobicmetabolisminskeletalmus-clepassestoliver,whichusesittosynthesizeglucose,THECYTOSKELETONPERFORMSwhichcanthenreturntomuscle(theCoricycle).MULTIPLECELLULARFUNCTIONS•Skeletalmusclecontainsphosphocreatine,whichactsasanenergystoreforshort-term(seconds)demands.Nonmusclecellsperformmechanicalwork,including•Freefattyacidsinplasmaareamajorsourceofenergy,par-self-propulsion,morphogenesis,cleavage,endocytosis,ticularlyundermarathonconditionsandinprolongedstar-exocytosis,intracellulartransport,andchangingcellvation.shape.Thesecellularfunctionsarecarriedoutbyanex-•Skeletalmusclecanutilizeketonebodiesduringstarvation.tensiveintracellularnetworkoffilamentousstructures•Skeletalmuscleistheprincipalsiteofmetabolismofconstitutingthecytoskeleton.Thecellcytoplasmisbranched-chainaminoacids,whichareusedasanenergynotasacoffluid,asoncethought.Essentiallyalleu-source.karyoticcellscontainthreetypesoffilamentousstruc-•Proteolysisofmuscleduringstarvationsuppliesaminoacidsforgluconeogenesis.tures:actinfilaments(7–9.5nmindiameter;also•Majoraminoacidsemanatingfrommusclearealanine(des-knownasmicrofilaments),microtubules(25nm),andtinedmainlyforgluconeogenesisinliverandformingpartintermediatefilaments(10–12nm).Eachtypeoffila-oftheglucose-alaninecycle)andglutamine(destinedmentcanbedistinguishedbiochemicallyandbythemainlyforthegutandkidneys).electronmicroscope.1Thistablebringstogethermaterialfromvariouschaptersinthisbook.NonmuscleCellsContainActinThatFormsMicrofilamentsG-actinispresentinmostifnotallcellsofthebody.Withappropriateconcentrationsofmagnesiumandpotassiumchloride,itspontaneouslypolymerizestoformdoublehelicalF-actinfilamentslikethoseseeninmuscle.Thereareatleasttwotypesofactininnonmus-
575MUSCLE&THECYTOSKELETON/577clecells:β-actinandγ-actin.Bothtypescancoexistindynamin,andmyosinsarereferredtoasmolecularthesamecellandprobablyevencopolymerizeinthemotors.samefilament.Inthecytoplasm,F-actinformsmicro-Anabsenceofdyneininciliaandflagellaresultsinfilamentsof7–9.5nmthatfrequentlyexistasbundlesimmotileciliaandflagella,leadingtomalesterilityandofatangled-appearingmeshwork.Thesebundlesarechronicrespiratoryinfection,aconditionknownasprominentjustunderlyingtheplasmamembraneofKartagenersyndrome.manycellsandaretherereferredtoasstressfibers.TheCertaindrugsbindtomicrotubulesandthusinter-stressfibersdisappearascellmotilityincreasesoruponferewiththeirassemblyordisassembly.Theseincludemalignanttransformationofcellsbychemicalsoronco-colchicine(usedfortreatmentofacutegoutyarthritis),genicviruses.vinblastine(avincaalkaloidusedfortreatingcertainAlthoughnotorganizedasinmuscle,actinfilamentstypesofcancer),paclitaxel(Taxol)(effectiveagainstinnonmusclecellsinteractwithmyosintocausecellu-ovariancancer),andgriseofulvin(anantifungalagent).larmovements.IntermediateFilamentsDifferFromMicrofilaments&MicrotubulesMicrotubulesContain-&-TubulinsAnintracellularfibroussystemexistsoffilamentswithMicrotubules,anintegralcomponentofthecellularcy-anaxialperiodicityof21nmandadiameterof8–10toskeleton,consistofcytoplasmictubes25nmindiam-nmthatisintermediatebetweenthatofmicrofilamentseterandoftenofextremelength.Microtubulesarenec-(6nm)andmicrotubules(23nm).Fourclassesofinter-essaryfortheformationandfunctionofthemitoticmediatefilamentsarefound,asindicatedinTablespindleandthusarepresentinalleukaryoticcells.49–13.Theyareallelongated,fibrousmolecules,withTheyarealsoinvolvedintheintracellularmovementofacentralroddomain,anaminoterminalhead,andaendocyticandexocyticvesiclesandformthemajorcarboxylterminaltail.Theyformastructurelikeastructuralcomponentsofciliaandflagella.Micro-rope,andthematurefilamentsarecomposedoftubulesareamajorcomponentofaxonsanddendrites,tetramerspackedtogetherinahelicalmanner.Theyareinwhichtheymaintainstructureandparticipateintheimportantstructuralcomponentsofcells,andmostareaxoplasmicflowofmaterialalongtheseneuronalrelativelystablecomponentsofthecytoskeleton,notprocesses.undergoingrapidassemblyanddisassemblyandnotMicrotubulesarecylindersof13longitudinallyarrangedprotofilaments,eachconsistingofdimersofα-tubulinandβ-tubulin,closelyrelatedproteinsofap-proximately50kDamolecularmass.ThetubulindimersassembleintoprotofilamentsandsubsequentlyTable49–13.Classesofintermediatefilamentsofintosheetsandthencylinders.Amicrotubule-organiz-eukaryoticcellsandtheirdistributions.ingcenter,locatedaroundapairofcentrioles,nucleatesthegrowthofnewmicrotubules.AthirdspeciesofMoleculartubulin,γ-tubulin,appearstoplayanimportantroleinProteinsMassDistributionsthisassembly.GTPisrequiredforassembly.Avarietyofproteinsareassociatedwithmicrotubules(micro-Keratinstubule-associatedproteins[MAPs],oneofwhichistau)TypeI(acidic)40–60kDaEpithelialcells,hair,andplayimportantrolesinmicrotubuleassemblyandTypeII(basic)50–70kDanailsstabilization.MicrotubulesareinastateofdynamicVimentin-likeinstability,constantlyassemblinganddisassembling.Vimentin54kDaVariousmesenchymalTheyexhibitpolarity(plusandminusends);thisisim-cellsportantintheirgrowthfromcentriolesandintheirDesmin53kDaMuscleabilitytodirectintracellularmovement.Forinstance,Glialfibrillaryacid50kDaGlialcellsinaxonaltransport,theproteinkinesin,withaproteinmyosin-likeATPaseactivity,useshydrolysisofATPtoPeripherin66kDaNeuronsmovevesiclesdowntheaxontowardthepositiveendofNeurofilamentsthemicrotubularformation.FlowofmaterialsintheLow(L),medium(M),60–130kDaNeuronsoppositedirection,towardthenegativeend,ispoweredandhigh(H)1bycytosolicdynein,anotherproteinwithATPaseac-tivity.Similarly,axonemaldyneinspowerciliaryandLaminsflagellarmovement.Anotherprotein,dynamin,usesA,B,andC65–75kDaNuclearlamina1GTPandisinvolvedinendocytosis.Kinesins,dyneins,Referstotheirmolecularmasses.
576578/CHAPTER49disappearingduringmitosis,asdoactinandmanymi-cle,thesarcoplasmicreticulumregulatesdistribution2+2+crotubularfilaments.AnimportantexceptiontothisisofCatothesarcomeres,whereasinflowofCavia2+providedbythelamins,which,subsequenttophosphor-Cachannelsinthesarcolemmaisofmajorimpor-ylation,disassembleatmitosisandreappearwhenitter-tanceincardiacandsmoothmuscle.minates.•ManycasesofmalignanthyperthermiainhumansKeratinsformalargefamily,withabout30mem-areduetomutationsinthegeneencodingtheCa2+bersbeingdistinguished.AsindicatedinTable49–13,releasechannel.twomajortypesofkeratinsarefound;allindividual•Anumberofdifferencesexistbetweenskeletalandkeratinsareheterodimersmadeupofonememberofcardiacmuscle;inparticular,thelattercontainsava-eachclass.rietyofreceptorsonitssurface.Vimentinsarewidelydistributedinmesodermal•Somecasesoffamilialhypertrophiccardiomyopathycells,anddesmin,glialfibrillaryacidicprotein,andpe-areduetomissensemutationsinthegenecodingforripherinarerelatedtothem.Allmembersofthevi-β-myosinheavychain.mentin-likefamilycancopolymerizewitheachother.Intermediatefilamentsareveryprominentinnerve•Smoothmuscle,unlikeskeletalandcardiacmuscle,cells;neurofilamentsareclassifiedaslow,medium,anddoesnotcontainthetroponinsystem;instead,phos-highonthebasisoftheirmolecularmasses.Laminsphorylationofmyosinlightchainsinitiatescontrac-formameshworkinappositiontotheinnernucleartion.membrane.Thedistributionofintermediatefilaments•Nitricoxideisaregulatorofvascularsmoothmuscle;innormalandabnormal(eg,cancer)cellscanbestud-blockageofitsformationfromargininecausesaniedbytheuseofimmunofluorescenttechniques,usingacuteelevationofbloodpressure,indicatingthatreg-antibodiesofappropriatespecificities.Theseantibodiesulationofbloodpressureisoneofitsmanyfunc-tospecificintermediatefilamentscanalsobeofusetotions.pathologistsinhelpingtodecidetheoriginofcertain•Duchenne-typemusculardystrophyisduetomuta-dedifferentiatedmalignanttumors.Thesetumorsmaytionsinthegene,locatedontheXchromosome,en-stillretainthetypeofintermediatefilamentsfoundincodingtheproteindystrophin.theircelloforigin.•Twomajortypesofmusclefibersarefoundinhu-Anumberofskindiseases,mainlycharacterizedbymans:white(anaerobic)andred(aerobic).Thefor-blistering,havebeenfoundtobeduetomutationsinmerareparticularlyusedinsprintsandthelatteringenesencodingvariouskeratins.Threeofthesedisor-prolongedaerobicexercise.Duringasprint,muscledersareepidermolysisbullosasimplex,epidermolyticusescreatinephosphateandglycolysisasenergyhyperkeratosis,andepidermolyticpalmoplantarkerato-sources;inthemarathon,oxidationoffattyacidsisderma.Theblisteringprobablyreflectsadiminishedca-ofmajorimportanceduringthelaterphases.pacityofvariouslayersoftheskintoresistmechanical•Nonmusclecellsperformvarioustypesofmechanicalstressesduetoabnormalitiesinmicrofilamentstructure.workcarriedoutbythestructuresconstitutingthecytoskeleton.ThesestructuresincludeactinfilamentsSUMMARY(microfilaments),microtubules(composedprimarily•Themyofibrilsofskeletalmusclecontainthickandofα-tubulinandβ-tubulin),andintermediatefila-thinfilaments.Thethickfilamentscontainmyosin.ments.Thelatterincludekeratins,vimentin-likepro-Thethinfilamentscontainactin,tropomyosin,andteins,neurofilaments,andlamins.thetroponincomplex(troponinsT,I,andC).•Theslidingfilamentcross-bridgemodelisthefoun-dationofcurrentthinkingaboutmusclecontraction.REFERENCESThebasisofthismodelisthattheinterdigitatingfila-AckermanMJ,ClaphamDE:Ionchannels—basicscienceandclin-mentsslidepastoneanotherduringcontractionandicaldisease.NEnglJMed1997;336:1575.cross-bridgesbetweenmyosinandactingenerateandAndreoliTE:Iontransportdisorders:introductorycomments.Amsustainthetension.JMed1998;104:85.(Firstofaseriesofarticlesoniontrans-•ThehydrolysisofATPisusedtodrivemovementofportdisorderspublishedbetweenJanuaryandAugust,1998.thefilaments.ATPbindstomyosinheadsandishy-Topicscoveredwerestructureandfunctionofionchannels,drolyzedtoADPandPibytheATPaseactivityofthearrhythmiasandantiarrhythmicdrugs,Liddlesyndrome,cholera,malignanthyperthermia,cysticfibrosis,theperiodicactomyosincomplex.paralysesandBarttersyndrome,andGittelmansyndrome.)2+•Caplaysakeyroleintheinitiationofmusclecon-FullerGM,ShieldsD:MolecularBasisofMedicalCellBiology.Ap-tractionbybindingtotroponinC.Inskeletalmus-pleton&Lange,1998.
577MUSCLE&THECYTOSKELETON/579GeevesMA,HolmesKC:Structuralmechanismofmusclecontrac-MayerB,HemmensB:Biosynthesisandactionofnitricoxideintion.AnnuRevBiochem1999;68:728.mammaliancells.TrendsBiochemSci1998;22:477.HilleB:IonChannelsofExcitableMembranes.Sinauer,2001.ScriverCRetal(editors):TheMetabolicandMolecularBasesofIn-HowardJ:MechanicsofMotorProteinsandtheCytoskeleton.Sin-heritedDisease,8thed.McGraw-Hill,2001.(Thiscompre-auer,2001.hensivefour-volumetextcontainscoverageofmalignanthy-perthermia[Chapter9],channelopathies[Chapter204],LodishHetal(editors):MolecularCellBiology,4thed.Freeman,hypertrophiccardiomyopathy[Chapter213],themuscular2000.(Chapters18and19ofthistextcontaincomprehen-dystrophies[Chapter216],anddisordersofintermediatefila-sivedescriptionsofcellmotilityandcellshape.)mentsandtheirassociatedproteins[Chapter221].)LokeJ,MacLennanDH:Malignanthyperthermiaandcentralcore2+disease:disordersofCareleasechannels.AmJMed1998;104:470.
578PlasmaProteins&Immunoglobulins50RobertK.Murray,MD,PhDBIOMEDICALIMPORTANCEsolventsorelectrolytes(orboth)toremovedifferentproteinfractionsinaccordancewiththeirsolubilityThefundamentalroleofbloodinthemaintenanceofcharacteristics.Thisisthebasisoftheso-calledsalting-homeostasisandtheeasewithwhichbloodcanbeob-outmethods,whichfindsomeusageinthedetermina-tainedhavemeantthatthestudyofitsconstituentshastionofproteinfractionsintheclinicallaboratory.beenofcentralimportanceinthedevelopmentofbio-Thus,onecanseparatetheproteinsoftheplasmaintochemistryandclinicalbiochemistry.Thebasicproper-threemajorgroups—fibrinogen,albumin,andglobu-tiesofanumberofplasmaproteins,includingthelins—bytheuseofvaryingconcentrationsofsodiumimmunoglobulins(antibodies),aredescribedinthisorammoniumsulfate.chapter.Changesintheamountsofvariousplasmapro-Themostcommonmethodofanalyzingplasmateinsandimmunoglobulinsoccurinmanydiseasesandproteinsisbyelectrophoresis.Therearemanytypesofcanbemonitoredbyelectrophoresisorothersuitableelectrophoresis,eachusingadifferentsupportingprocedures.Asindicatedinanearlierchapter,alterationsmedium.Inclinicallaboratories,celluloseacetateisoftheactivitiesofcertainenzymesfoundinplasmaarewidelyusedasasupportingmedium.Itsusepermitsofdiagnosticuseinanumberofpathologicconditions.resolution,afterstaining,ofplasmaproteinsintofivebands,designatedalbumin,α1,α2,β,andγfractions,THEBLOODHASMANYFUNCTIONSrespectively(Figure50–2).Thestainedstripofcellu-loseacetate(orothersupportingmedium)iscalledanThefunctionsofblood—exceptforspecificcellularelectrophoretogram.Theamountsofthesefivebandsonessuchasoxygentransportandcell-mediatedim-canbeconvenientlyquantifiedbyuseofdensitomet-munologicdefense—arecarriedoutbyplasmaanditsricscanningmachines.Characteristicchangesintheconstituents(Table50–1).amountsofoneormoreofthesefivebandsarefoundPlasmaconsistsofwater,electrolytes,metabolites,inmanydiseases.nutrients,proteins,andhormones.Thewaterandelec-trolytecompositionofplasmaispracticallythesameasthatofallextracellularfluids.Laboratorydetermina-tionsoflevelsofNa+,K+,Ca2+,Cl−,HCO−,PaCO,TheConcentrationofProteininPlasmaIs32ImportantinDeterminingtheDistributionandofbloodpHareimportantinthemanagementofmanypatients.ofFluidBetweenBlood&TissuesInarterioles,thehydrostaticpressureisabout37mmPLASMACONTAINSACOMPLEXHg,withaninterstitial(tissue)pressureof1mmHgMIXTUREOFPROTEINSopposingit.Theosmoticpressure(oncoticpressure)exertedbytheplasmaproteinsisapproximately25mmTheconcentrationoftotalproteininhumanplasmaisHg.Thus,anetoutwardforceofabout11mmHgapproximately7.0–7.5g/dLandcomprisesthemajordrivesfluidoutintotheinterstitialspaces.Invenules,partofthesolidsoftheplasma.Theproteinsofthethehydrostaticpressureisabout17mmHg,withtheplasmaareactuallyacomplexmixturethatincludesnotoncoticandinterstitialpressuresasdescribedabove;onlysimpleproteinsbutalsoconjugatedproteinssuchthus,anetforceofabout9mmHgattractswaterbackasglycoproteinsandvarioustypesoflipoproteins.intothecirculation.Theabovepressuresareoftenre-Thousandsofantibodiesarepresentinhumanplasma,ferredtoastheStarlingforces.Iftheconcentrationofthoughtheamountofanyoneantibodyisusuallyquiteplasmaproteinsismarkedlydiminished(eg,duetose-lowundernormalcircumstances.Therelativedimen-vereproteinmalnutrition),fluidisnotattractedbacksionsandmolecularmassesofsomeofthemostimpor-intotheintravascularcompartmentandaccumulatesintantplasmaproteinsareshowninFigure50–1.theextravasculartissuespaces,aconditionknownasTheseparationofindividualproteinsfromacom-edema.Edemahasmanycauses;proteindeficiencyisplexmixtureisfrequentlyaccomplishedbytheuseofoneofthem.580
579PLASMAPROTEINS&IMMUNOGLOBULINS/581Table50–1.Majorfunctionsofblood.PlasmaProteinsHaveBeenStudiedExtensively(1)Respiration—transportofoxygenfromthelungstotheBecauseoftherelativeeasewithwhichtheycanbeob-tissuesandofCO2fromthetissuestothelungstained,plasmaproteinshavebeenstudiedextensivelyin(2)Nutrition—transportofabsorbedfoodmaterialsbothhumansandanimals.Considerableinformationis(3)Excretion—transportofmetabolicwastetothekidneys,availableaboutthebiosynthesis,turnover,structure,lungs,skin,andintestinesforremovalandfunctionsofthemajorplasmaproteins.Alterations(4)Maintenanceofthenormalacid-basebalanceinthebodyoftheiramountsandoftheirmetabolisminmanydis-(5)Regulationofwaterbalancethroughtheeffectsofeasestateshavealsobeeninvestigated.Inrecentyears,bloodontheexchangeofwaterbetweenthecirculatingmanyofthegenesforplasmaproteinshavebeenclonedfluidandthetissuefluidandtheirstructuresdetermined.(6)RegulationofbodytemperaturebythedistributionofThepreparationofantibodiesspecificfortheindi-bodyheatvidualplasmaproteinshasgreatlyfacilitatedtheir(7)Defenseagainstinfectionbythewhitebloodcellsandstudy,allowingtheprecipitationandisolationofpurecirculatingantibodiesproteinsfromthecomplexmixturepresentintissuesor(8)Transportofhormonesandregulationofmetabolismplasma.Inaddition,theuseofisotopeshasmadepos-(9)Transportofmetabolitessiblethedeterminationoftheirpathwaysofbiosynthe-(10)Coagulationsisandoftheirturnoverratesinplasma.Thefollowinggeneralizationshaveemergedfromstudiesofplasmaproteins.A.MOSTPLASMAPROTEINSARESYNTHESIZEDINTHELIVERThishasbeenestablishedbyexperimentsatthewhole-Scaleanimallevel(eg,hepatectomy)andbyuseoftheiso-latedperfusedliverpreparation,ofliverslices,ofliver+–homogenates,andofinvitrotranslationsystemsusing10nmNaCIGlucosepreparationsofmRNAextractedfromliver.However,theγ-globulinsaresynthesizedinplasmacellsandcer-tainplasmaproteinsaresynthesizedinothersites,suchAlbuminHemoglobinasendothelialcells.69,00064,500B.PLASMAPROTEINSAREGENERALLYSYNTHESIZEDONMEMBRANE-BOUNDPOLYRIBOSOMESTheythentraversethemajorsecretoryrouteinthecellβ1-Globulinγ-Globulin90,000156,000(roughendoplasmicmembrane→smoothendoplasmicmembrane→Golgiapparatus→secretoryvesicles)priortoenteringtheplasma.Thus,mostplasmaproteinsaresynthesizedaspreproteinsandinitiallycontainaminoterminalsignalpeptides(Chapter46).Theyareusuallysubjectedtovariousposttranslationalmodifications(pro-α1-Lipoproteinteolysis,glycosylation,phosphorylation,etc)astheytravel200,000throughthecell.Transittimesthroughthehepatocyteβ1-Lipoprotein1,300,000fromthesiteofsynthesistotheplasmavaryfrom30min-utestoseveralhoursormoreforindividualproteins.C.MOSTPLASMAPROTEINSAREGLYCOPROTEINSFibrinogen340,000Accordingly,theygenerallycontaineitherN-orO-linkedoligosaccharidechains,orboth(Chapter47).Al-Figure50–1.Relativedimensionsandapproximatebuministhemajorexception;itdoesnotcontainsugarmolecularmassesofproteinmoleculesinthebloodresidues.Theoligosaccharidechainshavevariousfunc-(Oncley).tions(Table47–2).Removalofterminalsialicacid
580582/CHAPTER50AC+–Albuminα1α2βγBD+–Albuminα1α2βγFigure50–2.Techniqueofcelluloseacetatezoneelectrophoresis.A:Asmallamountofserumorotherfluidisappliedtoacelluloseacetatestrip.B:Electrophoresisofsampleinelectrolytebufferisperformed.C:Separatedproteinbandsarevisualizedincharacteristicpositionsafterbeingstained.D:Densitometerscanningfromcelluloseacetatestripconvertsbandstocharacteristicpeaksofalbumin,α1-globulin,α2-glob-ulin,β-globulin,andγ-globulin.(Reproduced,withpermission,fromParslowTGetal[editors]:MedicalImmunol-ogy,10thed.McGraw-Hill,2001.)residuesfromcertainplasmaproteins(eg,ceruloplas-nondenaturingconditions.Thisisotopeunitescovalentlymin)byexposuretoneuraminidasecanmarkedlywithtyrosineresiduesintheprotein.Thelabeledproteinshortentheirhalf-livesinplasma(Chapter47).isfreedofunbound131Ianditsspecificactivity(disinte-grationsperminutepermilligramofprotein)deter-D.MANYPLASMAPROTEINSEXHIBITPOLYMORPHISMmined.AknownamountoftheradioactiveproteinisApolymorphismisamendelianormonogenictraitthattheninjectedintoanormaladultsubject,andsamplesofexistsinthepopulationinatleasttwophenotypes,nei-bloodaretakenatvarioustimeintervalsfordetermina-therofwhichisrare(ie,neitherofwhichoccurswithtionsofradioactivity.Thevaluesforradioactivityarefrequencyoflessthan1–2%).TheABObloodgroupplottedagainsttime,andthehalf-lifeoftheprotein(thesubstances(Chapter52)arethebest-knownexamplestimefortheradioactivitytodeclinefromitspeakvalueofhumanpolymorphisms.Humanplasmaproteinstoone-halfofitspeakvalue)canbecalculatedfromthethatexhibitpolymorphismincludeα1-antitrypsin,hap-resultinggraph,discountingthetimesfortheinjectedtoglobin,transferrin,ceruloplasmin,andimmunoglob-proteintoequilibrate(mix)inthebloodandintheex-ulins.Thepolymorphicformsoftheseproteinscanbetravascularspaces.Thehalf-livesobtainedforalbumindistinguishedbydifferentprocedures(eg,varioustypesandhaptoglobininnormalhealthyadultsareapproxi-ofelectrophoresisorisoelectricfocusing),inwhicheachmately20and5days,respectively.Incertaindiseases,formmayshowacharacteristicmigration.Analysesofthehalf-lifeofaproteinmaybemarkedlyaltered.Forin-thesehumanpolymorphismshaveprovedtobeofge-stance,insomegastrointestinaldiseasessuchasregionalnetic,anthropologic,andclinicalinterest.ileitis(Crohndisease),considerableamountsofplasmaproteins,includingalbumin,maybelostintothebowelE.EACHPLASMAPROTEINHASACHARACTERISTICthroughtheinflamedintestinalmucosa.PatientswithHALF-LIFEINTHECIRCULATIONthisconditionhaveaprotein-losinggastroenteropathy,Thehalf-lifeofaplasmaproteincanbedeterminedbyandthehalf-lifeofinjectediodinatedalbumininthese131labelingtheisolatedpureproteinwithIundermild,subjectsmaybereducedtoaslittleas1day.
581PLASMAPROTEINS&IMMUNOGLOBULINS/583F.THELEVELSOFCERTAINPROTEINSINPLASMATable50–2.Somefunctionsofplasmaproteins.INCREASEDURINGACUTEINFLAMMATORYSTATESORSECONDARYTOCERTAINTYPESOFTISSUEDAMAGEFunctionPlasmaProteinsTheseproteinsarecalled“acutephaseproteins”(orre-AntiproteasesAntichymotrypsinactants)andincludeC-reactiveprotein(CRP,so-namedα1-Antitrypsin(α1-antiproteinase)becauseitreactswiththeCpolysaccharideofpneumo-α2-Macroglobulincocci),α1-antitrypsin,haptoglobin,α1-acidglycopro-Antithrombintein,andfibrinogen.TheelevationsofthelevelsoftheseBloodclottingVariouscoagulationfactors,fibrinogenproteinsvaryfromaslittleas50%toasmuchas1000-foldinthecaseofCRP.Theirlevelsarealsousuallyele-EnzymesFunctioninblood,eg,coagulationvatedduringchronicinflammatorystatesandinpa-factors,cholinesterasetientswithcancer.TheseproteinsarebelievedtoplayaLeakagefromcellsortissues,eg,amino-roleinthebody’sresponsetoinflammation.Forexam-transferasesple,C-reactiveproteincanstimulatetheclassiccomple-HormonesErythropoietin1mentpathway,andα1-antitrypsincanneutralizecertainproteasesreleasedduringtheacuteinflammatorystate.ImmunedefenseImmunoglobulins,complementproteins,CRPisusedasamarkeroftissueinjury,infection,andβ2-microglobulininflammation,andthereisconsiderableinterestinitsInvolvementinAcutephaseresponseproteins(eg,useasapredictorofcertaintypesofcardiovascularcon-inflammatoryC-reactiveprotein,α1-acidglyco-ditionssecondarytoatherosclerosis.Interleukin-1responsesprotein[orosomucoid])(IL-1),apolypeptidereleasedfrommononuclearphago-Oncofetalα1-Fetoprotein(AFP)cyticcells,istheprincipal—butnotthesole—stimula-torofthesynthesisofthemajorityofacutephasereac-TransportorAlbumin(variousligands,includingbili-2+tantsbyhepatocytes.AdditionalmoleculessuchasIL-6bindingrubin,freefattyacids,ions[Ca],2+2+areinvolved,andtheyaswellasIL-1appeartoworkatproteinsmetals[eg,Cu,Zn],metheme,thelevelofgenetranscription.steroids,otherhormones,andavari-Table50–2summarizesthefunctionsofmanyofetyofdrugs2+theplasmaproteins.TheremainderofthematerialinCeruloplasmin(containsCu;albuminprobablymoreimportantinphysio-thischapterpresentsbasicinformationregardingse-2+logictransportofCu)lectedplasmaproteins:albumin,haptoglobin,transfer-Corticosteroid-bindingglobulin(trans-rin,ceruloplasmin,α1-antitrypsin,α2-macroglobulin,cortin)(bindscortisol)theimmunoglobulins,andthecomplementsystem.Haptoglobin(bindsextracorpuscularThelipoproteinsarediscussedinChapter25.hemoglobin)Lipoproteins(chylomicrons,VLDL,LDL,AlbuminIstheMajorProteinHDL)inHumanPlasmaHemopexin(bindsheme)Retinol-bindingprotein(bindsretinol)Albumin(69kDa)isthemajorproteinofhumanSexhormone-bindingglobulin(bindsplasma(3.4–4.7g/dL)andmakesupapproximatelytestosterone,estradiol)60%ofthetotalplasmaprotein.About40%ofalbu-Thyroid-bindingglobulin(bindsT4,T3)minispresentintheplasma,andtheother60%ispre-Transferrin(transportiron)sentintheextracellularspace.TheliverproducesaboutTransthyretin(formerlyprealbumin;12gofalbuminperday,representingabout25%ofbindsT4andformsacomplexwithtotalhepaticproteinsynthesisandhalfitssecretedpro-retinol-bindingprotein)tein.Albuminisinitiallysynthesizedasaprepropro-1Variousotherproteinhormonescirculateinthebloodbutaretein.Itssignalpeptideisremovedasitpassesintothenotusuallydesignatedasplasmaproteins.Similarly,ferritinisalsocisternaeoftheroughendoplasmicreticulum,andafoundinplasmainsmallamounts,butittooisnotusuallycharac-hexapeptideattheresultingaminoterminalissubse-terizedasaplasmaprotein.quentlycleavedofffartheralongthesecretorypathway.Thesynthesisofalbuminisdepressedinavarietyofdiseases,particularlythoseoftheliver.Theplasmaofpatientswithliverdiseaseoftenshowsadecreaseintheratioofalbumintoglobulins(decreasedalbumin-globulinratio).Thesynthesisofalbumindecreasesrela-
582584/CHAPTER50tivelyearlyinconditionsofproteinmalnutrition,suchofthekidney,entersthetubules,andtendstoprecipi-askwashiorkor.tatetherein(ascanhappenafteramassiveincompatibleMaturehumanalbuminconsistsofonepolypeptidebloodtransfusion,whenthecapacityofhaptoglobintochainof585aminoacidsandcontains17disulfidebindhemoglobinisgrosslyexceeded)(Figure50–3).bonds.Bytheuseofproteases,albumincanbesubdi-However,theHb-Hpcomplexistoolargetopassvidedintothreedomains,whichhavedifferentfunc-throughtheglomerulus.ThefunctionofHpthusap-tions.Albuminhasanellipsoidalshape,whichmeanspearstobetopreventlossoffreehemoglobinintothethatitdoesnotincreasetheviscosityoftheplasmaaskidney.Thisconservesthevaluableironpresentinhe-muchasanelongatedmoleculesuchasfibrinogendoes.moglobin,whichwouldotherwisebelosttothebody.Becauseofitsrelativelylowmolecularmass(about69HumanhaptoglobinexistsinthreepolymorphickDa)andhighconcentration,albuministhoughttobeforms,knownasHp1-1,Hp2-1,andHp2-2.Hp1-1responsiblefor75–80%oftheosmoticpressureofmigratesinstarchgelelectrophoresisasasingleband,humanplasma.ElectrophoreticstudieshaveshownthatwhereasHp2-1andHp2-2exhibitmuchmorecom-12theplasmaofcertainhumanslacksalbumin.Theseplexbandpatterns.Twogenes,designatedHpandHp,subjectsaresaidtoexhibitanalbuminemia.Onecausedirectthesethreephenotypes,withHp2-1beingtheofthisconditionisamutationthataffectssplicing.heterozygousphenotype.IthasbeensuggestedthattheSubjectswithanalbuminemiashowonlymoderatehaptoglobinpolymorphismmaybeassociatedwithedema,despitethefactthatalbuministhemajordeter-theprevalenceofmanyinflammatorydiseases.minantofplasmaosmoticpressure.ItisthoughtthatThelevelsofhaptoglobininhumanplasmavaryandtheamountsoftheotherplasmaproteinsincreaseandareofsomediagnosticuse.Lowlevelsofhaptoglobinarecompensateforthelackofalbumin.foundinpatientswithhemolyticanemias.Thisisex-Anotherimportantfunctionofalbuminisitsabilityplainedbythefactthatwhereasthehalf-lifeofhaptoglo-tobindvariousligands.Theseincludefreefattyacidsbinisapproximately5days,thehalf-lifeoftheHb-Hp(FFA),calcium,certainsteroidhormones,bilirubin,complexisabout90minutes,thecomplexbeingrapidlyandsomeoftheplasmatryptophan.Inaddition,albu-removedfromplasmabyhepatocytes.Thus,whenhap-minappearstoplayanimportantroleintransportoftoglobinisboundtohemoglobin,itisclearedfromthecopperinthehumanbody(seebelow).Avarietyofplasmaabout80timesfasterthannormally.Accord-drugs,includingsulfonamides,penicillinG,dicumarol,ingly,thelevelofhaptoglobinfallsrapidlyinsituationsandaspirin,areboundtoalbumin;thisfindinghasim-wherehemoglobinisconstantlybeingreleasedfromredportantpharmacologicimplications.bloodcells,suchasoccursinhemolyticanemias.Hapto-Preparationsofhumanalbuminhavebeenwidelyglobinisanacutephaseprotein,anditsplasmalevelisusedinthetreatmentofhemorrhagicshockandofelevatedinavarietyofinflammatorystates.burns.However,thistreatmentisunderreviewbecauseCertainotherplasmaproteinsbindhemebutnotsomerecentstudieshavesuggestedthatadministrationofhemoglobin.Hemopexinisaβ1-globulinthatbindsalbuminintheseconditionsmayincreasemortalityrates.freeheme.Albuminwillbindsomemetheme(ferricheme)toformmethemalbumin,whichthentransfersHaptoglobinBindsExtracorpuscularthemethemetohemopexin.Hemoglobin,PreventingFreeHemoglobinFromEnteringtheKidneyAbsorptionofIronFromtheSmallIntestineIsTightlyRegulatedHaptoglobin(Hp)isaplasmaglycoproteinthatbindsextracorpuscularhemoglobin(Hb)inatightnoncova-Transferrin(Tf)isaplasmaproteinthatplaysacentrallentcomplex(Hb-Hp).Theamountofhaptoglobininroleintransportingironaroundthebodytositeswherehumanplasmarangesfrom40mgto180mgofhemo-globin-bindingcapacityperdeciliter.Approximately10%ofthehemoglobinthatisdegradedeachdayisre-A.Hb→Kidney→Excretedinurineorprecipitatesintubules;leasedintothecirculationandisthusextracorpuscular.(MW65,000)ironislosttobodyTheother90%ispresentinold,damagedredbloodB.Hb+Hp→Hb:Hpcomplex→Kidneycells,whicharedegradedbycellsofthehistiocyticsys-(MW65,000)(MW90,000)(MW155,000)tem.Themolecularmassofhemoglobinisapproxi-mately65kDa,whereasthemolecularmassofthesim-Catabolizedbylivercells;plestpolymorphicformofhaptoglobin(Hp1-1)foundironisconservedandreusedinhumansisapproximately90kDa.Thus,theHb-Hpcomplexhasamolecularmassofapproximately155Figure50–3.DifferentfatesoffreehemoglobinandkDa.Freehemoglobinpassesthroughtheglomerulusofthehemoglobin-haptoglobincomplex.
583PLASMAPROTEINS&IMMUNOGLOBULINS/585itisneeded.Beforewediscussitfurther,certainaspectsTable50–3.Distributionofironina70-kgofironmetabolismwillbereviewed.adultmale.1Ironisimportantinthehumanbodybecauseofitsoccurrenceinmanyhemoproteinssuchashemoglobin,Transferrin3–4mgmyoglobin,andthecytochromes.ItisingestedintheHemoglobininredbloodcells2500mgdieteitherashemeornonhemeiron(Figure50–4);asInmyoglobinandvariousenzymes300mgshown,thesedifferentformsinvolveseparatepathways.Instores(ferritinandhemosiderin)1000mgAbsorptionofironintheproximalduodenumistightlyAbsorption1mg/dregulated,asthereisnophysiologicpathwayforitsex-Losses1mg/dcretionfromthebody.Undernormalcircumstances,1Inanadultfemaleofsimilarweight,theamountinstoreswouldthebodyguardsitscontentofironzealously,sothatagenerallybeless(100–400mg)andthelosseswouldbegreaterhealthyadultmalelosesonlyabout1mg/d,whichisre-(1.5–2mg/d).placedbyabsorption.Adultfemalesaremorepronetostatesofirondeficiencybecausesomemayloseexcessivebloodduringmenstruation.Theamountsofironinvar-iousbodycompartmentsareshowninTable50–3.braneintotheplasma,whereitiscarriedbytransferrinEnterocytesintheproximalduodenumareresponsi-(seebelow).Passageacrossthebasolateralmembrane3+bleforabsorptionofiron.IncomingironintheFeappearstobecarriedoutbyanotherprotein,possibly2+stateisreducedtoFebyaferrireductasepresentonironregulatoryprotein1(IREG1).Thisproteinmaythesurfaceofenterocytes.VitaminCinfoodalsofavorsinteractwiththecopper-containingproteinhephaestin,reductionofferricirontoferrousiron.Thetransferofaproteinsimilartoceruloplasmin(seebelow).Hepha-ironfromtheapicalsurfacesofenterocytesintotheirin-estinisthoughttohaveaferroxidaseactivity,whichisteriorsisperformedbyaproton-coupleddivalentmetal2+importantinthereleaseofironfromcells.Thus,Feistransporter(DMT1).Thisproteinisnotspecificfor3+convertedbacktoFe,theforminwhichitistrans-iron,asitcantransportawidevarietyofdivalentcations.portedintheplasmabytransferrin.Onceinsideanenterocyte,ironcaneitherbestoredOverallregulationofironabsorptioniscomplexasferritinortransferredacrossthebasolateralmem-andnotwellunderstoodmechanistically.ItoccursatBrushborderIntestinalEnterocyteBloodlumenHemeHTHemeHOHPFe3+reductaseFe2+Fe2+FPFe2+Fe2++DMT1Fe2Fe3+-ferritinFe3+ShedFe3+−TF3+2+Figure50–4.Absorptionofiron.FeisconvertedtoFebyferricreductase,2+andFeistransportedintotheenterocytebytheapicalmembraneirontrans-porterDMT1.Hemeistransportedintotheenterocytebyaseparateheme2+transporter(HT),andhemeoxidase(HO)releasesFefromtheheme.Someof2+3+theintracellularFeisconvertedtoFeandboundbyferritin.Theremainder2+bindstothebasolateralFetransporter(FP)andistransportedintotheblood-3+stream,aidedbyhephaestin(HP).Inplasma,Feisboundtotheirontransportproteintransferrin(TF).(Reproduced,withpermission,fromGanongWF:ReviewofMedicalPhysiology,21sted.McGraw-Hill,2003.)
584586/CHAPTER50theleveloftheenterocyte,wherefurtherabsorptionoftheproteinisnormallyonlyone-thirdsaturatedwithironisblockedifasufficientamounthasbeentakenupiron.Inirondeficiencyanemia,theproteinisevenless(so-calleddietaryregulationexertedby“mucosalsaturatedwithiron,whereasinconditionsofstorageofblock”).Italsoappearstoberesponsivetotheoverallexcessironinthebody(eg,hemochromatosis)thesatu-requirementoferythropoiesisforiron(erythropoieticrationwithironismuchgreaterthanone-third.regulation).Absorptionisexcessiveinhereditaryhe-mochromatosis(seebelow).FerritinStoresIroninCellsTransferrinShuttlesIrontoSitesFerritinisanotherproteinthatisimportantintheme-tabolismofiron.Undernormalconditions,itstoresWhereItIsNeededironthatcanbecalleduponforuseasconditionsre-Transferrin(Tf)isaβ1-globulinwithamolecularmassquire.Inconditionsofexcessiron(eg,hemochromato-ofapproximately76kDa.Itisaglycoproteinandissis),bodystoresofironaregreatlyincreasedandmuchsynthesizedintheliver.About20polymorphicformsmoreferritinispresentinthetissues,suchastheliveroftransferrinhavebeenfound.Itplaysacentralroleinandspleen.Ferritincontainsapproximately23%iron,thebody’smetabolismofironbecauseittransportsironandapoferritin(theproteinmoietyfreeofiron)hasa3+molecularmassofapproximately440kDa.Ferritinis(2molofFepermoleofTf)inthecirculationtositeswhereironisrequired,eg,fromtheguttothebonecomposedof24subunitsof18.5kDa,whichsurroundmarrowandotherorgans.Approximately200billioninamicellarformsome3000–4500ferricatoms.Nor-redbloodcells(about20mL)arecatabolizedperday,mally,thereisalittleferritininhumanplasma.How-releasingabout25mgofironintothebody—mostofever,inpatientswithexcessiron,theamountofferritinwhichwillbetransportedbytransferrin.inplasmaismarkedlyelevated.TheamountofferritinTherearereceptors(TfRs)onthesurfacesofmanyinplasmacanbeconvenientlymeasuredbyasensitivecellsfortransferrin.Itbindstothesereceptorsandisin-andspecificradioimmunoassayandservesasanindexternalizedbyreceptor-mediatedendocytosis(compareofbodyironstores.thefateofLDL;Chapter25).TheacidpHinsidetheSynthesisofthetransferrinreceptor(TfR)andthatlysosomecausestheirontodissociatefromtheprotein.offerritinarereciprocallylinkedtocellularironcon-ThedissociatedironleavestheendosomeviaDMT1totent.SpecificuntranslatedsequencesofthemRNAsforenterthecytoplasm.Unliketheproteincomponentofbothproteins(namedironresponseelements)interactLDL,apoTfisnotdegradedwithinthelysosome.In-withacytosolicproteinsensitivetovariationsinlevelsstead,itremainsassociatedwithitsreceptor,returnstoofcellulariron(iron-responsiveelement-bindingpro-theplasmamembrane,dissociatesfromitsreceptor,tein).Whenironlevelsarehigh,cellsusestoredferritinreenterstheplasma,picksupmoreiron,andagainde-mRNAtosynthesizeferritin,andtheTfRmRNAisde-liverstheirontoneedycells.graded.Incontrast,whenironlevelsarelow,theTfRAbnormalitiesoftheglycosylationoftransferrinmRNAisstabilizedandincreasedsynthesisofreceptorsoccurinthecongenitaldisordersofglycosylationoccurs,whileferritinmRNAisapparentlystoredinan(Chapter47)andinchronicalcoholabuse.Theirdetec-inactiveform.Thisisanimportantexampleofcontroltionby,forexample,isoelectricfocusingisusedtohelpofexpressionofproteinsatthetranslationallevel.diagnosetheseconditions.Hemosiderinisasomewhatill-definedmolecule;itappearstobeapartlydegradedformofferritinbutstillIronDeficiencyAnemiacontainingiron.ItcanbedetectedbyhistologicstainsIsExtremelyPrevalent(eg,Prussianblue)foriron,anditspresenceisdeter-minedhistologicallywhenexcessivestorageofironAttentiontoironmetabolismisparticularlyimpor-occurs.tantinwomenforthereasonmentionedabove.Addi-tionally,inpregnancy,allowancesmustbemadeforHereditaryHemochromatosisIsDuethegrowingfetus.OlderpeoplewithpoordietarytoMutationsintheHFEGenehabits(“teaandtoasters”)maydevelopirondeficiency.Irondeficiencyanemiaduetoinadequateintake,inade-Hereditary(primary)hemochromatosisisaverypreva-quateutilization,orexcessivelossofironisoneofthelentautosomalrecessivedisorderincertainpartsofthemostprevalentconditionsseeninmedicalpractice.world(eg,Scotland,Ireland,andNorthAmerica).ItisTheconcentrationoftransferrininplasmaisapprox-characterizedbyexcessivestorageofironintissues,lead-imately300mg/dL.Thisamountoftransferrincaningtotissuedamage.Totalbodyironrangesbetweenbind300μgofironperdeciliter,sothatthisrepresents2.5gand3.5ginnormaladults;inprimaryhemochro-thetotaliron-bindingcapacityofplasma.However,matosisitusuallyexceeds15g.Theaccumulatediron
585PLASMAPROTEINS&IMMUNOGLOBULINS/587damagesorgansandtissuessuchastheliver,pancreaticMutationsinHFE,locatedonchromosome6p21.3,islets,andheart,perhapsinpartduetoeffectsonfreeleadingtoabnormalitiesinthestructureradicalproduction(Chapter52).Melaninandvariousofitsproteinproductamountsofironaccumulateintheskin,accountingfortheslate-graycoloroftenseen.Theprecisecauseofmelaninaccumulationisnotclear.Thefrequentcoexis-Lossofregulationofabsorptionofironinthesmallintestinetenceofdiabetesmellitus(duetoisletdamage)andtheskinpigmentationledtouseofthetermbronzedia-betesforthiscondition.In1995,FederandcolleaguesAccumulationofironinvarioustissues,butparticularlyisolatedagene,nowknownasHFE,locatedonchromo-liver,pancreaticislets,skin,andheartmusclesome6closetothemajorhistocompatibilitycomplexgenes.Theencodedprotein(HFE)wasfoundtobere-IrondirectlyorindirectlycausesdamagetothelatedtoMHCclass1antigens.Initially,twodifferentabovetissues,resultinginhepaticcirrhosis,diabetesmissensemutationswerefoundinHFEinindividualsmellitus,skinpigmentation,andcardiacproblemswithhereditaryhemochromatosis.Themorefrequentmutationwasonethatchangedcysteinylresidue282toFigure50–5.Tentativeschemeofthemaineventsatyrosylresidue(CY282Y),disruptingthestructureofincausationofprimaryhemochromatosis(MIMtheprotein.Theothermutationchangedhistidylresi-235200).ThetwoprincipalmutationsareCY282Yanddue63toanaspartylresidue(H63D).SomepatientsH63D(seetext).MutationsingenesotherthanHFEarewithhereditaryhemochromatosishaveneithermuta-alsoinvolvedinsomecases.tion,perhapsduetoothermutationsinHFEorbecauseoneormoreothergenesmaybeinvolvedinitscausa-tion.Geneticscreeningforthisconditionhasbeeneval-uatedbutisnotpresentlyrecommended.However,test-carries90%ofthecopperpresentinplasma.Eachmol-ingforHFEmutationsinindividualswithelevatedeculeofceruloplasminbindssixatomsofcopperveryserumironconcentrationsmaybeuseful.tightly,sothatthecopperisnotreadilyexchangeable.HFEhasbeenshowntobelocatedincellsintheAlbumincarriestheother10%oftheplasmacoppercryptsofthesmallintestine,thesiteofironabsorption.butbindsthemetallesstightlythandoesceruloplas-Thereisevidencethatitassociateswithβ2-microglobu-min.Albuminthusdonatesitscoppertotissuesmorelin,anassociationthatmaybenecessaryforitsstability,readilythanceruloplasminandappearstobemoreim-intracellularprocessing,andcellsurfaceexpression.Theportantthanceruloplasminincoppertransportinthecomplexinteractswiththetransferrinreceptor(TfR);humanbody.Ceruloplasminexhibitsacopper-depen-howthisleadstoexcessivestorageofironwhenHFEisdentoxidaseactivity,butitsphysiologicsignificancealteredbymutationisunderclosestudy.Themousehasnotbeenclarified.TheamountofceruloplasmininhomologofHFEhasbeenknockedout,resultinginaplasmaisdecreasedinliverdisease.Inparticular,lowpotentiallyusefulanimalmodelofhemochromatosis.levelsofceruloplasminarefoundinWilsondiseaseAschemeofthelikelymaineventsinthecausationof(hepatolenticulardegeneration),adiseaseduetoabnor-hereditaryhemochromatosisissetforthinFigure50–5.malmetabolismofcopper.Inordertoclarifythede-Secondaryhemochromatosiscanoccurafterre-scriptionofWilsondisease,weshallfirstconsiderthepeatedtransfusions(eg,fortreatmentofsicklecellane-metabolismofcopperinthehumanbodyandthenmia),excessiveoralintakeofiron(eg,byAfricanBantuMenkesdisease,anotherconditioninvolvingabnormalpeopleswhoconsumealcoholicbeveragesfermentedincoppermetabolism.containersmadeofiron),oranumberofothercondi-tions.Table50–4summarizeslaboratorytestsusefulintheassessmentofpatientswithabnormalitiesofironme-Table50–4.Laboratorytestsforassessingtabolism.patientswithdisordersofironmetabolism.CeruloplasminBindsCopper,&LowLevels•Redbloodcellcountandestimationofhemoglobin•Determinationsofplasmairon,totaliron-bindingcapacityofThisPlasmaProteinAreAssociated(TIBC),and%transferrinsaturationWithWilsonDisease•DeterminationofferritininplasmabyradioimmunoassayCeruloplasmin(about160kDa)isanα2-globulin.It•Prussianbluestainoftissuesectionshasabluecolorbecauseofitshighcoppercontentand•Determinationofamountofiron(μg/g)inatissuebiopsy
586588/CHAPTER50CopperIsaCofactorforCertainEnzymesonlymaleinfants,involvesthenervoussystem,connec-tivetissue,andvasculature,andisusuallyfatalinin-Copperisanessentialtraceelement.Itisrequiredinfancy.In1993,itwasreportedthatthebasisofMenkesthedietbecauseitisthemetalcofactorforavarietyofdiseasewasmutationsinthegeneforacopper-bindingenzymes(seeTable50–5).CopperacceptsanddonatesP-typeATPase.Interestingly,theenzymeshowedstruc-electronsandisinvolvedinreactionsinvolvingdismu-turalsimilaritytocertainmetal-bindingproteinsinmi-tation,hydroxylation,andoxygenation.However,ex-croorganisms.ThisATPaseisthoughttoberesponsiblecesscoppercancauseproblemsbecauseitcanoxidizefordirectingtheeffluxofcopperfromcells.Whenal-proteinsandlipids,bindtonucleicacids,andenhanceteredbymutation,copperisnotmobilizednormallytheproductionoffreeradicals.Itisthusimportanttofromtheintestine,inwhichitaccumulates,asitdoesinhavemechanismsthatwillmaintaintheamountofavarietyofothercellsandtissues,fromwhichitcannotcopperinthebodywithinnormallimits.Thebodyofexit.Despitetheaccumulationofcopper,theactivitiesthenormaladultcontainsabout100mgofcopper,lo-ofmanycopper-dependentenzymesaredecreased,per-catedmostlyinbone,liver,kidney,andmuscle.Thehapsbecauseofadefectofitsincorporationintothedailyintakeofcopperisabout2–4mg,withaboutapoenzymes.Normalliverexpressesverylittleofthe50%beingabsorbedinthestomachanduppersmallATPase,whichexplainstheabsenceofhepaticinvolve-intestineandtheremainderexcretedinthefeces.Cop-mentinMenkesdisease.Thisworkledtothesugges-periscarriedtotheliverboundtoalbumin,takenuptionthatlivermightcontainadifferentcopper-bindingbylivercells,andpartofitisexcretedinthebile.Cop-ATPase,whichcouldbeinvolvedinthecausationofperalsoleavestheliverattachedtoceruloplasmin,Wilsondisease.Asdescribedbelow,thisturnedouttowhichissynthesizedinthatorgan.bethecase.TheTissueLevelsofCopper&ofCertainWilsonDiseaseIsAlsoDuetoMutationsOtherMetalsAreRegulatedininaGeneEncodingaCopper-BindingPartbyMetallothioneinsP-TypeATPaseMetallothioneinsareagroupofsmallproteins(about6.5kDa),foundinthecytosolofcells,particularlyofWilsondiseaseisageneticdiseaseinwhichcopperfailsliver,kidney,andintestine.Theyhaveahighcontentoftobeexcretedinthebileandaccumulatesinliver,cysteineandcanbindcopper,zinc,cadmium,andmer-brain,kidney,andredbloodcells.Itcanberegardedascury.TheSHgroupsofcysteineareinvolvedinbindinganinabilitytomaintainanear-zerocopperbalance,re-themetals.Acuteintake(eg,byinjection)ofcopperandsultingincoppertoxicosis.Theincreaseofcopperinofcertainothermetalsincreasestheamount(induction)livercellsappearstoinhibitthecouplingofcoppertooftheseproteinsintissues,asdoesadministrationofapoceruloplasminandleadstolowlevelsofceruloplas-certainhormonesorcytokines.Theseproteinsmaymininplasma.Astheamountofcopperaccumulates,functiontostoretheabovemetalsinanontoxicformpatientsmaydevelopahemolyticanemia,chronicliverandareinvolvedintheiroverallmetabolisminthedisease(cirrhosis,hepatitis),andaneurologicsyndromebody.Sequestrationofcopperalsodiminishestheowingtoaccumulationofcopperinthebasalgangliaamountofthismetalavailabletogeneratefreeradicals.andothercenters.AfrequentclinicalfindingistheKayser-Fleischerring.Thisisagreenorgoldenpig-MenkesDiseaseIsDuetoMutationsmentringaroundthecorneaduetodepositionofcop-intheGeneEncodingaCopper-perinDescemet’smembrane.ThemajorlaboratorytestsofcoppermetabolismarelistedinTable50–6.IfBindingP-TypeATPaseWilsondiseaseissuspected,aliverbiopsyshouldbeMenkesdisease(“kinky”or“steely”hairdisease)isaperformed;avalueforlivercopperofover250μgperdisorderofcoppermetabolism.ItisX-linked,affectsgramdryweightalongwithaplasmalevelofcerulo-plasminofunder20mg/dLisdiagnostic.ThecauseofWilsondiseasewasalsorevealedinTable50–5.Someimportantenzymesthat1993,whenitwasreportedthatavarietyofmutationsinageneencodingacopper-bindingP-typeATPasecontaincopper.wereresponsible.Thegeneisestimatedtoencodeaproteinof1411aminoacids,whichishighlyhomolo-•AmineoxidasegoustotheproductofthegeneaffectedinMenkesdis-•Copper-dependentsuperoxidedismutaseease.Inamannernotyetfullyexplained,anonfunc-•CytochromeoxidasetionalATPasecausesdefectiveexcretionofcopperinto•Tyrosinasethebile,areductionofincorporationofcopperinto
587PLASMAPROTEINS&IMMUNOGLOBULINS/589Table50–6.Majorlaboratorytestsusedintheotherproteasesbyformingcomplexeswiththem.Atinvestigationofdiseasesofcoppermetabolism.1least75polymorphicformsoccur,manyofwhichcanbeseparatedbyelectrophoresis.ThemajorgenotypeisMM,anditsphenotypicproductisPiM.TherearetwoNormalAdultTestRangeareasofclinicalinterestconcerningα1-antitrypsin.Ade-ficiencyofthisproteinhasaroleincertaincases(ap-Serumcopper10–22μmol/Lproximately5%)ofemphysema.ThisoccursmainlyinCeruloplasmin200–600mg/LsubjectswiththeZZgenotype,whosynthesizePiZ,andalsoinPiSZheterozygotes,bothofwhomsecretecon-Urinarycopper<1μmol/24hsiderablylessproteinthanPiMMindividuals.Consider-Livercopper20–50μg/gdryweightablylessofthisproteinissecretedascomparedwith1PiM.Whentheamountofα1-antitrypsinisdeficientBasedonGawAetal:ClinicalBiochemistry.ChurchillLivingstone,andpolymorphonuclearwhitebloodcellsincreaseinthe1995.lung(eg,duringpneumonia),theaffectedindividuallacksacounterchecktoproteolyticdamageofthelungbyproteasessuchaselastase(Figure50–6).Itisofcon-apoceruloplasmin,andtheaccumulationofcopperinsiderableinterestthataparticularmethionine(residueliverandsubsequentlyinotherorganssuchasbrain.358)ofα1-antitrypsinisinvolvedinitsbindingtopro-TreatmentforWilsondiseaseconsistsofadietlowteases.Smokingoxidizesthismethioninetomethionineincopperalongwithlifelongadministrationofpenicil-sulfoxideandthusinactivatesit.Asaresult,affectedlamine,whichchelatescopper,isexcretedintheurine,moleculesofα1-antitrypsinnolongerneutralizeandthusdepletesthebodyoftheexcessofthismineral.proteases.ThisisparticularlydevastatinginpatientsAnotherconditioninvolvingceruloplasminisaceru-(eg,PiZZphenotype)whoalreadyhavelowlevelsofloplasminemia.Inthisgeneticdisorder,levelsofcerulo-α1-antitrypsin.Thefurtherdiminutioninα1-antitrypsinplasminareverylowandconsequentlyitsferroxidaseac-broughtaboutbysmokingresultsinincreasedproteolytictivityismarkedlydeficient.Thisleadstofailureofreleasedestructionoflungtissue,acceleratingthedevelopmentofironfromcells,andironaccumulatesincertainbrainofemphysema.Intravenousadministrationofα1-anti-cells,hepatocytes,andpancreaticisletcells.Affectedindi-trypsin(augmentationtherapy)hasbeenusedasanad-vidualsshowsevereneurologicsignsandhavediabetesjunctinthetreatmentofpatientswithemphysemaduemellitus.Useofachelatingagentoradministrationoftoα1-antitrypsindeficiency.Attemptsarebeingmade,plasmaorceruloplasminconcentratemaybebeneficial.usingthetechniquesofproteinengineering,toreplacemethionine358byanotherresiduethatwouldnotbesubjecttooxidation.Theresulting“mutant”α1-anti-Deficiencyof1-Antiproteinasetrypsinwouldthusaffordprotectionagainstproteases(1-Antitrypsin)IsAssociatedforamuchlongerperiodoftimethanwouldnativeWithEmphysema&OneTypeα1-antitrypsin.Attemptsarealsobeingmadetodevelopgenetherapyforthiscondition.OneapproachistouseofLiverDiseaseamodifiedadenovirus(apathogenoftherespiratoryα1-Antiproteinase(about52kDa)wasformerlycalledtract)intowhichthegeneforα1-antitrypsinhasbeenα1-antitrypsin,andthisnameisretainedhere.Itisainserted.Theviruswouldthenbeintroducedintothesingle-chainproteinof394aminoacids,containsthreerespiratorytract(eg,byanaerosol).Thehopeisthatoligosaccharidechains,andisthemajorcomponentpulmonaryepithelialcellswouldexpressthegeneand(>90%)oftheα1fractionofhumanplasma.Itissyn-secreteα1-antitrypsinlocally.Experimentsinanimalsthesizedbyhepatocytesandmacrophagesandisthehaveindicatedthefeasibilityofthisapproach.principalserineproteaseinhibitor(serpin,orPi)ofDeficiencyofα1-antitrypsinisalsoimplicatedinhumanplasma.Itinhibitstrypsin,elastase,andcertainonetypeofliverdisease(α1-antitrypsindeficiencyliverA.Activeelastase+α1–AT→Inactiveelastase:α1–ATcomplex→Noproteolysisoflung→Notissuedamage→B.Activeelastase+ornoα1–AT→Activeelastase→Proteolysisoflung→TissuedamageFigure50–6.Schemeillustrating(A)normalinactivationofelastasebyα1-antitrypsinand(B)situationinwhichtheamountofα1-antitrypsinissubstantiallyreduced,resultinginpro-teolysisbyelastaseandleadingtotissuedamage.
588590/CHAPTER50disease).Inthiscondition,moleculesoftheZZpheno-comprises8–10%ofthetotalplasmaproteininhu-typeaccumulateandaggregateinthecisternaeofthemans.Approximately10%ofthezincinplasmaisendoplasmicreticulumofhepatocytes.Aggregationistransportedbyα2-macroglobulin,theremainderbeingduetoformationofpolymersofmutantα1-antitrypsin,transportedbyalbumin.Theproteinissynthesizedbyathepolymersformingviaastronginteractionbetweenavarietyofcelltypes,includingmonocytes,hepatocytes,specificloopinonemoleculeandaprominentβ-andastrocytes.Itisthemajormemberofagroupofpleatedsheetinanother(loop-sheetpolymerization).plasmaproteinsthatincludecomplementproteinsC3Bymechanismsthatarenotunderstood,hepatitisre-andC4.Theseproteinscontainauniqueinternalcyclicsultswithconsequentcirrhosis(accumulationofmas-thiolesterbond(formedbetweenacysteineandaglut-siveamountsofcollagen,resultinginfibrosis).Itispos-amineresidue)andforthisreasonhavebeendesignatedsiblethatadministrationofasyntheticpeptideasthethiolesterplasmaproteinfamily.resemblingtheloopsequencecouldinhibitloop-sheetα2-Macroglobulinbindsmanyproteinasesandispolymerization.Diseasessuchasα1-antitrypsindefi-thusanimportantpanproteinaseinhibitor.Theα2-ciency,inwhichcellularpathologyisprimarilycausedmacroglobulin-proteinasecomplexesarerapidlyclearedbythepresenceofaggregatesofaberrantformsofindi-fromtheplasmabyareceptorlocatedonmanycellvidualproteins,havebeennamedconformationaldis-types.Inaddition,α2-macroglobulinbindsmanycy-eases.Mostappeartobeduetotheformationbycon-tokines(platelet-derivedgrowthfactor,transformingformationallyunstableproteinsofβsheets,whichingrowthfactor-β,etc)andappearstobeinvolvedintar-turnleadstoformationofaggregates.Othermembersgetingthemtowardparticulartissuesorcells.OnceofthisgroupofconditionsincludeAlzheimerdisease,takenupbycells,thecytokinescandissociatefromα2-Parkinsondisease,andHuntingtondisease.macroglobulinandsubsequentlyexertavarietyofef-Atpresent,severeα1-antitrypsindeficiencyliverdis-fectsoncellgrowthandfunction.Thebindingofpro-easecanbesuccessfullytreatedbylivertransplantation.teinasesandcytokinesbyα2-macroglobulininvolvesInthefuture,introductionofthegenefornormalα1-differentmechanismsthatwillnotbeconsideredhere.antitrypsinintohepatocytesmaybecomepossible,butthiswouldnotstopproductionofthePiZprotein.Fig-AmyloidosisOccursbytheDepositionure50–7isaschemeofthecausationofthisdisease.ofFragmentsofVariousPlasmaProteinsinTissues2-MacroglobulinNeutralizesManyAmyloidosisistheaccumulationofvariousinsolubleProteases&TargetsCertainfibrillarproteinsbetweenthecellsoftissuestoanextentCytokinestoTissuesthataffectsfunction.Thefibrilsgenerallyrepresentα2-Macroglobulinisalargeplasmaglycoprotein(720proteolyticfragmentsofvariousplasmaproteinsandkDa)madeupoffouridenticalsubunitsof180kDa.Itpossessaβ-pleatedsheetstructure.Theterm“amyloi-dosis”isamisnomer,asitwasoriginallythoughtthatthefibrilswerestarch-likeinnature.Amongthemostcommonprecursorproteinsareimmunoglobulinlightchains(seebelow),amyloid-associatedproteinderivedGAGtoAAGmutationinexon5ofgeneforα1-ATfromserumamyloid-associatedprotein(aplasmaglyco-onchromosome14protein),andtransthyretin(Table50–2).Theprecursorproteinsinplasmaaregenerallyeitherincreasedin342342amount(eg,immunoglobulinlightchainsinmultipleResultsinGlutoLyssubstitutioninα1-AT,myelomaorβ2-microglobulininpatientsbeingmain-causingformationofPiZZtainedonchronicdialysis)ormutantforms(eg,oftransthyretininfamilialamyloidoticneuropathies).PiZZaccumulatesincisternaeTheprecisefactorsthatdeterminethedepositionofofendoplasmicreticulumandaggregatesproteolyticfragmentsintissuesawaitelucidation.vialoop-sheetpolymerizationOtherproteinshavebeenfoundinamyloidfibrils,suchascalcitoninandamyloidβprotein(notderivedfromaLeadstohepatitis(mechanismunknown)plasmaprotein)inAlzheimerdisease;atotalofaboutandcirrhosisin~10%ofZZhomozygotes15differentproteinshavebeenfound.AllfibrilshaveaPcomponentassociatedwiththem,whichisderivedFigure50–7.Schemeofcausationofα1-antitrypsin-fromserumamyloidPcomponent,aplasmaproteindeficiencyliverdisease.ThemutationshowncausescloselyrelatedtoC-reactiveprotein.TissuesectionsformationofPiZZ(MIM107400).(α1-AT,α1-antitrypsin.)containingamyloidfibrilsinteractwithCongoredstain
589PLASMAPROTEINS&IMMUNOGLOBULINS/591anddisplaystrikinggreenbirefringencewhenviewedmunoglobulinsotherthandirectbindingofantigens.bypolarizingmicroscopy.Depositionofamyloidoc-BecausetherearetwoFabregions,IgGmoleculesbindcursinpatientswithavarietyofdisorders;treatmentoftwomoleculesofantigenandaretermeddivalent.Thetheunderlyingdisordershouldbeprovidedifpossible.siteontheantigentowhichanantibodybindsistermedanantigenicdeterminant,orepitope.ThePLASMAIMMUNOGLOBULINSPLAYareainwhichpapaincleavestheimmunoglobulinmol-AMAJORROLEINTHEBODY’Secule—ie,theregionbetweentheCH1andCH2do-mains—isreferredtoasthe“hingeregion.”ThehingeDEFENSEMECHANISMSregionconfersflexibilityandallowsbothFabarmstoTheimmunesystemofthebodyconsistsoftwomajormoveindependently,thushelpingthemtobindtocomponents:BlymphocytesandTlymphocytes.Theantigenicsitesthatmaybevariabledistancesapart(eg,Blymphocytesaremainlyderivedfrombonemarrowonbacterialsurfaces).FcandhingeregionsdifferinthecellsinhigheranimalsandfromthebursaofFabriciusdifferentclassesofantibodies,buttheoverallmodelofinbirds.TheTlymphocytesareofthymicorigin.TheantibodystructureforeachclassissimilartothatBcellsareresponsibleforthesynthesisofcirculating,showninFigure50–8forIgG.humoralantibodies,alsoknownasimmunoglobulins.TheTcellsareinvolvedinavarietyofimportantcell-AllLightChainsAreEitherKappamediatedimmunologicprocessessuchasgraftrejec-orLambdainTypetion,hypersensitivityreactions,anddefenseagainstma-lignantcellsandmanyviruses.ThissectionconsidersTherearetwogeneraltypesoflightchains,kappa(κ)onlytheplasmaimmunoglobulins,whicharesynthe-andlambda(λ),whichcanbedistinguishedonthesizedmainlyinplasmacells.ThesearespecializedcellsbasisofstructuraldifferencesintheirCLregions.AofBcelllineagethatsynthesizeandsecreteimmuno-givenimmunoglobulinmoleculealwayscontainstwoκglobulinsintotheplasmainresponsetoexposuretoaortwoλlightchains—neveramixtureofκandλ.Invarietyofantigens.humans,theκchainsaremorefrequentthanλchainsinimmunoglobulinmolecules.AllImmunoglobulinsContainaMinimumofTwoLight&TwoHeavyChainsTheFiveTypesofHeavyChainDetermineImmunoglobulinClassImmunoglobulinscontainaminimumoftwoiden-ticallight(L)chains(23kDa)andtwoidenticalheavyFiveclassesofHchainhavebeenfoundinhumans(H)chains(53–75kDa),heldtogetherasatetramer(Table50–7),distinguishedbydifferencesintheirCH(L2H2)bydisulfidebondsThestructureofIgGisregions.Theyaredesignatedγ,α,μδ,andε.TheμshowninFigure50–8;itisY-shaped,withbindingofandεchainseachhavefourCHdomainsratherthanantigenoccurringatbothtipsoftheY.Eachchaincantheusualthree.ThetypeofHchaindeterminesthebedividedconceptuallyintospecificdomains,orre-classofimmunoglobulinandthusitseffectorfunction.gions,thathavestructuralandfunctionalsignificance.Therearethusfiveimmunoglobulinclasses:IgG,IgA,Thehalfofthelight(L)chaintowardthecarboxylter-IgM,IgD,andIgE.Thebiologicfunctionsoftheseminalisreferredtoastheconstantregion(CL),whilefiveclassesaresummarizedinTable50–8.theaminoterminalhalfisthevariableregionofthelightchain(VL).Approximatelyone-quarteroftheNoTwoVariableRegionsAreIdenticalheavy(H)chainattheaminoterminalsisreferredtoasitsvariableregion(VH),andtheotherthree-quartersofThevariableregionsofimmunoglobulinmoleculestheheavychainarereferredtoastheconstantregionsconsistoftheVLandVHdomainsandarequitehetero-(CH1,CH2,CH3)ofthatHchain.Theportionofthegeneous.Infact,notwovariableregionsfromdifferentimmunoglobulinmoleculethatbindsthespecificanti-humanshavebeenfoundtohaveidenticalaminoacidgenisformedbytheaminoterminalportions(variablesequences.However,aminoacidanalyseshaveshownregions)ofboththeHandLchains—ie,theVHandthatthevariableregionsarecomprisedofrelativelyin-VLdomains.Thedomainsoftheproteinchainsconsistvariableregionsandotherhypervariableregions(Figureoftwosheetsofantiparalleldistinctstretchesofamino50–9).Lchainshavethreehypervariableregions(inacidsthatbindantigen.VL)andHchainshavefour(inVH).Thesehypervari-AsdepictedinFigure50–8,digestionofanim-ableregionscomprisetheantigen-bindingsite(locatedmunoglobulinbytheenzymepapainproducestwoatthetipsoftheYshowninFigure50–8)anddictateantigen-bindingfragments(Fab)andonecrystallizabletheamazingspecificityofantibodies.Forthisreason,fragment(Fc),whichisresponsibleforfunctionsofim-hypervariableregionsarealsotermedcomplementar-
590592/CHAPTER50+HN3VL+FabHNS3SSCLchainLSHchainSHingeregionVSHFCSCH2CH3SSSCH1SSSSCOO—SSHchainSSHchain—COOSSSSSSSSPepsinCleavagesitesPapainSSHchainSLchainSS+HNS3Fab+HN3Figure50–8.StructureofIgG.Themoleculeconsistsoftwolight(L)chainsandtwoheavy(H)chains.Eachlightchainconsistsofavariable(VL)andaconstant(CL)region.Eachheavychainconsistsofavariableregion(VH)andaconstantregionthatisdividedintothreedomains(CH1,CH2,andCH3).TheCH2domaincontainsthecomplement-bindingsiteandtheCH3domaincontainsasitethatattachestoreceptorsonneutrophilsandmacrophages.Theantigen-bindingsiteisformedbythehypervariableregionsofboththelightandheavychains,whicharelocatedinthevariableregionsofthesechains(seeFigure50–9).Thelightandheavychainsarelinkedbydisulfidebonds,andtheheavychainsarealsolinkedtoeachotherbydisulfidebonds.(Reproduced,withpermission,fromParslowTGetal[editors]:MedicalImmunology,10thed.McGraw-Hill,2001.)ity-determiningregions(CDRs).Aboutfivetotenspecificities,afeaturethatcontributestothetremen-aminoacidsineachhypervariableregion(CDR)con-dousdiversityofantibodymoleculesandistermedtributetotheantigen-bindingsite.CDRsarelocatedcombinatorialdiversity.Largeantigensinteractwithonsmallloopsofthevariabledomains,thesurroundingalloftheCDRsofanantibody,whereassmallligandspolypeptideregionsbetweenthehypervariableregionsmayinteractwithonlyoneorafewCDRsthatformabeingtermedframeworkregions.CDRsfrombothpocketorgrooveintheantibodymolecule.TheessenceVHandVLdomains,broughttogetherbyfoldingoftheofantigen-antibodyinteractionsismutualcomplemen-polypeptidechainsinwhichtheyarecontained,formataritybetweenthesurfacesofCDRsandepitopes.Thesinglehypervariablesurfacecomprisingtheantigen-interactionsbetweenantibodiesandantigensinvolvebindingsite.VariouscombinationsofHandLchainnoncovalentforcesandbonds(electrostaticandvanderCDRscangiverisetomanyantibodiesofdifferentWaalsforcesandhydrogenandhydrophobicbonds).
591PLASMAPROTEINS&IMMUNOGLOBULINS/5931Table50–7.Propertiesofhumanimmunoglobulins.PropertyIgGIgAIgMIgDIgEPercentageoftotalimmunoglo-751590.20.004bulininserum(approximate)Serumconcentration100020012030.05(mg/dL)(approximate)2Sedimentationcoefficient7S7Sor11S19S7S8SMolecularweight150170or9001801902(×1000)400StructureMonomerMonomerordimerMonomerordimerMonomerMonomerH-chainsymbolγαμδεComplementfixation+−+−−Transplacentalpassage+−−?−Mediationofallergicresponses−−−−+Foundinsecretions−+−−−3Opsonization+−−−−AntigenreceptoronBcell−−+?−PolymericformcontainsJchain−++−−1Reproduced,withpermission,fromLevinsonW,JawetzE:MedicalMicrobiologyandImmunology,7thed.McGraw-Hill,2002.2The11Sformisfoundinsecretions(eg,saliva,milk,tears)andfluidsoftherespiratory,intestinal,andgenitaltracts.3IgMopsonizesindirectlybyactivatingcomplement.ThisproducesC3b,whichisanopsonin.TheConstantRegionsDetermine(VL)gene,ajoiningregion(J)gene(bearingnorela-Class-SpecificEffectorFunctionstionshiptotheJchainofIgAorIgM),andaconstantregion(CL)gene.EachheavychainistheproductofatTheconstantregionsoftheimmunoglobulinmolecules,leastfourdifferentgenes:avariableregion(VH)gene,aparticularlytheCH2andCH3(andCH4ofIgManddiversityregion(D)gene,ajoiningregion(J)gene,andIgE),whichconstitutetheFcfragment,areresponsibleaconstantregion(CH)gene.Thus,the“onegene,onefortheclass-specificeffectorfunctionsofthedifferentprotein”conceptisnotvalid.Themolecularmecha-immunoglobulinmolecules(Table50–7,bottompart),nismsresponsibleforthegenerationofthesingleim-eg,complementfixationortransplacentalpassage.munoglobulinchainsfrommultiplestructuralgenesareSomeimmunoglobulinssuchasimmuneIgGexistdiscussedinChapters36and39.onlyinthebasictetramericstructure,whileotherssuchasIgAandIgMcanexistashigherorderpolymersoftwo,three(IgA),orfive(IgM)tetramericunits(FigureAntibodyDiversityDepends50–10).onGeneRearrangementsTheLchainsandHchainsaresynthesizedassepa-ratemoleculesandaresubsequentlyassembledwithinEachpersoniscapableofgeneratingantibodiesdirectedtheBcellorplasmacellintomatureimmunoglobulinagainstperhaps1milliondifferentantigens.Thegener-molecules,allofwhichareglycoproteins.ationofsuchimmenseantibodydiversitydependsuponanumberoffactorsincludingtheexistenceofBothLight&HeavyChainsAreProductsmultiplegenesegments(V,C,J,andDsegments),theirrecombinations(seeChapters36and39),theofMultipleGenescombinationsofdifferentLandHchains,ahighfre-Eachimmunoglobulinlightchainistheproductofatquencyofsomaticmutationsinimmunoglobulingenes,leastthreeseparatestructuralgenes:avariableregionandjunctionaldiversity.Thelatterreflectstheaddi-
592594/CHAPTER50Table50–8.MajorfunctionsofLightchainimmunoglobulins.1hypervariableregionsImmunoglobulinMajorFunctionsVIgGMainantibodyinthesecondaryre-Lsponse.Opsonizesbacteria,makingVInterchainHthemeasiertophagocytose.Fixescom-disulfideplement,whichenhancesbacterialCbondsLkilling.NeutralizesbacterialtoxinsandCviruses.Crossestheplacenta.HHeavychainIgASecretoryIgApreventsattachmentofhypervariableregionsbacteriaandvirusestomucousmem-Intrachainbranes.Doesnotfixcomplement.disulfidebondsIgMProducedintheprimaryresponsetoanantigen.Fixescomplement.Doesnotcrosstheplacenta.AntigenreceptoronthesurfaceofBcells.IgDUncertain.FoundonthesurfaceofmanyBcellsaswellasinserum.IgEMediatesimmediatehypersensitivitybycausingreleaseofmediatorsfrommastcellsandbasophilsuponexposuretoFigure50–9.SchematicmodelofanIgGmoleculeantigen(allergen).Defendsagainstshowingapproximatepositionsofthehypervariablere-worminfectionsbycausingreleaseofgionsinheavyandlightchains.Theantigen-bindingenzymesfromeosinophils.Doesnotfixsiteisformedbythesehypervariableregions.Thehy-complement.Mainhostdefenseagainstpervariableregionsarealsocalledcomplementarity-helminthicinfections.determiningregions(CDRs).(Modifiedandreproduced,1Reproduced,withpermission,fromLevinsonW,JawetzE:Med-withpermission,fromParslowTGetal[editors]:MedicalicalMicrobiologyandImmunology,7thed.McGraw-Hill,2002.Immunology,10thed.McGraw-Hill,2001.)tionordeletionofarandomnumberofnucleotidesgenerateanIgGmoleculewithantigenspecificityiden-whencertaingenesegmentsarejoinedtogether,andin-ticaltothatoftheoriginalIgMmolecule.Thesametroducesanadditionaldegreeofdiversity.Thus,thelightchaincanalsocombinewithanαheavychain,abovefactorsensurethatavastnumberofantibodiesagaincontainingtheidenticalVHregion,toformanIgAcanbesynthesizedfromseveralhundredgenesegments.moleculewithidenticalantigenspecificity.Thesethreeclasses(IgM,IgG,andIgA)ofimmunoglobulinmole-Class(Isotype)SwitchingOccursculesagainstthesameantigenhaveidenticalvariabledo-DuringImmuneResponsesmainsofboththeirlight(VL)chainsandheavy(VH)chainsandaresaidtoshareanidiotype.(IdiotypesareInmosthumoralimmuneresponses,antibodieswiththeantigenicdeterminantsformedbythespecificaminoidenticalspecificitybutofdifferentclassesaregeneratedacidsinthehypervariableregions.)Thedifferentclassesinaspecificchronologicorderinresponsetotheim-ofthesethreeimmunoglobulins(calledisotypes)aremunogen(immunizingantigen).Forinstance,antibod-thusdeterminedbytheirdifferentCHregions,whichareiesoftheIgMclassnormallyprecedemoleculesofthecombinedwiththesameantigen-specificVHregions.IgGclass.Theswitchfromoneclasstoanotherisdesig-nated“classorisotypeswitching,”anditsmolecularBothOver-&Underproductionbasishasbeeninvestigatedextensively.AsingletypeofofImmunoglobulinsMayResultimmunoglobulinlightchaincancombinewithananti-inDiseaseStatesgen-specificμchaintogenerateaspecificIgMmolecule.Subsequently,thesameantigen-specificlightchainDisordersofimmunoglobulinsincludeincreasedpro-combineswithaγchainwithanidenticalVHregiontoductionofspecificclassesofimmunoglobulinsoreven
593PLASMAPROTEINS&IMMUNOGLOBULINS/595LLHHMonomerDimerA.SerumlgAHJchainHLLLHB.SecretorylgA(dimer)JchainHSecretoryLcomponentLHJchainFigure50–10.Schematicrepre-sentationofserumIgA,secretoryIgA,andIgM.BothIgAandIgMhaveC.lgMaJchain,butonlysecretoryIgAhas(pentamer)Hasecretorycomponent.PolypeptideLchainsarerepresentedbythicklines;disulfidebondslinkingdifferentpolypeptidechainsarerepresentedbythinlines.(Reproduced,withper-mission,fromParslowTGetal[edi-tors]:MedicalImmunology,10thed.McGraw-Hill,2001.)specificimmunoglobulinmolecules,thelatterbyclonalHybridomasProvideLong-TermSourcestumorsofplasmacellscalledmyelomas.MultipleofHighlyUsefulMonoclonalAntibodiesmyelomaisaneoplasticcondition;electrophoresisofserumorurinewillusuallyrevealalargeincreaseofoneWhenanantigenisinjectedintoananimal,theresult-particularimmunoglobulinoroneparticularlightchainingantibodiesarepolyclonal,beingsynthesizedbya(thelattertermedaBenceJonesprotein).DecreasedmixtureofBcells.Polyclonalantibodiesaredirectedproductionmayberestrictedtoasingleclassofim-againstanumberofdifferentsites(epitopesordetermi-munoglobulinmolecules(eg,IgAorIgG)ormayin-nants)ontheantigenandthusarenotmonospecific.volveunderproductionofallclassesofimmunoglobu-However,bymeansofamethoddevelopedbyKohlerlins(IgA,IgD,IgE,IgG,andIgM).AseverereductionandMilstein,largeamountsofasinglemonoclonalan-insynthesisofanimmunoglobulinclassduetoage-tibodyspecificforoneepitopecanbeobtained.neticabnormalitycanresultinaseriousimmunodefi-Themethodinvolvescellfusion,andtheresultingciencydisease—eg,agammaglobulinemia,inwhichpermanentcelllineiscalledahybridoma.Typically,BproductionofIgGismarkedlyaffected—becauseofcellsareobtainedfromthespleenofamouse(orotherimpairmentofthebody’sdefenseagainstmicroorgan-suitableanimal)previouslyinjectedwithanantigenorisms.mixtureofantigens(eg,foreigncells).TheBcellsare
594596/CHAPTER50mixedwithmousemyelomacellsandexposedtopoly-teryofmonoclonalantibodiescanbeobtained,manyofethyleneglycol,whichcausescellfusion.Asummaryofwhicharespecificforindividualcomponentsoftheim-theprinciplesinvolvedingeneratinghybridomacellsismunogenicmixture.ThehybridomacellscanbefrozengiveninFigure50–11.Undertheconditionsused,onlyandstoredandsubsequentlythawedwhenmoreofthethehybridomacellsmultiplyincellculture.Thisin-antibodyisrequired;thisensuresitslong-termsupply.volvesplatingthehybridcellsintohypoxanthine-Thehybridomacellscanalsobegrownintheabdomenaminopterin-thymidine(HAT)-containingmediumatofmice,providingrelativelylargesuppliesofanti-aconcentrationsuchthateachdishcontainsapproxi-bodies.matelyonecell.Thus,acloneofhybridomacellsmul-Becauseoftheirspecificity,monoclonalantibodiestipliesineachdish.Theculturemediumisharvestedhavebecomeusefulreagentsinmanyareasofbiologyandscreenedforantibodiesthatreactwiththeoriginalandmedicine.Forexample,theycanbeusedtomea-antigenorantigens.Iftheimmunogenisamixtureofsuretheamountsofmanyindividualproteins(eg,manyantigens(eg,acellmembranepreparation),anplasmaproteins),todeterminethenatureofinfectiousindividualculturedishwillcontainacloneofhy-agents(eg,typesofbacteria),andtosubclassifybothbridomacellssynthesizingamonoclonalantibodytonormal(eg,lymphocytes)andtumorcells(eg,leukemiconespecificantigenicdeterminantofthemixture.Bycells).Inaddition,theyarebeingusedtodirectthera-harvestingthemediafrommanyculturedishes,abat-peuticagentstotumorcellsandalsotoacceleratere-movalofdrugsfromthecirculationwhentheyreachtoxiclevels(eg,digoxin).MyelomacellBcellTheComplementSystemComprisesAbout20PlasmaProteins&IsInvolvedinCellLysis,Inflammation,&OtherProcessesPlasmacontainsapproximately20proteinsthatareFusedinpresenceofPEGmembersofthecomplementsystem.ThissystemwasdiscoveredwhenitwasobservedthatadditionoffreshHybridomacellserumcontainingantibodiesdirectedtoabacteriumcauseditslysis.Unlikeantibodies,thefactorwaslabileGrowninpresenceofHATmediumwhenheatedat56°C.SubsequentworkhasresolvedHybridomamultiplies;myelomaandBcellsdietheproteinsofthesystemandhowtheyfunction;mosthavebeenclonedandsequenced.ThemajorproteincomponentsaredesignatedC1–9,withC9associatedHybridomacellwiththeC5–8complex(togetherconstitutingtheFigure50–11.Schemeofproductionofahy-membraneattackcomplex)beinginvolvedingenerat-bridomacell.Themyelomacellsareimmortalized,doingalipid-solubleporeinthecellmembranethatnotproduceantibody,andareHGPRT–(renderingthecausesosmoticlysis.salvagepathwayofpurinesynthesis[Chapter34]inac-Thedetailsofthissystemarerelativelycomplex,andtive).TheBcellsarenotimmortalized,eachproducesaatextbookofimmunologyshouldbeconsulted.The+basicconceptisthatthenormallyinactiveproteinsofspecificantibody,andtheyareHGPRT.Polyethylenethesystem,whentriggeredbyastimulus,becomeacti-glycol(PEG)stimulatescellfusion.Theresultinghy-vatedbyproteolysisandinteractinaspecificsequencebridomacellsareimmortalized(viatheparental+withoneormoreoftheotherproteinsofthesystem.myelomacells),produceantibody,andareHGPRTThisresultsincelllysisandgenerationofpeptideor(bothlatterpropertiesgainedfromtheparentalBcells).polypeptidefragmentsthatareinvolvedinvariousas-TheBcellswilldieinthemediumbecausetheyarenotpectsofinflammation(chemotaxis,phagocytosis,etc).immortalized.InthepresenceofHAT,themyelomaThesystemhasotherfunctions,suchasclearanceofcellswillalsodie,sincetheaminopterininHATsup-antigen-antibodycomplexesfromthecirculation.Acti-pressespurinesynthesisbythedenovopathwaybyin-vationofthecomplementsystemistriggeredbyoneofhibitingreutilizationoftetrahydrofolate(Chapter34).tworoutes,calledtheclassicandthealternativepath-However,thehybridomacellswillsurvive,grow(be-ways.ThefirstinvolvesinteractionofC1withantigen-causetheyareHGPRT+),and—ifcloned—produceantibodycomplexes,andthesecond(notinvolvingan-monoclonalantibody.(HAT,hypoxanthine,tibody)involvesdirectinteractionofbacterialcellaminopterin,andthymidine;HGPRT,hypoxanthine-surfacesorpolysaccharideswithacomponentdesig-guaninephosphoribosyltransferase.)natedC3b.
595PLASMAPROTEINS&IMMUNOGLOBULINS/597Thecomplementsystemresemblesbloodcoagula-trophils.Geneticdeficiencyofthisproteinisacausetion(Chapter51)inthatitinvolvesbothconversionofofemphysemaandcanalsoleadtoliverdisease.inactiveprecursorstoactiveproductsbyproteasesanda•α2-Macroglobulinisamajorplasmaproteinthatcascadewithamplification.neutralizesmanyproteasesandtargetscertaincy-tokinestospecificorgans.SUMMARY•Immunoglobulinsplayakeyroleinthedefensemechanismsofthebody,asdoproteinsofthecom-•Plasmacontainsmanyproteinswithavarietyofplementsystem.Someoftheprincipalfeaturesoffunctions.Mostaresynthesizedintheliverandaretheseproteinsaredescribed.glycosylated.•Albumin,whichisnotglycosylated,isthemajorpro-teinandistheprincipaldeterminantofintravascularosmoticpressure;italsobindsmanyligands,suchasREFERENCESdrugsandbilirubin.•Haptoglobinbindsextracorpuscularhemoglobin,AndrewsNC:Disordersofironmetabolism.NEnglJMedpreventsitslossintothekidneyandurine,andhence1999;341:1986.preservesitsironforreutilization.CarrellRW,LomasDA:Alpha1-antitrypsindeficiency—amodelforconformationaldiseases.NEnglJMed2002;346:45.•Transferrinbindsiron,transportingittositeswhereGabayC,KushnerI:Acute-phaseproteinsandothersystemicre-itisrequired.Ferritinprovidesanintracellularstoresponsestoinflammation.NewEnglJMed1999;340:448.ofiron.IrondeficiencyanemiaisaveryprevalentHarrisED:Cellularcoppertransportandmetabolism.AnnuRevdisorder.HereditaryhemochromatosishasbeenNutr2000;20:291.showntobeduetomutationsinHFE,ageneencod-LangloisMR,DelangheJR:BiologicalandclinicalsignificanceofingtheproteinHFE,whichappearstoplayanim-haptoglobinpolymorphisminhumans.ClinChem1996;portantroleinabsorptionofiron.2:1589.•Ceruloplasmincontainssubstantialamountsofcop-LevinsonW,JawetzE:MedicalMicrobiologyandImmunology,6thper,butalbuminappearstobemoreimportantwithed.Appleton&Lange,2000.regardtoitstransport.BothWilsondiseaseandParslowTGetal(editors):MedicalImmunology,10thed.Appleton&Lange,2001.Menkesdisease,whichreflectabnormalitiesofcoppermetabolism,havebeenfoundtobeduetomutationsPepysMB,BergerA:TherenaissanceofCreactiveprotein.BMJ2001;322:4.ingenesencodingcopper-bindingP-typeATPases.WaheedAetal:Regulationoftransferrin-mediatedironuptakeby•α1-AntitrypsinisthemajorserineproteaseinhibitorHFE,theproteindefectiveinhereditaryhemochromatosis.ofplasma,inparticularinhibitingtheelastaseofneu-ProcNatlAcadUSA2002;99:3117.
596Hemostasis&Thrombosis51MargaretL.Rand,PhD,&RobertK.Murray,MD,PhDBIOMEDICALIMPORTANCEThereAreThreeTypesofThrombiBasicaspectsoftheproteinsofthebloodcoagulationThreetypesofthrombiorclotsaredistinguished.Allsystemandoffibrinolysisaredescribedinthischapter.threecontainfibrininvariousproportions.Somefundamentalaspectsofplateletbiologyarealso(1)Thewhitethrombusiscomposedofplateletsandpresented.Hemorrhagicandthromboticstatescanfibrinandisrelativelypoorinerythrocytes.Itcauseseriousmedicalemergencies,andthrombosesinformsatthesiteofaninjuryorabnormalvesselthecoronaryandcerebralarteriesaremajorcausesofwall,particularlyinareaswherebloodflowisdeathinmanypartsoftheworld.Rationalmanage-rapid(arteries).mentoftheseconditionsrequiresaclearunderstandingofthebasesofbloodclottingandfibrinolysis.(2)Theredthrombusconsistsprimarilyofredcellsandfibrin.ItmorphologicallyresemblestheclotformedinatesttubeandmayforminvivoinHEMOSTASIS&THROMBOSISHAVEareasofretardedbloodfloworstasis(eg,veins)THREECOMMONPHASESwithorwithoutvascularinjury,oritmayformatasiteofinjuryorinanabnormalvesselincon-Hemostasisisthecessationofbleedingfromacutorjunctionwithaninitiatingplateletplug.severedvessel,whereasthrombosisoccurswhentheen-(3)Athirdtypeisadisseminatedfibrindepositindotheliumliningbloodvesselsisdamagedorremovedverysmallbloodvesselsorcapillaries.(eg,uponruptureofanatheroscleroticplaque).Theseprocessesencompassbloodclotting(coagulation)andWeshallfirstdescribethecoagulationpathwaylead-involvebloodvessels,plateletaggregation,andplasmaingtotheformationoffibrin.Thenweshallbrieflyde-proteinsthatcauseformationordissolutionofplateletscribesomeaspectsoftheinvolvementofplateletsandaggregates.bloodvesselwallsintheoverallprocess.ThisseparationInhemostasis,thereisinitialvasoconstrictionoftheofclottingfactorsandplateletsisartificial,sincebothinjuredvessel,causingdiminishedbloodflowdistaltoplayintimateandoftenmutuallyinterdependentrolestheinjury.Thenhemostasisandthrombosissharethreeinhemostasisandthrombosis,butitfacilitatesdescrip-phases:tionoftheoverallprocessesinvolved.(1)Formationofalooseandtemporaryplateletag-gregateatthesiteofinjury.Plateletsbindtocol-BothIntrinsic&ExtrinsicPathwaysResultlagenatthesiteofvesselwallinjuryandareacti-intheFormationofFibrinvatedbythrombin(themechanismofactivationTwopathwaysleadtofibrinclotformation:theintrin-ofplateletsisdescribedbelow),formedintheco-sicandtheextrinsicpathways.Thesepathwaysarenotagulationcascadeatthesamesite,orbyADPre-independent,aspreviouslythought.However,thisarti-leasedfromotheractivatedplatelets.Uponacti-ficialdistinctionisretainedinthefollowingtexttofa-vation,plateletschangeshapeand,inthecilitatetheirdescription.presenceoffibrinogen,aggregatetoformthehe-Initiationofthefibrinclotinresponsetotissuein-mostaticplug(inhemostasis)orthrombus(injuryiscarriedoutbytheextrinsicpathway.Howthethrombosis).intrinsicpathwayisactivatedinvivoisunclear,butit(2)Formationofafibrinmeshthatbindstotheinvolvesanegativelychargedsurface.Theintrinsicandplateletaggregate,formingamorestablehemosta-extrinsicpathwaysconvergeinafinalcommonpath-ticplugorthrombus.wayinvolvingtheactivationofprothrombintothrom-(3)Partialorcompletedissolutionofthehemostaticbinandthethrombin-catalyzedcleavageoffibrinogenplugorthrombusbyplasmin.toformthefibrinclot.Theintrinsic,extrinsic,andfinalcommonpathwaysarecomplexandinvolvemanydifferentproteins(Figure51–1andTable51–1).In598
597HEMOSTASIS&THROMBOSIS/599IntrinsicpathwayPKHKXIIXIIaHKCa2+ExtrinsicpathwayXIXIaVIICa2+IXIXaVIIa/TissuefactorCa2+VIIIVIIIaPLXXaXFigure51–1.Thepathwaysofbloodcoagulation.TheintrinsicCa2+andextrinsicpathwaysareindi-VVaPLcated.TheeventsdepictedbelowfactorXaaredesignatedthefinalcommonpathway,culminatingintheformationofcross-linkedfibrin.ProthrombinThrombinNewobservations(dottedarrow)includethefindingthatcomplexesoftissuefactorandfactorVIIaacti-FibrinogenXIIIvatenotonlyfactorX(intheclassicextrinsicpathway)butalsofactorIXintheintrinsicpathway.Inad-Classiccoagulationcascadedition,thrombinandfactorXaFibrinmonomerXIIIaPositivefeedbackfeedback-activateatthetwosites(hypothesized)indicated(dashedarrows).(PK,Extrinsic-to-intrinsicprekallikrein;HK,HMWkininogen;activationFibrinpolymerPL,phospholipids.)(Reproduced,withpermission,fromRobertsHR,LozierJN:Newperspectivesonthecoagulationcascade.HospPract[OffCross-linkedEd]1992Jan;27:97.)fibrinpolymer
598600/CHAPTER51Table51–1.NumericalsystemfornomenclatureTable51–2.Thefunctionsoftheproteinsofbloodclottingfactors.Thenumbersindicateinvolvedinbloodcoagulation.theorderinwhichthefactorshavebeendiscoveredandbearnorelationshiptotheorderZymogensofserineproteasesinwhichtheyact.FactorXIIBindstonegativelychargedsurfaceatsiteofvesselwallinjury;activatedbyhigh-MWkininogenandkallikrein.FactorCommonNameFactorXIActivatedbyfactorXIIa.2+IFibrinogenThesefactorsareusuallyreferredFactorIXActivatedbyfactorXlainpresenceofCa.IIProthrombintobytheircommonnames.FactorVIIActivatedthrombininpresenceofCa2+.IIITissuefactorThesefactorsareusuallynotre-FactorXActivatedonsurfaceofactivatedplateletsbyIVCa2+ferredtoascoagulationfactors.tenasecomplex(Ca2+,factorsVIIIaandIXa)VProaccelerin,labilefactor,accelerator(Ac-)andbyfactorVIIainpresenceoftissuefac-2+globulintorandCa.VII1Proconvertin,serumprothrombinconversionFactorIIActivatedonsurfaceofactivatedplateletsby2+accelerator(SPCA),cothromboplastinprothrombinasecomplex(Ca,factorsVaVIIIAntihemophilicfactorA,antihemophilicglobulinandXa).(AHG)[FactorsII,VII,IX,andXareGla-containingIXAntihemophilicfactorB,Christmasfactor,plasmazymogens.](Gla=γ-carboxyglutamate.)thromboplastincomponent(PTC)CofactorsXStuart-ProwerfactorFactorVIIIActivatedbythrombin;factorVIIIaisaco-XIPlasmathromboplastinantecedent(PTA)factorintheactivationoffactorXbyXIIHagemanfactorfactorIXa.XIIIFibrinstabilizingfactor(FSF),fibrinoligaseFactorVActivatedbythrombin;factorVaisaco-1factorintheactivationofprothrombinbyThereisnofactorVI.factorXa.TissuefactorAglycoproteinexpressedonthesurfaceofgeneral,asshowninTable51–2,theseproteinscanbe(factorIII)injuredorstimulatedendothelialcellstoclassifiedintofivetypes:(1)zymogensofserine-depen-actasacofactorforfactorVIIa.dentproteases,whichbecomeactivatedduringtheFibrinogenprocessofcoagulation;(2)cofactors;(3)fibrinogen;FactorICleavedbythrombintoformfibrinclot.(4)atransglutaminase,whichstabilizesthefibrinclot;Thiol-dependenttransglutaminaseand(5)regulatoryandotherproteins.2+FactorXIIIActivatedbythrombininpresenceofCa;stabilizesfibrinclotbycovalentcross-TheIntrinsicPathwayLeadslinking.toActivationofFactorXRegulatoryandotherproteinsTheintrinsicpathway(Figure51–1)involvesfactorsProteinCActivatedtoproteinCabythrombinboundXII,XI,IX,VIII,andXaswellasprekallikrein,high-tothrombomodulin;thendegradesfac-2+torsVIIIaandVa.molecular-weight(HMW)kininogen,Ca,andplate-ProteinSActsasacofactorofproteinC;bothproteinsletphospholipids.Itresultsintheproductionoffac-containGla(γ-carboxyglutamate)torXa(byconvention,activatedclottingfactorsareresidues.referredtobyuseofthesuffixa).Thrombo-ProteinonthesurfaceofendothelialThispathwaycommenceswiththe“contactphase”modulincells;bindsthrombin,whichthenacti-inwhichprekallikrein,HMWkininogen,factorXII,vatesproteinC.andfactorXIareexposedtoanegativelychargedacti-vatingsurface.Invivo,theproteinsprobablyassembleXIaandalsoreleasesbradykinin(anonapeptidewithonendothelialcellmembranes,whereasglassorkaolinpotentvasodilatoraction)fromHMWkininogen.2+canbeusedforinvitrotestsoftheintrinsicpathway.FactorXIainthepresenceofCaactivatesfactorIXWhenthecomponentsofthecontactphaseassemble(55kDa,azymogencontainingvitaminK-dependentontheactivatingsurface,factorXIIisactivatedtofac-γ-carboxyglutamate[Gla]residues;seeChapter45),totorXIIauponproteolysisbykallikrein.Thisfactortheserineprotease,factorIXa.ThisinturncleavesanXIIa,generatedbykallikrein,attacksprekallikreintoArg-IlebondinfactorX(56kDa)toproducethetwo-generatemorekallikrein,settingupareciprocalactiva-chainserineprotease,factorXa.Thislatterreactionre-tion.FactorXIIa,onceformed,activatesfactorXItoquirestheassemblyofcomponents,calledthetenase
599HEMOSTASIS&THROMBOSIS/601complex,onthesurfaceofactivatedplatelets:Ca2+andcleaveplasminogenandkallikreincanactivatesingle-factorVIIIa,aswellasfactorsIXaandX.Itshouldbechainurokinase.notedthatinallreactionsinvolvingtheGla-containingTissuefactorpathwayinhibitor(TFPI)isamajorzymogens(factorsII,VII,IX,andX),theGlaresiduesphysiologicinhibitorofcoagulation.Itisaproteinthatintheaminoterminalregionsofthemoleculesserveascirculatesinthebloodassociatedwithlipoproteins.high-affinitybindingsitesforCa2+.ForassemblyoftheTFPIdirectlyinhibitsfactorXabybindingtotheen-tenasecomplex,theplateletsmustfirstbeactivatedtozymenearitsactivesite.ThisfactorXa-TFPIcomplexexposetheacidic(anionic)phospholipids,phos-theninhibitsthefactorVIIa-tissuefactorcomplex.phatidylserineandphosphatidylinositol,thatarenormallyontheinternalsideoftheplasmamembraneTheFinalCommonPathwayofBloodofresting,nonactivatedplatelets.FactorVIII(330ClottingInvolvesActivationofkDa),aglycoprotein,isnotaproteaseprecursorbutaProthrombintoThrombincofactorthatservesasareceptorforfactorsIXaandXontheplateletsurface.FactorVIIIisactivatedbyInthefinalcommonpathway,factorXa,producedbyminutequantitiesofthrombintoformfactorVIIIa,eithertheintrinsicortheextrinsicpathway,activateswhichisinturninactivateduponfurthercleavagebyprothrombin(factorII)tothrombin(factorIIa),thrombin.whichthenconvertsfibrinogentofibrin(Figure51–1).Theactivationofprothrombin,likethatoffactorX,occursonthesurfaceofactivatedplateletsandrequiresTheExtrinsicPathwayAlsoLeadstheassemblyofaprothrombinasecomplex,consistingtoActivationofFactorXBut2+ofplateletanionicphospholipids,Ca,factorVa,fac-byaDifferentMechanismtorXa,andprothrombin.FactorXaoccursatthesitewheretheintrinsicandex-FactorV(330kDa),aglycoproteinwithhomologytrinsicpathwaysconverge(Figure51–1)andleadintotofactorVIIIandceruloplasmin,issynthesizedinthethefinalcommonpathwayofbloodcoagulation.Theliver,spleen,andkidneyandisfoundinplateletsasextrinsicpathwayinvolvestissuefactor,factorsVIIandwellasinplasma.ItfunctionsasacofactorinamannerX,andCa2+andresultsintheproductionoffactorXa.similartothatoffactorVIIIinthetenasecomplex.Itisinitiatedatthesiteoftissueinjurywiththeexpo-WhenactivatedtofactorVabytracesofthrombin,itsureoftissuefactor(Figure51–1)onsubendothelialbindstospecificreceptorsontheplateletmembranecells.Tissuefactorinteractswithandactivatesfactor(Figure51–2)andformsacomplexwithfactorXaandVII(53kDa),acirculatingGla-containingglycoproteinprothrombin.Itissubsequentlyinactivatedbyfurthersynthesizedintheliver.Tissuefactoractsasacofactoractionofthrombin,therebyprovidingameansoflimit-forfactorVIIa,enhancingitsenzymaticactivitytoacti-ingtheactivationofprothrombintothrombin.Pro-vatefactorX.Theassociationoftissuefactorandfactorthrombin(72kDa;Figure51–3)isasingle-chaingly-VIIaiscalledtissuefactorcomplex.FactorVIIacoproteinsynthesizedintheliver.TheaminoterminalcleavesthesameArg-IlebondinfactorXthatiscleavedregionofprothrombin(1inFigure51–3)containstenbythetenasecomplexoftheintrinsicpathway.Activa-Glaresidues,andtheserine-dependentactiveproteasetionoffactorXprovidesanimportantlinkbetweenthesite(indicatedbythearrowhead)isinthecarboxylter-intrinsicandextrinsicpathways.minalregionofthemolecule.UponbindingtotheAnotherimportantinteractionbetweentheextrinsiccomplexoffactorsVaandXaontheplateletmem-andintrinsicpathwaysisthatcomplexesoftissuefactorbrane,prothrombiniscleavedbyfactorXaattwositesandfactorVIIaalsoactivatefactorIXintheintrinsic(Figure51–2)togeneratetheactive,two-chainthrom-pathway.Indeed,theformationofcomplexesbe-binmolecule,whichisthenreleasedfromtheplatelettweentissuefactorandfactorVIIaisnowconsid-surface.TheAandBchainsofthrombinareheldto-eredtobethekeyprocessinvolvedininitiationofgetherbyadisulfidebond.bloodcoagulationinvivo.Thephysiologicsignifi-canceoftheinitialstepsoftheintrinsicpathway,inConversionofFibrinogentoFibrinwhichfactorXII,prekallikrein,andHMWkininogenIsCatalyzedbyThrombinareinvolved,hasbeencalledintoquestionbecausepa-tientswithahereditarydeficiencyofthesecomponentsFibrinogen(factorI,340kDa;seeFigures51–1anddonotexhibitbleedingproblems.Similarly,patients51–4andTables51–1and51–2)isasolubleplasmawithadeficiencyoffactorXImaynothavebleedingglycoproteinthatconsistsofthreenonidenticalpairsofproblems.Theintrinsicpathwaymayactuallybemorepolypeptidechains(Aα,Bβγ)2covalentlylinkedbyimportantinfibrinolysis(seebelow)thanincoagula-disulfidebonds.TheBβandγchainscontainas-tion,sincekallikrein,factorXIIa,andfactorXIacanparagine-linkedcomplexoligosaccharides.Allthree
600602/CHAPTER51PrethrombinFigure51-2.Diagrammaticrepresentation(nottoscale)ofthebindingoffactorsVa,Xa,F-1.2Ca2+,andprothrombintotheplasmamembrane+S-SCOO–oftheactivatedplatelet.ThesitesofcleavageofNH3prothrombinbyfactorXaareindicatedbytwoCa2+Ca2+VPlateletarrows.ThepartofprothrombindestinedtoaXaplasma2+formthrombinislabeledprethrombin.TheCamembraneIndicatesnegativechargesisboundtoanionicphospholipidsoftheplasmatowhichCa2+binds.membraneoftheactivatedplatelet.chainsaresynthesizedintheliver;thethreestructuralture(α,β,γ)2.SinceFPAandFPBcontainonly16andgenesinvolvedareonthesamechromosome,andtheir14residues,respectively,thefibrinmoleculeretainsexpressioniscoordinatelyregulatedinhumans.The98%oftheresiduespresentinfibrinogen.Theremovalaminoterminalregionsofthesixchainsareheldinofthefibrinopeptidesexposesbindingsitesthatallowcloseproximitybyanumberofdisulfidebonds,whilethemoleculesoffibrinmonomerstoaggregatesponta-thecarboxylterminalregionsarespreadapart,givingneouslyinaregularlystaggeredarray,forminganinsol-risetoahighlyasymmetric,elongatedmolecule(Figureublefibrinclot.Itistheformationofthisinsolublefib-51–4).TheAandBportionsoftheAαandBβchains,rinpolymerthattrapsplatelets,redcells,andotherdesignatedfibrinopeptidesA(FPA)andB(FPB),re-componentstoformthewhiteorredthrombi.Thisspectively,attheaminoterminalendsofthechains,initialfibrinclotisratherweak,heldtogetheronlybybearexcessnegativechargesasaresultofthepresencethenoncovalentassociationoffibrinmonomers.ofaspartateandglutamateresidues,aswellasanun-Inadditiontoconvertingfibrinogentofibrin,usualtyrosineO-sulfateinFPB.ThesenegativechargesthrombinalsoconvertsfactorXIIItofactorXIIIa.Thiscontributetothesolubilityoffibrinogeninplasmaandfactorisahighlyspecifictransglutaminasethatcova-alsoservetopreventaggregationbycausingelectrosta-lentlycross-linksfibrinmoleculesbyformingpeptideticrepulsionbetweenfibrinogenmolecules.bondsbetweentheamidegroupsofglutamineandtheThrombin(34kDa),aserineproteaseformedbyε-aminogroupsoflysineresidues(Figure51–5B),theprothrombinasecomplex,hydrolyzesthefourArg-yieldingamorestablefibrinclotwithincreasedresis-Glybondsbetweenthefibrinopeptidesandtheαandβtancetoproteolysis.portionsoftheAαandBβchainsoffibrinogen(Figure51–5A).Thereleaseofthefibrinopeptidesbythrombingeneratesfibrinmonomer,whichhasthesubunitstruc-LevelsofCirculatingThrombinMustBeCarefullyControlledorClotsMayFormXaOnceactivethrombinisformedinthecourseofhemo-S–Sstasisorthrombosis,itsconcentrationmustbecarefullyGlancontrolledtopreventfurtherfibrinformationor12ABplateletactivation.Thisisachievedintwoways.Thrombincirculatesasitsinactiveprecursor,pro-Xathrombin,whichisactivatedastheresultofacascadeofenzymaticreactions,eachconvertinganinactivezy-F-12Prethrombin(beforeXacleavage)mogentoanactiveenzymeandleadingfinallytotheThrombin(afterXacleavage)conversionofprothrombintothrombin(Figure51–1).Ateachpointinthecascade,feedbackmechanismsFigure51–3.Diagrammaticrepresentation(nottoproduceadelicatebalanceofactivationandinhibition.scale)ofprothrombin.Theaminoterminalistotheleft;TheconcentrationoffactorXIIinplasmaisapproxi-region1containsalltenGlaresidues.Thesitesofcleav-mately30μg/mL,whilethatoffibrinogenis3mg/mL,agebyfactorXaareshownandtheproductsnamed.withintermediateclottingfactorsincreasinginconcen-Thesiteofthecatalyticallyactiveserineresidueisindi-trationasoneproceedsdownthecascade,showingthatcatedbythesolidtriangle.TheAandBchainsofactivetheclottingcascadeprovidesamplification.Thesecondthrombin(shaded)areheldtogetherbythedisulfidemeansofcontrollingthrombinactivityistheinactiva-bridge.tionofanythrombinformedbycirculatinginhibi-
601HEMOSTASIS&THROMBOSIS/603FPAFPBAαchainFigure51–4.Diagrammaticrepresentation(nottoscale)offibrinogenshowingpairsofAα,BβchainBβ,andγchainslinkedbydisulfidebonds.(FPA,γchain–NH+–fibrinopeptideA;FPB,fibrinopeptideB.)COO3COOtors,themostimportantofwhichisantithrombinIIIthrombinaswellastoitsothersubstrates.Thisisthe(seebelow).basisfortheuseofheparininclinicalmedicinetoin-hibitcoagulation.TheanticoagulanteffectsofheparinTheActivityofAntithrombinIII,canbeantagonizedbystronglycationicpolypeptidessuchasprotamine,whichbindstronglytoheparin,anInhibitorofThrombin,thusinhibitingitsbindingtoantithrombinIII.Individ-IsIncreasedbyHeparinualswithinheriteddeficienciesofantithrombinIIIareFournaturallyoccurringthrombininhibitorsexistinpronetodevelopvenousthrombosis,providingevi-normalplasma.ThemostimportantisantithrombindencethatantithrombinIIIhasaphysiologicfunctionIII(oftencalledsimplyantithrombin),whichcon-andthatthecoagulationsysteminhumansisnormallytributesapproximately75%oftheantithrombinactiv-inadynamicstate.ity.AntithrombinIIIcanalsoinhibittheactivitiesofThrombinisinvolvedinanadditionalregulatoryfactorsIXa,Xa,XIa,XIIa,andVIIacomplexedwithmechanismthatoperatesincoagulation.Itcombinestissuefactor.2-Macroglobulincontributesmostofwiththrombomodulin,aglycoproteinpresentonthetheremainderoftheantithrombinactivity,withhep-surfacesofendothelialcells.Thecomplexactivatespro-arincofactorIIand1-antitrypsinactingasminorin-teinC.IncombinationwithproteinS,activatedpro-hibitorsunderphysiologicconditions.teinC(APC)degradesfactorsVaandVIIIa,limitingTheendogenousactivityofantithrombinIIIistheiractionsincoagulation.Ageneticdeficiencyofei-greatlypotentiatedbythepresenceofacidicproteogly-therproteinCorproteinScancausevenousthrombo-canssuchasheparin(Chapter48).Thesebindtoasis.Furthermore,patientswithfactorVLeiden(whichspecificcationicsiteofantithrombinIII,inducingahasaglutamineresidueinplaceofanarginineatposi-conformationalchangeandpromotingitsbindingtotion506)haveanincreasedriskofvenousthromboticAThrombin––+ArgGlyCOO–NH3––FibrinopeptideFibrinchain(AorB)(αorβ)BFigure51-5.FormationofafibrinOclot.A:Thrombin-inducedcleavage+FibrinCH2CH2CH2CH2NH3H2NCCH2CH2FibrinofArg-GlybondsoftheAαandBβ(Lysyl)(Glutaminyl)chainsoffibrinogentoproducefi-brinopeptides(left-handside)and+theαandβchainsoffibrinmono-NH4FactorXIIIa(Transglutaminase)mer(right-handside).B:Cross-Olinkingoffibrinmoleculesbyacti-vatedfactorXIII(factorXIIIa).FibrinCH2CH2CH2CH2NHCCH2CH2Fibrin
602604/CHAPTER51diseasebecausefactorVLeidenisresistanttoinactiva-Inpastyears,treatmentforpatientswithhemophiliationbyAPC.ThisconditionistermedAPCresistance.Ahasconsistedofadministrationofcryoprecipitates(enrichedinfactorVIII)preparedfromindividualdonorsorlyophilizedfactorVIIIconcentratespreparedCoumarinAnticoagulantsInhibitthefromplasmapoolsofupto5000donors.Itisnowpos-sibletopreparefactorVIIIbyrecombinantDNAVitaminK-DependentCarboxylationoftechnology.Suchpreparationsarefreeofcontaminat-FactorsII,VII,IX,&Xingviruses(eg,hepatitisA,B,C,orHIV-1)foundinThecoumarindrugs(eg,warfarin),whichareusedashumanplasmabutareatpresentexpensive;theiruseanticoagulants,inhibitthevitaminK-dependentcar-mayincreaseifcostofproductiondecreases.boxylationofGlutoGlaresidues(seeChapter45)intheaminoterminalregionsoffactorsII,VII,IX,andXFibrinClotsAreDissolvedbyPlasminandalsoproteinsCandS.Theseproteins,allofwhich2+Asstatedabove,thecoagulationsystemisnormallyinaaresynthesizedintheliver,aredependentontheCa-bindingpropertiesoftheGlaresiduesfortheirnormalstateofdynamicequilibriuminwhichfibrinclotsarefunctioninthecoagulationpathways.Thecoumarinsconstantlybeinglaiddownanddissolved.Thislatteractbyinhibitingthereductionofthequinonederiva-processistermedfibrinolysis.Plasmin,theserinepro-tivesofvitaminKtotheactivehydroquinoneformsteasemainlyresponsiblefordegradingfibrinandfi-(Chapter45).Thus,theadministrationofvitaminKbrinogen,circulatesintheformofitsinactivezymogen,willbypassthecoumarin-inducedinhibitionandallowplasminogen(90kDa),andanysmallamountsofplas-maturationoftheGla-containingfactors.Reversalofminthatareformedinthefluidphaseunderphysio-coumarininhibitionbyvitaminKrequires12–24logicconditionsarerapidlyinactivatedbythefast-hours,whereasreversaloftheanticoagulanteffectsofactingplasmininhibitor,α2-antiplasmin.Plasminogenheparinbyprotamineisalmostinstantaneous.bindstofibrinandthusbecomesincorporatedinclotsHeparinandwarfarinarewidelyusedinthetreat-astheyareproduced;sinceplasminthatisformedmentofthromboticandthromboembolicconditions,whenboundtofibrinisprotectedfromα2-antiplasmin,suchasdeepveinthrombosisandpulmonaryembolus.itremainsactive.ActivatorsofplasminogenofvariousHeparinisadministeredfirst,becauseofitsprompttypesarefoundinmostbodytissues,andallcleavetheonsetofaction,whereaswarfarintakesseveraldaystosameArg-Valbondinplasminogentoproducethetwo-reachfulleffect.Theireffectsarecloselymonitoredbychainserineprotease,plasmin(Figure51–6).useofappropriatetestsofcoagulation(seebelow)be-Tissueplasminogenactivator(alteplase;t-PA)isacauseoftheriskofproducinghemorrhage.serineproteasethatisreleasedintothecirculationfromvascularendotheliumunderconditionsofinjuryorHemophiliaAIsDuetoaGeneticallyDeterminedDeficiencyofFactorVIIIPlasminogenPLASMINOGENactivatorsInheriteddeficienciesoftheclottingsystemthatresultinbleedingarefoundinhumans.Themostcommonis+–NH3Arg–ValCOOdeficiencyoffactorVIII,causinghemophiliaA,anXSSchromosome-linkeddiseasethathasplayedamajorroleinthehistoryoftheroyalfamiliesofEurope.Hemo-philiaBisduetoadeficiencyoffactorIX;itsclinicalfeaturesarealmostidenticaltothoseofhemophiliaA,buttheconditionscanbeseparatedonthebasisofspe-+–cificassaysthatdistinguishbetweenthetwofactors.NH3ArgValCOOThegeneforhumanfactorVIIIhasbeenclonedSSandisoneofthelargestsofarstudied,measuring186PLASMINkbinlengthandcontaining26exons.Avarietyofmu-tationshavebeendetectedleadingtodiminishedactiv-Figure51–6.Activationofplasminogen.ThesameityoffactorVIII;theseincludepartialgenedeletionsArg-Valbondiscleavedbyallplasminogenactivatorsandpointmutationsresultinginprematurechainter-togivethetwo-chainplasminmolecule.Thesolidtrian-mination.PrenataldiagnosisbyDNAanalysisaftergleindicatestheserineresidueoftheactivesite.Thechorionicvillussamplingisnowpossible.twochainsofplasminareheldtogetherbyadisulfidebridge.
603HEMOSTASIS&THROMBOSIS/605stressandiscatalyticallyinactiveunlessboundtofibrin.slightlybettersurvivalrate.Table51–3comparessomeUponbindingtofibrin,t-PAcleavesplasminogenthrombolyticfeaturesofstreptokinaseandt-PA.withintheclottogenerateplasmin,whichinturndi-Thereareanumberofdisorders,includingcancergeststhefibrintoformsolubledegradationproductsandshock,inwhichtheconcentrationsofplasmino-andthusdissolvestheclot.Neitherplasminnorthegenactivatorsincrease.Inaddition,theantiplasminplasminogenactivatorcanremainboundtotheseactivitiescontributedbyα1-antitrypsinandα2-antiplas-degradationproducts,andsotheyarereleasedintotheminmaybeimpairedindiseasessuchascirrhosis.Sincefluidphase,wheretheyareinactivatedbytheirnaturalcertainbacterialproducts,suchasstreptokinase,areca-inhibitors.Prourokinaseistheprecursorofasecondac-pableofactivatingplasminogen,theymayberesponsi-tivatorofplasminogen,urokinase.Originallyisolatedbleforthediffusehemorrhagesometimesobservedinfromurine,itisnowknowntobesynthesizedbycellpatientswithdisseminatedbacterialinfections.typessuchasmonocytesandmacrophages,fibroblasts,andepithelialcells.Itsmainactionisprobablyinthedegradationofextracellularmatrix.Figure51–7indi-ActivationofPlateletsInvolvescatesthesitesofactionoffiveproteinsthatinfluenceStimulationofthetheformationandactionofplasmin.PolyphosphoinositidePathwayRecombinantt-PA&StreptokinaseArePlateletsnormallycirculateinanunstimulateddisk-shapedform.Duringhemostasisorthrombosis,theyUsedasClotBustersbecomeactivatedandhelpformhemostaticplugsorAlteplase(t-PA),producedbyrecombinantDNAtech-thrombi.Threemajorstepsareinvolved:(1)adhesionnology,isusedtherapeuticallyasafibrinolyticagent,astoexposedcollageninbloodvessels,(2)releaseoftheisstreptokinase.However,thelatterislessselectivecontentsoftheirgranules,and(3)aggregation.thant-PA,activatingplasminogeninthefluidphasePlateletsadheretocollagenviaspecificreceptorson(whereitcandegradecirculatingfibrinogen)aswellastheplateletsurface,includingtheglycoproteincomplexplasminogenthatisboundtoafibrinclot.TheamountGPIa–IIa(α2β1integrin;Chapter52),inareactionofplasminproducedbytherapeuticdosesofstreptoki-thatinvolvesvonWillebrandfactor.Thisisaglyco-nasemayexceedthecapacityofthecirculatingα2-protein,secretedbyendothelialcellsintotheplasma,antiplasmin,causingfibrinogenaswellasfibrintobewhichstabilizesfactorVIIIandbindstocollagenanddegradedandresultinginthebleedingoftenencoun-thesubendothelium.PlateletsbindtovonWillebrandteredduringfibrinolytictherapy.Becauseofitsselec-factorviaaglycoproteincomplex(GPIb–V–IX)onthetivityfordegradingfibrin,thereisconsiderablethera-plateletsurface;thisinteractionisespeciallyimportantpeuticinterestintheuseofrecombinantt-PAtorestoreinplateletadherencetothesubendotheliumundercon-thepatencyofcoronaryarteriesfollowingthrombosis.ditionsofhighshearstressthatoccurinsmallvesselsIfadministeredearlyenough,beforeirreversibledam-andstenosedarteries.ageofheartmuscleoccurs(about6hoursafteronsetofPlateletsadherenttocollagenchangeshapeandthrombosis),t-PAcansignificantlyreducethemortalityspreadoutonthesubendothelium.Theyreleasetheratefrommyocardialdamagefollowingcoronarycontentsoftheirstoragegranules(thedensegranulesthrombosis.t-PAismoreeffectivethanstreptokinaseatandthealphagranules);secretionisalsostimulatedbyrestoringfullpatencyandalsoappearstoresultinathrombin.Streptokinase–+PlasminogenStreptokinase-plasminogencomplexFigure51–7.SchemeofsitesofactionPlasminogent-PAUrokinaseofstreptokinase,tissueplasminogenacti-activatorvator(t-PA),urokinase,plasminogenacti-inhibitor–vatorinhibitor,andα2-antiplasmin(thelasttwoproteinsexertinhibitoryactions).Streptokinaseformsacomplexwithplas-Plasminα2-Antiplasminminogen,whichexhibitsproteolyticactiv-ity;thiscleavessomeplasminogentoplas-min,initiatingfibrinolysis.FibrinFibrindegradationproducts
604606/CHAPTER51Table51–3.ComparisonofsomepropertiesofThrombin,formedfromthecoagulationcascade,isstreptokinase(SK)andtissueplasminogenthemostpotentactivatorofplateletsandinitiatesactivator(t-PA)withregardtotheiruseasplateletactivationbyinteractingwithitsreceptoron1theplasmamembrane(Figure51–8).Thefurtherthrombolyticagents.eventsleadingtoplateletactivationareexamplesoftransmembranesignaling,inwhichachemicalmes-SKt-PAsengeroutsidethecellgenerateseffectormoleculesin-Selectiveforfibrinclot−+sidethecell.Inthisinstance,thrombinactsastheex-Producesplasminemia+−ternalchemicalmessenger(stimulusoragonist).TheReducesmortality++interactionofthrombinwithitsreceptorstimulatestheCausesallergicreaction+−activityofanintracellularphospholipaseC.Thisen-Causeshypotension+−zymehydrolyzesthemembranephospholipidphos-CostpertreatmentRelativelylowRelativelyhighphatidylinositol4,5-bisphosphate(PIP2,apolyphospho-(approximate)inositide)toformthetwointernaleffectormolecules,1DatafromWebbJ,ThompsonC:Thrombolysisforacutemyocar-1,2-diacylglyceroland1,4,5-inositoltrisphosphate.dialinfarction.CanFamPhysician1992;38:1415.HydrolysisofPIP2isalsoinvolvedintheactionofmanyhormonesanddrugs.DiacylglycerolstimulatesCollagenProstacyclinTxA2ThrombinADPAggregationFibrinogenGPllb-lllaR1R2R3R4R5++++Plasmamembrane+PLCβPIP2ACPLA2PL+PKCSignalingcAMPArachidonicacideventsIP3DAGPhosphorylationTxA2ofpleckstrin–2+CaReleaseofcontentsofplateletgranulesPhosphorylation(denseandalpha),oflightchainofincludingADP;ActinmyosinsignalingeventsActomyosinChangeofshapeFigure51–8.Diagrammaticrepresentationofplateletactivation.Theexternalenviron-ment,theplasmamembrane,andtheinsideofaplateletaredepictedfromtoptobottom.Thrombinandcollagenarethetwomostimportantplateletactivators.ADPisconsidered15aweakagonist;itcausesaggregationbutnotgranulerelease.(GP,glycoprotein;R–R,variousreceptors;AC,adenylylcyclase;PLA2,phospholipaseA2;PL,phospholipids;PLCβ,phospholipaseCβ;PIP2,phosphatidylinositol4,5-bisphosphate;cAMP,cyclicAMP;PKC,proteinkinaseC;TxA2,thromboxaneA2;IP3,inositol1,4,5-trisphosphate;DAG,1,2-diacyl-glycerol.TheGproteinsthatareinvolvedarenotshown.)
605HEMOSTASIS&THROMBOSIS/607proteinkinaseC,whichphosphorylatestheproteintors,whichmayhelpdissolvethrombi.Table51–4listspleckstrin(47kDa).Thisresultsinaggregationandre-somemoleculesproducedbyendothelialcellsthataf-leaseofthecontentsofthestoragegranules.ADPre-fectthrombosisandfibrinolysis.Endothelium-derivedleasedfromdensegranulescanalsoactivateplatelets,relaxingfactor(nitricoxide)isdiscussedinChapter49.resultinginaggregationofadditionalplatelets.IP3Analysisofthemechanismsofuptakeofatherogenic2+causesreleaseofCaintothecytosolmainlyfromthelipoproteins,suchasLDL,byendothelial,smoothmus-densetubularsystem(orresidualsmoothendoplasmiccle,andmonocyticcellsofarteries,alongwithdetailedreticulumfromthemegakaryocyte),whichtheninter-studiesofhowtheselipoproteinsdamagesuchcellsisaactswithcalmodulinandmyosinlightchainkinase,keyareaofstudyinelucidatingthemechanismsofath-leadingtophosphorylationofthelightchainsoferosclerosis(Chapter26).myosin.Thesechainstheninteractwithactin,causingchangesofplateletshape.AspirinIsanEffectiveAntiplateletDrugCollagen-inducedactivationofaplateletphospholi-paseAbyincreasedlevelsofcytosolicCa2+resultsinCertaindrugs(antiplateletdrugs)modifythebehavior2liberationofarachidonicacidfromplateletphospho-ofplatelets.Themostimportantisaspirin(acetylsali-lipids,leadingtotheformationofthromboxaneA2cylicacid),whichirreversiblyacetylatesandthusin-(Chapter23),whichinturn,inareceptor-mediatedhibitstheplateletcyclooxygenasesysteminvolvedinfashion,canfurtheractivatephospholipaseC,promot-formationofthromboxaneA2(Chapter14),apotentingplateletaggregation.aggregatorofplateletsandalsoavasoconstrictor.Activatedplatelets,besidesformingaplateletaggre-Plateletsareverysensitivetoaspirin;aslittleas30mg/dgate,arerequired,vianewlyexpressedanionicphos-(oneaspirintabletusuallycontains325mg)effectivelypholipidsonthemembranesurface,foraccelerationofeliminatesthesynthesisofthromboxaneA2.AspirintheactivationoffactorsXandIIinthecoagulationcas-alsoinhibitsproductionofprostacyclin(PGI2,whichcade(Figure51–1).opposesplateletaggregationandisavasodilator)byen-Alloftheaggregatingagents,includingthrombin,collagen,ADP,andotherssuchasplatelet-activatingfactor,modifytheplateletsurfacesothatfibrinogencanbindtoaglycoproteincomplex,GPIIb–IIIaTable51–4.Moleculessynthesizedby(αIIbβ3integrin;Chapter52),ontheactivatedplateletendothelialcellsthatplayaroleintheregulation1surface.Moleculesofdivalentfibrinogenthenlinkadja-ofthrombosisandfibrinolysis.centactivatedplateletstoeachother,formingaplateletaggregate.Someagents,includingepinephrine,sero-MoleculeActiontonin,andvasopressin,exertsynergisticeffectswithotheraggregatingagents.ADPase(anectoenzyme)DegradesADP(anaggregatingagentofplatelets)toAMP+PiEndothelium-derivedrelax-InhibitsplateletadhesionandEndothelialCellsSynthesizeProstacycliningfactor(nitricoxide)aggregationbyelevatinglev-&OtherCompoundsThatAffectelsofcGMPClotting&ThrombosisHeparansulfate(aglycos-Anticoagulant;combineswithaminoglycan)antithrombinIIItoinhibitTheendothelialcellsinthewallsofbloodvesselsmakethrombinimportantcontributionstotheoverallregulationofhe-Prostacyclin(PGI2,aprosta-Inhibitsplateletaggregationbymostasisandthrombosis.AsdescribedinChapter23,glandin)increasinglevelsofcAMPthesecellssynthesizeprostacyclin(PGI2),apotentin-Thrombomodulin(aglyco-BindsproteinC,whichisthenhibitorofplateletaggregation,opposingtheactionofprotein)cleavedbythrombintoyieldthromboxaneA2.ProstacyclinactsbystimulatingtheactivatedproteinC;thisinactivityofadenylylcyclaseinthesurfacemembranesofcombinationwithproteinSplatelets.TheresultingincreaseofintraplateletcAMPdegradesfactorsVaandVIIIa,2+limitingtheiractionsopposestheincreaseinthelevelofintracellularCaproducedbyIP3andthusinhibitsplateletactivationTissueplasminogenactiva-Activatesplasminogentoplas-(Figure51–8).Endothelialcellsplayotherrolesinthetor(t-PA,aprotease)min,whichdigestsfibrin;theregulationofthrombosis.Forinstance,thesecellspos-actionoft-PAisopposedbysessanADPase,whichhydrolyzesADP,andthusop-plasminogenactivatorin-posesitsaggregatingeffectonplatelets.Inaddition,hibitor-1(PAI-1)thesecellsappeartosynthesizeheparansulfate,ananti-1AdaptedfromWuKK:Endothelialcellsinhemostasis,thrombosiscoagulant,andtheyalsosynthesizeplasminogenactiva-andinflammation.HospPract(OffEd)1992Apr;27:145.
606608/CHAPTER51dothelialcells,butunlikeplatelets,thesecellsregenerate•Foractivity,factorsII,VII,IX,andXandproteinsCcyclooxygenasewithinafewhours.Thus,theoverallandSrequirevitaminK-dependentγ-carboxylationbalancebetweenthromboxaneA2andprostacyclincanofcertainglutamateresidues,aprocessthatisinhib-beshiftedinfavorofthelatter,opposingplateletaggre-itedbytheanticoagulantwarfarin.gation.Indicationsfortreatmentwithaspirinthusin-•Fibrinisdissolvedbyplasmin.Plasminexistsasancludemanagementofanginaandevolvingmyocardialinactiveprecursor,plasminogen,whichcanbeacti-infarctionandalsopreventionofstrokeanddeathinvatedbytissueplasminogenactivator(t-PA).Bothpatientswithtransientcerebralischemicattacks.t-PAandstreptokinasearewidelyusedtotreatearlythrombosisinthecoronaryarteries.LaboratoryTestsMeasureCoagulation•Thrombinandotheragentscauseplateletaggrega-&Thrombolysistion,whichinvolvesavarietyofbiochemicalandmorphologicevents.StimulationofphospholipaseCAnumberoflaboratorytestsareavailabletomeasureandthepolyphosphoinositidepathwayisakeyeventthephasesofhemostasisdescribedabove.Thetestsin-inplateletactivation,butotherprocessesarealsoin-cludeplateletcount,bleedingtime,activatedpartialvolved.thromboplastintime(aPTTorPTT),prothrombintime(PT),thrombintime(TT),concentrationoffibrin-•Aspirinisanimportantantiplateletdrugthatactsbyogen,fibrinclotstability,andmeasurementoffibrininhibitingproductionofthromboxaneA2.degradationproducts.Theplateletcountquantitatesthenumberofplatelets,andthebleedingtimeisanREFERENCESoveralltestofplateletfunction.aPTTisameasureoftheintrinsicpathwayandPToftheextrinsicpathway.BennettJS:Mechanismsofplateletadhesionandaggregation:anPTisusedtomeasuretheeffectivenessoforalanticoag-update.HospPract(OffEd)1992;27:124.ulantssuchaswarfarin,andaPTTisusedtomonitorBrozeGJ:Tissuefactorpathwayinhibitorandtherevisedtheoryofheparintherapy.Thereaderisreferredtoatextbookofcoagulation.AnnuRevMed1995;46:103.hematologyforadiscussionofthesetests.ClemetsonKJ:Plateletactivation:signaltransductionviamem-branereceptors.ThrombHaemost1995;74:111.SUMMARYCollenD,LijnenHR:Basicandclinicalaspectsoffibrinolysisandthrombolysis.Blood1991;78:3114.•HemostasisandthrombosisarecomplexprocessesHandinRI:Anticoagulant,fibrinolyticandantiplatelettherapy.involvingcoagulationfactors,platelets,andbloodIn:Harrison’sPrinciplesofInternalMedicine,15thed.Braun-vessels.waldEetal(editors).McGraw-Hill,2001.HandinRI:Disordersofcoagulationandthrombosis.In:Harri-•Manycoagulationfactorsarezymogensofserinepro-son’sPrinciplesofInternalMedicine,15thed.BraunwaldEetteases,becomingactivatedduringtheoverallprocess.al(editors).McGraw-Hill,2001.•BothintrinsicandextrinsicpathwaysofcoagulationHandinRI:Disordersoftheplateletandvesselwall.In:Harrison’sexist,thelatterinitiatedbytissuefactor.Thepath-PrinciplesofInternalMedicine,15thed.BraunwaldEetalwaysconvergeatfactorXa,embarkingonthecom-(editors).McGraw-Hill,2001.monfinalpathwayresultinginthrombin-catalyzedKrollMH,SchaferAI:Biochemicalmechanismsofplateletactiva-conversionoffibrinogentofibrin,whichisstrength-tion.Blood1989;74:1181.enedbycross-linking,catalyzedbyfactorXIII.RobertsHR,LozierJN:Newperspectivesonthecoagulationcas-cade.HospPract(OffEd)1992;27:97.•Geneticdisordersofcoagulationfactorsoccur,andRothGJ,CalverleyDC:Aspirin,platelets,andthrombosis:theorythetwomostcommoninvolvefactorsVIII(hemo-andpractice.Blood1994;83:885.philiaA)andIX(hemophiliaB).SchmaierAH:Contactactivation:arevision.ThrombHaemost•Animportantnaturalinhibitorofcoagulationisan-1997;78:101.tithrombinIII;geneticdeficiencyofthisproteincanWuKK:Endothelialcellsinhemostasis,thrombosisandinflamma-resultinthrombosis.tion.HospPract(OffEd)1992;27:145.
607Red&WhiteBloodCells52RobertK.Murray,MD,PhDBIOMEDICALIMPORTANCEintracellularorganelles,suchasmitochondria,lyso-somes,orGolgiapparatus.Humanredbloodcells,likeBloodcellshavebeenstudiedintensivelybecausetheymostredcellsofanimals,arenonnucleated.However,areobtainedeasily,becauseoftheirfunctionalimpor-theredcellisnotmetabolicallyinert.ATPissynthe-tance,andbecauseoftheirinvolvementinmanydiseasesizedfromglycolysisandisimportantinprocessesthatprocesses.Thestructureandfunctionofhemoglobin,helptheredbloodcellmaintainitsbiconcaveshapetheporphyrias,jaundice,andaspectsofironmetabo-andalsointheregulationofthetransportofions(eg,lismarediscussedinpreviouschapters.ReductionofbytheNa+-K+ATPaseandtheanionexchangeproteinthenumberofredbloodcellsandoftheircontentof[seebelow])andofwaterinandoutofthecell.Thebi-hemoglobinisthecauseoftheanemias,adiverseandconcaveshapeincreasesthesurface-to-volumeratioofimportantgroupofconditions,someofwhichareseentheredbloodcell,thusfacilitatinggasexchange.Theverycommonlyinclinicalpractice.Certainoftheredcellcontainscytoskeletalcomponents(seebelow)bloodgroupsystems,presentonthemembranesofthatplayanimportantroleindeterminingitsshape.erythrocytesandotherbloodcells,areofextremeim-portanceinrelationtobloodtransfusionandtissueAboutTwoMillionRedBloodCellsEntertransplantation.Table52–1summarizesthecausesofanumberofimportantdiseasesaffectingredbloodcells;theCirculationperSecondsomearediscussedinthischapter,andtheremainderThelifespanofthenormalredbloodcellis120days;arediscussedelsewhereinthistext.Everyorganinthethismeansthatslightlylessthan1%ofthepopulationbodycanbeaffectedbyinflammation;neutrophilsplayofredcells(200billioncells,or2millionpersecond)isacentralroleinacuteinflammation,andotherwhitereplaceddaily.Thenewredcellsthatappearinthecir-bloodcells,suchaslymphocytes,playimportantrolesculationstillcontainribosomesandelementsoftheen-inchronicinflammation.Leukemias,definedasmalig-doplasmicreticulum.TheRNAoftheribosomescanbenantneoplasmsofblood-formingtissues,canaffectdetectedbysuitablestains(suchascresylblue),andcellsprecursorcellsofanyofthemajorclassesofwhitecontainingitaretermedreticulocytes;theynormallybloodcells;commontypesareacuteandchronicmy-numberabout1%ofthetotalredbloodcellcount.Theelocyticleukemia,affectingprecursorsoftheneu-lifespanoftheredbloodcellcanbedramaticallyshort-trophils;andacuteandchroniclymphocyticleukemias.enedinavarietyofhemolyticanemias.ThenumberofCombinationchemotherapy,usingcombinationsofreticulocytesismarkedlyincreasedintheseconditions,variouschemotherapeuticagents,allofwhichactatoneasthebonemarrowattemptstocompensateforrapidormorebiochemicalloci,hasbeenremarkablyeffectivebreakdownofredbloodcellsbyincreasingtheamountinthetreatmentofcertainofthesetypesofleukemias.ofnew,youngredcellsinthecirculation.Understandingtheroleofredandwhitecellsinhealthanddiseaserequiresaknowledgeofcertainfundamen-ErythropoietinRegulatesProductiontalaspectsoftheirbiochemistry.ofRedBloodCellsTHEREDBLOODCELLISSIMPLEINHumanerythropoietinisaglycoproteinof166aminoTERMSOFITSSTRUCTURE&FUNCTIONacids(molecularmassabout34kDa).Itsamountinplasmacanbemeasuredbyradioimmunoassay.ItistheThemajorfunctionsoftheredbloodcellarerelativelymajorregulatorofhumanerythropoiesis.Erythropoietinsimple,consistingofdeliveringoxygentothetissuesissynthesizedmainlybythekidneyandisreleasedinre-andofhelpinginthedisposalofcarbondioxideandsponsetohypoxiaintothebloodstream,inwhichitprotonsformedbytissuemetabolism.Thus,ithasatravelstothebonemarrow.Thereitinteractswithpro-muchsimplerstructurethanmosthumancells,beinggenitorsofredbloodcellsviaaspecificreceptor.There-essentiallycomposedofamembranesurroundingaso-ceptorisatransmembraneproteinconsistingoftwodif-lutionofhemoglobin(thisproteinformsabout95%offerentsubunitsandanumberofdomains.Itisnotatheintracellularproteinoftheredcell).Therearenotyrosinekinase,butitstimulatestheactivitiesofspecific609
608610/CHAPTER52Table52–1.Summaryofthecausesofsomemembersofthisclassofenzymesinvolvedindown-importantdisordersaffectingredbloodcells.streamsignaltransduction.Erythropoietininteractswitharedcellprogenitor,knownastheburst-formingunit-erythroid(BFU-E),causingittoproliferateandDisorderSoleorMajorCausedifferentiate.Inaddition,itinteractswithalaterpro-IrondeficiencyanemiaInadequateintakeorexcessivelossgenitoroftheredbloodcell,calledthecolony-formingofironunit-erythroid(CFU-E),alsocausingittoproliferateMethemoglobinemiaIntakeofexcessoxidants(variousandfurtherdifferentiate.Fortheseeffects,erythropoi-chemicalsanddrugs)etinrequiresthecooperationofotherfactors(eg,inter-GeneticdeficiencyintheNADH-leukin-3andinsulin-likegrowthfactor;Figure52–1).dependentmethemoglobinre-TheavailabilityofacDNAforerythropoietinhasductasesystem(MIM250800)madeitpossibletoproducesubstantialamountsofthisInheritanceofHbM(MIM141800)hormoneforanalysisandfortherapeuticpurposes;pre-viouslytheisolationoferythropoietinfromhumanSicklecellanemiaSequenceofcodon6oftheβchainurineyieldedverysmallamountsoftheprotein.The(MIM141900)changedfromGAGinthenormalmajoruseofrecombinanterythropoietinhasbeeningenetoGTGinthesicklecellgene,resultinginsubstitutionofthetreatmentofasmallnumberofanemicstates,suchvalineforglutamicacidasthatduetorenalfailure.α-ThalassemiasMutationsintheα-globingenes,MANYGROWTHFACTORSREGULATE(MIM141800)mainlyunequalcrossing-overandlargedeletionsandlesscom-PRODUCTIONOFWHITEBLOODCELLSmonlynonsenseandframeshiftAlargenumberofhematopoieticgrowthfactorshavemutationsbeenidentifiedinrecentyearsinadditiontoerythro-β-ThalassemiaAverywidevarietyofmutationsinpoietin.Thisareaofstudyaddstoknowledgeaboutthe(MIM141900)theβ-globingene,includingdele-differentiationofbloodcells,providesfactorsthatmaytions,nonsenseandframeshiftbeusefulintreatment,andalsohasimplicationsforun-mutations,andothersaffectingderstandingoftheabnormalgrowthofbloodcells(eg,everyaspectofitsstructure(eg,theleukemias).Likeerythropoietin,mostofthegrowthsplicesites,promotermutants)factorsisolatedhavebeenglycoproteins,areveryactiveMegaloblasticanemiasDecreasedabsorptionofB12,ofteninvivoandinvitro,interactwiththeirtargetcellsviaDeficiencyofduetoadeficiencyofintrinsicfac-specificcellsurfacereceptors,andultimately(viaintra-vitaminB12tor,normallysecretedbygastriccellularsignals)affectgeneexpression,therebypromot-parietalcellsingdifferentiation.Manyhavebeencloned,permittingtheirproductioninrelativelylargeamounts.TwoofDeficiencyoffolicDecreasedintake,defectiveabsorp-particularinterestaregranulocyte-andgranulocyte-acidtion,orincreaseddemand(eg,inpregnancy)forfolatemacrophagecolony-stimulatingfactors(G-CSFandGM-CSF,respectively).G-CSFisrelativelyspecific,in-HereditaryDeficienciesintheamountorintheducingmainlygranulocytes.GM-CSFaffectsavariety1spherocytosisstructureofαorβspectrin,ofprogenitorcellsandinducesgranulocytes,macro-ankyrin,band3orband4.1phages,andeosinophils.Whentheproductionofneu-Glucose-6-phosphateAvarietyofmutationsinthegenetrophilsisseverelydepressed,thisconditionisreferreddehydrogenase(X-linked)forG6PD,mostlysingletoasneutropenia.Itisparticularlylikelytooccurin1(G6PD)deficiencypointmutationspatientstreatedwithcertainchemotherapeuticregi-(MIM305900)mensandafterbonemarrowtransplantation.Thesepa-tientsareliabletodevelopoverwhelminginfections.Pyruvatekinase(PK)Presumablyavarietyofmutationsdeficiency1inthegenefortheR(redcell)iso-G-CSFhasbeenadministeredtosuchpatientstoboost(MIM255200)zymeofPKproductionofneutrophils.ParoxysmalnocturnalMutationsinthePIG-Agene,affect-hemoglobinemia1ingsynthesisofGPI-anchoredTHEREDBLOODCELLHASAUNIQUE&(MIM311770)proteinsRELATIVELYSIMPLEMETABOLISM1Thelastfourdisorderscausehemolyticanemias,asdoanumberVariousaspectsofthemetabolismoftheredcell,manyoftheotherdisorderslisted.Mostoftheaboveconditionsaredis-ofwhicharediscussedinotherchaptersofthistext,arecussedinotherchaptersofthistext.MIMnumbersapplyonlytosummarizedinTable52–2.disorderswithageneticbasis.
609RED&WHITEBLOODCELLS/611InterleukinsInterleukinsGM-CSFPluripotentGM-CSFEpoErythroidMatureCFU-GEMMBFU-EstemcellprecursorRBCs(earlyandlate)Figure52–1.Greatlysimplifiedschemeofdifferentiationofstemcellstoredbloodcells.Variousinterleukins(ILs),suchasIL-3,IL-4,IL-9,andIL-11,areinvolvedatdifferentstepsoftheoverallprocess.Erythroidprecursorsincludethepronor-moblast,basophilic,polychromatophilic,andorthochromatophilicnormoblasts,andthereticulocyte.Epoactsonbasophilicnormoblastsbutnotonlaterery-throidcells.(CFU-GEMM,colony-formingunitwhosecellsgiverisetogranulo-cytes,erythrocytes,macrophages,andmegakaryocytes;BFU-E,burst-formingunit-erythroid;GM-CSF,granulocyte-macrophagecolony-stimulatingfactor;Epo,erythropoietin;RBC,redbloodcell.)TheRedBloodCellHasaGlucosegrammedbyaddingpurifiedmRNAsorwhole-cellex-TransporterinItsMembranetractsofmRNAs,andradioactiveproteinsaresynthe-35sizedinthepresenceofS-labeledL-methionineorTheentryrateofglucoseintoredbloodcellsisfarotherradiolabeledaminoacids.Theradioactivepro-greaterthanwouldbecalculatedforsimplediffusion.teinssynthesizedareseparatedbySDS-PAGEandde-Rather,itisanexampleoffacilitateddiffusion(Chap-tectedbyradioautography.ter41).Thespecificproteininvolvedinthisprocessiscalledtheglucosetransporterorglucosepermease.SomeofitspropertiesaresummarizedinTable52–3.SuperoxideDismutase,Catalase,Theprocessofentryofglucoseintoredbloodcellsisof&GlutathioneProtectBloodCellsmajorimportancebecauseitisthemajorfuelsupplyforFromOxidativeStress&Damagethesecells.Aboutsevendifferentbutrelatedglucosetransportershavebeenisolatedfromvarioustissues;un-Severalpowerfuloxidantsareproducedduringtheliketheredcelltransporter,someoftheseareinsulin-courseofmetabolism,inbothbloodcellsandmost−⋅dependent(eg,inmuscleandadiposetissue).Thereisothercellsofthebody.Theseincludesuperoxide(O2),•considerableinterestinthelattertypesoftransporterhydrogenperoxide(H2O2),peroxylradicals(ROO),•becausedefectsintheirrecruitmentfromintracellularandhydroxylradicals(OH).Thelastisaparticularlysitestothesurfaceofskeletalmusclecellsmayhelpex-reactivemoleculeandcanreactwithproteins,nucleicplaintheinsulinresistancedisplayedbypatientswithacids,lipids,andothermoleculestoaltertheirstructuretype2diabetesmellitus.andproducetissuedamage.ThereactionslistedinTable52–4playanimportantroleinformingtheseox-ReticulocytesAreActiveidantsandindisposingofthem;eachofthesereactionswillnowbeconsideredinturn.inProteinSynthesisSuperoxideisformed(reaction1)intheredbloodThematureredbloodcellcannotsynthesizeprotein.cellbytheauto-oxidationofhemoglobintomethemo-Reticulocytesareactiveinproteinsynthesis.Onceretic-globin(approximately3%ofhemoglobininhumanredulocytesenterthecirculation,theylosetheirintracellu-bloodcellshasbeencalculatedtoauto-oxidizeperday);larorganelles(ribosomes,mitochondria,etc)withininothertissues,itisformedbytheactionofenzymesabout24hours,becomingyoungredbloodcellsandsuchascytochromeP450reductaseandxanthineoxi-concomitantlylosingtheirabilitytosynthesizeprotein.dase.Whenstimulatedbycontactwithbacteria,neu-Extractsofrabbitreticulocytes(obtainedbyinjectingtrophilsexhibitarespiratoryburst(seebelow)andpro-rabbitswithachemical—phenylhydrazine—thatcausesducesuperoxideinareactioncatalyzedbyNADPHaseverehemolyticanemia,sothattheredcellsareal-oxidase(reaction2).Superoxidespontaneouslydismu-mostcompletelyreplacedbyreticulocytes)arewidelytatestoformH2O2andO2;however,therateofthisusedasaninvitrosystemforsynthesizingproteins.En-samereactionisspeededuptremendouslybytheactiondogenousmRNAspresentinthesereticulocytesareoftheenzymesuperoxidedismutase(reaction3).Hy-destroyedbyuseofanuclease,whoseactivitycanbein-drogenperoxideissubjecttoanumberoffates.Theen-2+zymecatalase,presentinmanytypesofcells,convertshibitedbyadditionofCa.Thesystemisthenpro-
610612/CHAPTER52Table52–2.SummaryofimportantaspectsofTable52–3.Somepropertiesoftheglucosethemetabolismoftheredbloodcell.transporterofthemembraneoftheredbloodcell.•TheRBCishighlydependentuponglucoseasitsenergysource;itsmembranecontainshighaffinityglucosetrans-•Itaccountsforabout2%oftheproteinofthemembraneofporters.theRBC.•Glycolysis,producinglactate,isthesiteofproductionof•ItexhibitsspecificityforglucoseandrelatedD-hexosesATP.(L-hexosesarenottransported).•BecausetherearenomitochondriainRBCs,thereisnopro-•Thetransporterfunctionsatapproximately75%ofitsVmaxductionofATPbyoxidativephosphorylation.atthephysiologicconcentrationofbloodglucose,issat-•TheRBChasavarietyoftransportersthatmaintainionicurableandcanbeinhibitedbycertainanalogsofglucose.andwaterbalance.•Atleastsevensimilarbutdistinctglucosetransportershave•Productionof2,3-bisphosphoglycerate,byreactionscloselybeendetectedtodateinmammaliantissues,ofwhichtheassociatedwithglycolysis,isimportantinregulatingtheredcelltransporterisone.abilityofHbtotransportoxygen.•Itisnotdependentuponinsulin,unlikethecorresponding•ThepentosephosphatepathwayisoperativeintheRBC(itcarrierinmuscleandadiposetissue.metabolizesabout5–10%ofthetotalfluxofglucose)and•Itscompleteaminoacidsequence(492aminoacids)hasproducesNADPH;hemolyticanemiaduetoadeficiencyofbeendetermined.theactivityofglucose-6-phosphatedehydrogenaseiscom-•Ittransportsglucosewheninsertedintoartificialliposomes.mon.•Itisestimatedtocontain12transmembranehelicalseg-•Reducedglutathione(GSH)isimportantinthemetabolismments.oftheRBC,inparttocounteracttheactionofpotentially•Itfunctionsbygeneratingagatedporeinthemembranetotoxicperoxides;theRBCcansynthesizeGSHandrequirespermitpassageofglucose;theporeisconformationallyde-NADPHtoreturnoxidizedglutathione(G-S-S-G)tothere-pendentonthepresenceofglucoseandcanoscillateducedstate.rapidly(about900times/s).•TheironofHbmustbemaintainedintheferrousstate;fer-ricironisreducedtotheferrousstatebytheactionofanNADH-dependentmethemoglobinreductasesystemin-•−volvingcytochromeb5reductaseandcytochromeb5.8),whichalsoproducesOHandOH.Superoxidecan•Synthesisofglycogen,fattyacids,protein,andnucleicacidsreleaseironionsfromferritin.Thus,productionof•doesnotoccurintheRBC;however,somelipids(eg,choles-OHmaybeoneofthemechanismsinvolvedintissueterol)intheredcellmembranecanexchangewithcorre-injuryduetoironoverload(eg,hemochromatosis;spondingplasmalipids.Chapter50).•TheRBCcontainscertainenzymesofnucleotidemetabo-Chemicalcompoundsandreactionscapableofgen-lism(eg,adenosinedeaminase,pyrimidinenucleotidase,eratingpotentialtoxicoxygenspeciescanbereferredtoandadenylylkinase);deficienciesoftheseenzymesarein-aspro-oxidants.Ontheotherhand,compoundsandvolvedinsomecasesofhemolyticanemia.reactionsdisposingofthesespecies,scavengingthem,•WhenRBCsreachtheendoftheirlifespan,theglobinisde-suppressingtheirformation,oropposingtheiractionsgradedtoaminoacids(whicharereutilizedinthebody),areantioxidantsandincludecompoundssuchastheironisreleasedfromhemeandalsoreutilized,andtheNADPH,GSH,ascorbicacid,andvitaminE.Inanor-tetrapyrrolecomponentofhemeisconvertedtobilirubin,malcell,thereisanappropriatepro-oxidant:antioxi-whichismainlyexcretedintothebowelviathebile.dantbalance.However,thisbalancecanbeshiftedto-wardthepro-oxidantswhenproductionofoxygenspeciesisincreasedgreatly(eg,followingingestionofittoH2OandO2(reaction4).Neutrophilspossessacertainchemicalsordrugs)orwhenlevelsofantioxi-uniqueenzyme,myeloperoxidase,thatusesH2O2anddantsarediminished(eg,byinactivationofenzymesin-halidestoproducehypohalousacids(reaction5);thisvolvedindisposalofoxygenspeciesandbyconditionssubjectisdiscussedfurtherbelow.Theselenium-thatcauselowlevelsoftheantioxidantsmentionedcontainingenzymeglutathioneperoxidase(Chapter20)above).Thisstateiscalled“oxidativestress”andcanwillalsoactonreducedglutathione(GSH)andH2O2resultinseriouscelldamageifthestressismassiveortoproduceoxidizedglutathione(GSSG)andH2O(re-prolonged.action6);thisenzymecanalsouseotherperoxidesasOxygenspeciesarenowthoughttoplayanimpor-•−tantroleinmanytypesofcellularinjury(eg,resultingsubstrates.OHandOHcanbeformedfromH2O2in2+fromadministrationofvarioustoxicchemicalsorfromanonenzymaticreactioncatalyzedbyFe(theFenton−⋅ischemia),someofwhichcanresultincelldeath.Indi-reaction,reaction7).O2andH2O2arethesubstratesintheiron-catalyzedHaber-Weissreaction(reactionrectevidencesupportingaroleforthesespeciesingen-
611RED&WHITEBLOODCELLS/613Table52–4.Reactionsofimportanceinrelationtooxidativestressinbloodcellsandvarioustissues.−−⋅(1)Productionofsuperoxide(by-productofvariousreactions)O2+e→O2(2)NADPH-oxidase2O+NADPH→2O−⋅+NADP+H+22(3)SuperoxidedismutaseO−⋅+O−⋅+2H+→HO+O22222(4)CatalaseH2O2→2H2O+O2(5)MyeloperoxidaseHO+X−+H+→HOX+HO(X−=Cl−,Br−,SCN−)222(6)Glutathioneperoxidase(Se-dependent)2GSH+R-O-OH→GSSG+H2O+ROH(7)FentonreactionFe2++HO→Fe3++OH⋅+OH−22(8)Iron-catalyzedHaber-WeissreactionO−⋅+HO→O+OH⋅+OH−2222(9)Glucose-6-phosphatedehydrogenase(G6PD)G6P+NADP→6Phosphogluconate+NADPH+H+(10)GlutathionereductaseG-S-S-G+NADPH+H+→2GSH+NADPeratingcellinjuryisprovidedifadministrationofansensitivehemolyticanemia]andsulfonamides)andenzymesuchassuperoxidedismutaseorcatalaseischemicals(eg,naphthalene)precipitateanattack,be-−⋅foundtoprotectagainstcellinjuryinthesituationcausetheirintakeleadstogenerationofH2O2orO2.understudy.Normally,H2O2isdisposedofbycatalaseandglu-tathioneperoxidase(Table52–4,reactions4and6),DeficiencyofGlucose-6-PhosphatethelattercausingincreasedproductionofGSSG.GSHDehydrogenaseIsFrequentinCertainisregeneratedfromGSSGbytheactionoftheenzymeAreas&IsanImportantCauseglutathionereductase,whichdependsontheavailabil-ityofNADPH(reaction10).Theredbloodcellsofin-ofHemolyticAnemiadividualswhoaredeficientintheactivityofglucose-6-NADPH,producedinthereactioncatalyzedbythephosphatedehydrogenasecannotgeneratesufficientX-linkedglucose-6-phosphatedehydrogenase(TableNADPHtoregenerateGSHfromGSSG,whichin52–4,reaction9)inthepentosephosphatepathwayturnimpairstheirabilitytodisposeofH2O2andof(Chapter20),playsakeyroleinsupplyingreducingoxygenradicals.ThesecompoundscancauseoxidationequivalentsintheredcellandinothercellssuchastheofcriticalSHgroupsinproteinsandpossiblyperoxida-hepatocyte.Becausethepentosephosphatepathwayistionoflipidsinthemembraneoftheredcell,causingvirtuallyitssolemeansofproducingNADPH,theredlysisoftheredcellmembrane.SomeoftheSHgroupsbloodcellisverysensitivetooxidativedamageiftheofhemoglobinbecomeoxidized,andtheproteinpre-functionofthispathwayisimpaired(eg,byenzymede-cipitatesinsidetheredbloodcell,formingHeinzbod-ficiency).OnefunctionofNADPHistoreduceGSSGies,whichstainpurplewithcresylviolet.ThepresencetoGSH,areactioncatalyzedbyglutathionereductaseofHeinzbodiesindicatesthatredbloodcellshavebeen(reaction10).subjectedtooxidativestress.Figure52–2summarizesDeficiencyoftheactivityofglucose-6-phosphatethepossiblechainofeventsinhemolyticanemiaduetodehydrogenase,owingtomutation,isextremelyfre-deficiencyofglucose-6-phosphatedehydrogenase.quentinsomeregionsoftheworld(eg,tropicalAfrica,theMediterranean,certainpartsofAsia,andinNorthMethemoglobinIsUselessAmericaamongblacks).ItisthemostcommonofallinTransportingOxygenenzymopathies(diseasescausedbyabnormalitiesofen-zymes),andover300geneticvariantsoftheenzymeTheferrousironofhemoglobinissusceptibletooxida-havebeendistinguished;atleast100millionpeoplearetionbysuperoxideandotheroxidizingagents,formingdeficientinthisenzymeowingtothesevariants.Themethemoglobin,whichcannottransportoxygen.Onlydisorderresultingfromdeficiencyofglucose-6-phos-averysmallamountofmethemoglobinispresentinphatedehydrogenaseishemolyticanemia.Consump-normalblood,astheredbloodcellpossessesaneffec-tionofbroadbeans(Viciafaba)byindividualsdeficienttivesystem(theNADH-cytochromeb5methemoglobin3+inactivityoftheenzymecanprecipitateanattackofreductasesystem)forreducinghemeFebacktothe2+hemolyticanemia(mostlikelybecausethebeanscon-Festate.ThissystemconsistsofNADH(generatedtainpotentialoxidants).Inaddition,anumberofdrugsbyglycolysis),aflavoproteinnamedcytochromeb5re-(eg,theantimalarialdrugprimaquine[theconditionductase(alsoknownasmethemoglobinreductase),and3+causedbyintakeofprimaquineiscalledprimaquine-cytochromeb5.TheFeofmethemoglobinisreduced
612614/CHAPTER52MutationsinthegeneforG6PDbranesduetoincreasedamountsofdeoxygenatedhe-moglobininarterialblood,orinthiscaseduetoin-creasedamountsofmethemoglobin)isusuallythepre-DecreasedactivityofG6PDsentingsigninbothtypesandisevidentwhenover10%ofhemoglobinisinthe“met”form.DiagnosisismadeDecreasedlevelsofNADPHbyspectroscopicanalysisofblood,whichrevealsthecharacteristicabsorptionspectrumofmethemoglobin.Additionally,asampleofbloodcontainingmethemo-DecreasedregenerationofGSHfromGSSGbyglutathionereductase(whichusesNADPH)globincannotbefullyreoxygenatedbyflushingoxygenthroughit,whereasnormaldeoxygenatedbloodcan.ElectrophoresiscanbeusedtoconfirmthepresenceofOxidation,duetodecreasedlevelsofGSHandanabnormalhemoglobin.Ingestionofmethyleneblue–increasedlevelsofintracellularoxidants(eg,O•),2orascorbicacid(reducingagents)isusedtotreatmildofSHgroupsofHb(formingHeinzbodies),andofmethemoglobinemiaduetoenzymedeficiency.Acutemembraneproteins,alteringmembranestructuremassivemethemoglobinemia(duetoingestionofchem-andincreasingsusceptibilitytoingestionicals)shouldbetreatedbyintravenousinjectionofbymacrophages(peroxidativedamagetolipidsmethyleneblue.inthemembranealsopossible)HemolysisMOREISKNOWNABOUTTHEMEMBRANEOFTHEHUMANREDBLOODCELLTHANFigure52–2.SummaryofprobableeventscausingABOUTTHESURFACEMEMBRANEOFhemolyticanemiaduetodeficiencyoftheactivityofANYOTHERHUMANCELLglucose-6-phosphatedehydrogenase(G6PD)(MIM305900).Avarietyofbiochemicalapproacheshavebeenusedtostudythemembraneoftheredbloodcell.Thesein-cludeanalysisofmembraneproteinsbySDS-PAGE,2+theuseofspecificenzymes(proteinases,glycosidases,backtotheFestatebytheactionofreducedcyto-andothers)todeterminethelocationofproteinsandchromeb5:glycoproteinsinthemembrane,andvarioustechniquestostudyboththelipidcompositionanddispositionofHbF--e3+++→+CytbbHbFe2Cyt5red5oxindividuallipids.Morphologic(eg,electronmicros-copy,freeze-fractureelectronmicroscopy)andotherReducedcytochromeb5isthenregeneratedbytheac-techniques(eg,useofantibodiestospecificcompon-tionofcytochromeb5reductase:ents)havealsobeenwidelyused.Whenredbloodcellsarelysedunderspecificconditions,theirmem-braneswillresealintheiroriginalorientationtoformCytbb5ox+→NADHCyt5red+NADghosts(right-side-outghosts).Byalteringthecondi-tions,ghostscanalsobemadetoresealwiththeircy-tosolicaspectexposedontheexterior(inside-outMethemoglobinemiaIsInheritedghosts).Bothtypesofghostshavebeenusefulinana-orAcquiredlyzingthedispositionofspecificproteinsandlipidsinMethemoglobinemiacanbeclassifiedaseitherinheritedthemembrane.Inrecentyears,cDNAsformanypro-oracquiredbyingestionofcertaindrugsandchemicals.teinsofthismembranehavebecomeavailable,permit-Neithertypeoccursfrequently,butphysiciansmustbetingthedeductionoftheiraminosequencesanddo-awareofthem.Theinheritedformisusuallyduetode-mains.Allinall,moreisknownaboutthemembraneficientactivityofmethemoglobinreductase,transmittedoftheredbloodcellthanaboutanyothermembraneofinanautosomalrecessivemanner.Certainabnormalhe-humancells(Table52–5).moglobins(HbM)arealsorarecausesofmethemoglo-binemia.InHbM,mutationchangestheaminoacidAnalysisbySDS-PAGEResolvesresiduetowhichhemeisattached,thusalteringitsaffin-theProteinsoftheMembraneityforoxygenandfavoringitsoxidation.IngestionofoftheRedBloodCellcertaindrugs(eg,sulfonamides)orchemicals(eg,ani-line)cancauseacquiredmethemoglobinemia.CyanosisWhenthemembranesofredbloodcellsareanalyzed(bluishdiscolorationoftheskinandmucousmem-bySDS-PAGE,abouttenmajorproteinsareresolved
613RED&WHITEBLOODCELLS/615Table52–5.Summaryofbiochemical1informationaboutthemembraneofthehumanSpectrin2redbloodcell.2.1Ankyrin2.2and2.3•Themembraneisabilayercomposedofabout50%lipidisoforms2.6and50%protein.•Themajorlipidclassesarephospholipidsandcholesterol;Anionexchangeprotein34.1themajorphospholipidsarephosphatidylcholine(PC),4.2phosphatidylethanolamine(PE),andphosphatidylserine(PS)alongwithsphingomyelin(Sph).Glycophorins•Thecholine-containingphospholipids,PCandSph,pre-Actin5dominateintheouterleafletandtheamino-containingG3PD6phospholipids(PEandPS)intheinnerleaflet.7•Glycosphingolipids(GSLs)(neutralGSLs,gangliosides,andcomplexspecies,includingtheABObloodgroupsub-Globinstances)constituteabout5–10%ofthetotallipid.•AnalysisbySDS-PAGEshowsthatthemembranecontainsabout10majorproteinsandmorethan100minorspecies.Membrane•Themajorproteins(whichincludespectrin,ankyrin,theanionexchangeprotein,actin,andband4.1)havebeenSkeletonstudiedintensively,andtheprincipalfeaturesoftheirdis-Membraneposition(eg,integralorperipheral),structure,andfunctionCoomassiePASstainhavebeenestablished.bluestain•Manyoftheproteinsareglycoproteins(eg,theglyco-phorins)containingO-orN-linked(orboth)oligosaccharideFigure52–3.Diagrammaticrepresentationofthechainslocatedontheexternalsurfaceofthemembrane.majorproteinsofthemembraneofthehumanredbloodcellseparatedbySDS-PAGE.ThebandsdetectedbystainingwithCoomassieblueareshowninthetwo(Figure52–3),severalofwhichhavebeenshowntobeleft-handchannels,andtheglycoproteinsdetectedbyglycoproteins.TheirmigrationonSDS-PAGEwasstainingwithperiodicacid-Schiff(PAS)reagentareusedtonametheseproteins,withtheslowestmigratingshownintheright-handchannel.(Reproduced,with(andhencehighestmolecularmass)beingdesignatedpermission,fromBeckWS,TepperRI:Hemolyticanemiasband1orspectrin.AllthesemajorproteinshavebeenIII:membranedisorders.In:Hematology,5thed.BeckWSisolated,mostofthemhavebeenidentified,andcon-[editor].TheMITPress,1991.)siderableinsighthasbeenobtainedabouttheirfunc-tions(Table52–6).Manyoftheiraminoacidse-quencesalsohavebeenestablished.Inaddition,ithasbeendeterminedwhichareintegralorperipheralmem-bilayeratleasttentimes.Itprobablyexistsasadimerinbraneproteins,whicharesituatedontheexternalsur-themembrane,inwhichitformsatunnel,permittingface,whichareonthecytosolicsurface,andwhichspantheexchangeofchlorideforbicarbonate.Carbondiox-themembrane(Figure52–4).Manyminorcompo-ide,formedinthetissues,enterstheredcellasbicar-nentscanalsobedetectedintheredcellmembranebybonate,whichisexchangedforchlorideinthelungs,useofsensitivestainingmethodsortwo-dimensionalwherecarbondioxideisexhaled.Theaminoterminalgelelectrophoresis.Oneoftheseistheglucosetrans-endbindsmanyproteins,includinghemoglobin,pro-porterdescribedabove.teins4.1and4.2,ankyrin,andseveralglycolyticen-zymes.Purifiedband3hasbeenaddedtolipidvesiclesTheMajorIntegralProteinsoftheRedinvitroandhasbeenshowntoperformitstransportfunctionsinthisreconstitutedsystem.BloodCellMembraneAretheAnionGlycophorinsA,B,andCarealsotransmembraneExchangeProtein&theGlycophorinsglycoproteinsbutofthesingle-passtype,extendingTheanionexchangeprotein(band3)isatransmem-acrossthemembraneonlyonce.Aisthemajorgly-braneglycoprotein,withitscarboxylterminalendoncophorin,ismadeupof131aminoacids,andisheavilytheexternalsurfaceofthemembraneanditsaminoter-glycosylated(about60%ofitsmass).Itsaminoterminalminalendonthecytoplasmicsurface.Itisanexampleend,whichcontains16oligosaccharidechains(15ofofamultipassmembraneprotein,extendingacrossthewhichareO-glycans),extrudesoutfromthesurfaceof
614616/CHAPTER521Table52–6.Principalproteinsoftheredcellmembrane.Integral(I)orApproximate2BandNumberProteinPeripheral(P)MolecularMass(kDa)1Spectrin(α)P2402Spectrin(β)P2202.1AnkyrinP2102.2“P1952.3“P1752.6“P1453AnionexchangeproteinI1004.1UnnamedP805ActinP436Glyceraldehyde-3-phosphatedehydrogenaseP357TropomyosinP298UnnamedP23GlycophorinsA,B,andCI31,23,and281AdaptedfromLuxDE,BeckerPS:Disordersoftheredcellmembraneskeleton:hereditaryspherocytosisandhereditaryelliptocytosis.Chapter95in:TheMetabolicBasisofInheritedDisease,6thed.ScriverCRetal(editors).McGraw-Hill,1989.2ThebandnumberreferstothepositionofmigrationonSDS-PAGE(seeFigure52–3).Theglycophorinsarede-tectedbystainingwiththeperiodicacid-Schiffreagent.Anumberofothercomponents(eg,4.2and4.9)arenotlisted.Nativespectrinisα2β2.theredbloodcell.Approximately90%ofthesialicacidoftheredcellmembraneislocatedinthisprotein.Itstransmembranesegment(23aminoacids)isα-helical.Thecarboxylterminalendextendsintothecytosolandbindstoprotein4.1,whichinturnbindstospectrin.PolymorphismofthisproteinisthebasisoftheMNSPECTRIN-SPECTRIN-bloodgroupsystem(seebelow).GlycophorinAcon-ANKYRIN-3ACTIN-4.1tainsbindingsitesforinfluenzavirusandforPlasmo-INTERACTIONINTERACTIONdiumfalciparum,thecauseofoneformofmalaria.In-triguingly,thefunctionofredbloodcellsofindividualswholackglycophorinAdoesnotappeartobeaffected.GlycophorinOutsideLipidbilayerSpectrin,Ankyrin,&OtherPeripheralInside3Alpha4.1MembraneProteinsHelpDeterminetheSpectrinShape&FlexibilityoftheRedBloodCellAnkyrinTheredbloodcellmustbeabletosqueezethroughActinsometightspotsinthemicrocirculationduringitsnu-Betamerouspassagesaroundthebody;thesinusoidsoftheSPECTRINSELF-spleenareofspecialimportanceinthisregard.FortheASSOCIATIONredcelltobeeasilyandreversiblydeformable,itsmem-branemustbebothfluidandflexible;itshouldalsoFigure52–4.Diagrammaticrepresentationofthepreserveitsbiconcaveshape,sincethisfacilitatesgasex-interactionofcytoskeletalproteinswitheachotherandchange.Membranelipidshelpdeterminemembranewithcertainintegralproteinsofthemembraneofthefluidity.Attachedtotheinneraspectofthemembraneredbloodcell.(Reproduced,withpermission,fromBeckoftheredbloodcellareanumberofperipheralcy-WS,TepperRI:HemolyticanemiasIII:membranedisor-toskeletalproteins(Table52–6)thatplayimportantders.In:Hematology,5thed.BeckWS[editor].TheMITrolesinrespecttopreservingshapeandflexibility;thesePress,1991.)willnowbedescribed.
615RED&WHITEBLOODCELLS/617Spectrinisthemajorproteinofthecytoskeleton.It0.85g/dL.WhenexposedtoaconcentrationofNaCliscomposedoftwopolypeptides:spectrin1(αchain)of0.5g/dL,veryfewnormalredbloodcellsarehe-andspectrin2(βchain).Thesechains,measuringap-molyzed,whereasapproximately50%ofspherocytesproximately100nminlength,arealignedinanan-wouldlyseundertheseconditions.Theexplanationistiparallelmannerandarelooselyintertwined,formingathatthespherocyte,beingalmostcircular,haslittlepo-dimer.Bothchainsaremadeupofsegmentsof106tentialextravolumetoaccommodateadditionalwateraminoacidsthatappeartofoldintotriple-strandedandthuslysesreadilywhenexposedtoaslightlylowerα-helicalcoilsjoinedbynonhelicalsegments.Oneosmoticpressurethanisnormal.dimerinteractswithanother,formingahead-to-headOnecauseofhereditaryspherocytosis(Figure52–5)tetramer.Theoverallshapeconfersflexibilityontheisadeficiencyintheamountofspectrinorabnormali-proteinandinturnonthemembraneoftheredbloodtiesofitsstructure,sothatitnolongertightlybindsthecell.Atleastfourbindingsitescanbedefinedinspec-otherproteinswithwhichitnormallyinteracts.Thistrin:(1)forself-association,(2)forankyrin(bands2.1,weakensthemembraneandleadstothespherocyticetc),(3)foractin(band5),and(4)forprotein4.1.shape.Abnormalitiesofankyrinandofbands3and4.1Ankyrinisapyramid-shapedproteinthatbindsareinvolvedinothercases.spectrin.Inturn,ankyrinbindstightlytoband3,se-Hereditaryelliptocytosisisageneticdisorderthatcuringattachmentofspectrintothemembrane.Anky-issimilartohereditaryspherocytosisexceptthataf-rinissensitivetoproteolysis,accountingfortheappear-fectedredbloodcellsassumeanelliptic,disk-likeshape,anceofbands2.2,2.3,and2.6,allofwhicharederivedrecognizablebymicroscopy.Itisalsoduetoabnormali-fromband2.1.tiesinspectrin;somecasesreflectabnormalitiesofbandActin(band5)existsinredbloodcellsasshort,dou-4.1orofglycophorinC.ble-helicalfilamentsofF-actin.Thetailendofspectrindimersbindstoactin.Actinalsobindstoprotein4.1.THEBIOCHEMICALBASESOFTHEProtein4.1,aglobularprotein,bindstightlytotheABOBLOODGROUPSYSTEMtailendofspectrin,neartheactin-bindingsiteofthelatter,andthusispartofaprotein4.1-spectrin-actinHAVEBEENESTABLISHEDternarycomplex.Protein4.1alsobindstotheintegralAtleast21humanbloodgroupsystemsarerecognized,proteins,glycophorinsAandC,therebyattachingthethebestknownofwhicharetheABO,Rh(Rhesus),ternarycomplextothemembrane.Inaddition,proteinandMNsystems.Theterm“bloodgroup”appliestoa4.1mayinteractwithcertainmembranephospholipids,definedsystemofredbloodcellantigens(bloodgroupthusconnectingthelipidbilayertothecytoskeleton.substances)controlledbyageneticlocushavingavari-Certainotherproteins(4.9,adducin,andtropo-ablenumberofalleles(eg,A,B,andOintheABOsys-myosin)alsoparticipateincytoskeletalassembly.tem).Theterm“bloodtype”referstotheantigenicphenotype,usuallyrecognizedbytheuseofappropriateAbnormalitiesintheAmountorStructureofSpectrinCauseHereditarySpherocytosis&ElliptocytosisMutationsinDNAaffectingtheamountorstructureofαorβspectrinorofcertainothercytoskeletalHereditaryspherocytosisisageneticdisease,transmittedproteins(eg,ankyrin,band3,band4.1)asanautosomaldominant,thataffectsabout1:5000NorthAmericans.Itischaracterizedbythepresenceofspherocytes(sphericalredbloodcells,withalowsur-Weakensinteractionsamongtheperipheralandface-to-volumeratio)intheperipheralblood,byahe-integralproteinsoftheredcellmembranemolyticanemia,andbysplenomegaly.Thespherocytesarenotasdeformableasarenormalredbloodcells,andWeakensthestructureoftheredcellmembranetheyaresubjecttodestructioninthespleen,thusgreatlyshorteningtheirlifeinthecirculation.Hereditarysphe-rocytosisiscurablebysplenectomybecausethesphero-Adoptsspherocyticshapeandissubjecttocytescanpersistinthecirculationifthespleenisabsent.destructioninthespleenThespherocytesaremuchmoresusceptibletoos-moticlysisthanarenormalredbloodcells.Thisisas-Hemolyticanemiasessedintheosmoticfragilitytest,inwhichredbloodcellsareexposedinvitrotodecreasingconcentrationsFigure52–5.Summaryofthecausationofheredi-ofNaCl.ThephysiologicconcentrationofNaClistaryspherocytosis(MIM182900).
616618/CHAPTER52antibodies.Forpurposesofbloodtransfusion,itispar-golipids,whereasinsecretionsthesameoligosaccha-ticularlyimportanttoknowthebasicsoftheABOandridesarepresentinglycoproteins.Theirpresenceinse-Rhsystems.However,knowledgeofbloodgroupsys-cretionsisdeterminedbyagenedesignatedSe(forse-temsisalsoofbiochemical,genetic,immunologic,an-cretor),whichcodesforaspecificfucosyl(Fuc)thropologic,obstetric,pathologic,andforensicinterest.transferaseinsecretoryorgans,suchastheexocrineHere,weshalldiscussonlysomekeyfeaturesoftheglands,butwhichisnotactiveinredbloodcells.Indi-ABOsystem.Fromabiochemicalviewpoint,themajorvidualsofSeSeorSesegenotypessecreteAorBantigensinterestsintheABOsubstanceshavebeeninisolating(orboth),whereasindividualsofthesesegenotypedoanddeterminingtheirstructures,elucidatingtheirnotsecreteAorBsubstances,buttheirredbloodcellspathwaysofbiosynthesis,anddeterminingthenaturescanexpresstheAandBantigens.oftheproductsoftheA,B,andOgenes.HSubstanceIstheBiosyntheticPrecursorTheABOSystemIsofCrucialImportanceofBoththeA&BSubstancesinBloodTransfusionTheABOsubstanceshavebeenisolatedandtheirstruc-ThissystemwasfirstdiscoveredbyLandsteinerin1900turesdetermined;simplifiedversions,showingonlywheninvestigatingthebasisofcompatibleandincom-theirnonreducingends,arepresentedinFigure52–6.patibletransfusionsinhumans.ThemembranesoftheItisimportanttofirstappreciatethestructureoftheHredbloodcellsofmostindividualscontainonebloodsubstance,sinceitistheprecursorofboththeAandBgroupsubstanceoftypeA,typeB,typeAB,ortypeO.substancesandisthebloodgroupsubstancefoundinIndividualsoftypeAhaveanti-BantibodiesintheirpersonsoftypeO.HsubstanceitselfisformedbytheplasmaandwillthusagglutinatetypeBortypeABactionofafucosyltransferase,whichcatalyzesthead-blood.IndividualsoftypeBhaveanti-Aantibodiesandditionoftheterminalfucoseinα1→2linkageontowillagglutinatetypeAortypeABblood.TypeABtheterminalGalresidueofitsprecursor:bloodhasneitheranti-Anoranti-Bantibodiesandhasbeendesignatedtheuniversalrecipient.TypeObloodGDPFucGal--+→βαβ--RFuc12,-Gal--RGDP+hasneitherAnorBsubstancesandhasbeendesignatedPrecursorHsubstancetheuniversaldonor.Theexplanationofthesefindingsisrelatedtothefactthatthebodydoesnotusuallypro-TheHlocuscodesforthisfucosyltransferase.Thehal-duceantibodiestoitsownconstituents.Thus,individ-leleoftheHlocuscodesforaninactivefucosyltrans-ualsoftypeAdonotproduceantibodiestotheirownferase;therefore,individualsofthehhgenotypecannotbloodgroupsubstance,A,butdopossessantibodiestogenerateHsubstance,theprecursoroftheAandBtheforeignbloodgroupsubstance,B,possiblybecauseantigens.Thus,individualsofthehhgenotypewillhavesimilarstructuresarepresentinmicroorganismstoredbloodcellsoftypeO,eventhoughtheymaypossesswhichthebodyisexposedearlyinlife.Sinceindividu-theenzymesnecessarytomaketheAorBsubstancesalsoftypeOhaveneitherAnorBsubstances,theypos-(seebelow).sessantibodiestoboththeseforeignsubstances.Theabovedescriptionhasbeensimplifiedconsiderably;eg,TheAGeneEncodesaGalNAcTransferase,therearetwosubgroupsoftypeA:A1andA2.theBGeneaGalTransferase,&theOGeneThegenesresponsibleforproductionoftheABOanInactiveProductsubstancesarepresentonthelongarmofchromo-some9.Therearethreealleles,twoofwhichareIncomparisonwithHsubstance(Figure52–6),Asub-codominant(AandB)andthethird(O)recessive;stancecontainsanadditionalGalNAcandBsubstancetheseultimatelydeterminethefourphenotypicprod-anadditionalGal,linkedasindicated.Anti-Aantibod-ucts:theA,B,AB,andOsubstances.iesaredirectedtotheadditionalGalNAcresiduefoundintheAsubstance,andanti-BantibodiesaredirectedTheABOSubstancesAretowardtheadditionalGalresiduefoundintheBsub-stance.Thus,GalNAcistheimmunodominantsugarGlycosphingolipids&Glycoproteins(ie,theonedeterminingthespecificityoftheantibodySharingCommonOligosaccharideChainsformed)ofbloodgroupAsubstance,whereasGalistheTheABOsubstancesarecomplexoligosaccharidespre-immunodominantsugaroftheBsubstance.Inviewofsentinmostcellsofthebodyandincertainsecretions.thestructuralfindings,itisnotsurprisingthatAsub-Onmembranesofredbloodcells,theoligosaccharidesstancecanbesynthesizedinvitrofromOsubstanceinathatdeterminethespecificnaturesoftheABOsub-reactioncatalyzedbyaGalNActransferase,employingstancesappeartobemostlypresentinglycosphin-UDP-GalNAcasthesugardonor.Similarly,blood
617RED&WHITEBLOODCELLS/619Fucα12Galβ14GlcNAc–Rα13GalNActransferaseFucα12Galβ14GlcNAc–RGaltransferaseGalNAcAsubstanceH(orO)substanceFucα12Galβ14GlcNAc–Rα13GalBsubstanceFigure52–6.DiagrammaticrepresentationofthestructuresoftheH,A,andBbloodgroupsubstances.Rrepresentsalongcomplexoligosaccharidechain,joinedeithertoceramidewherethesubstancesareglycosphingolipids,ortothepolypeptidebackboneofaproteinviaaserineorthreonineresiduewherethesubstancesareglycoproteins.Notethatthebloodgroupsubstancesarebianten-nary;ie,theyhavetwoarms,formedatabranchpoint(notindicated)betweentheGlcNAc—R,andonlyonearmofthebranchisshown.Thus,theH,A,andBsub-stanceseachcontaintwooftheirrespectiveshortoligosaccharidechainsshownabove.TheABsubstancecontainsonetypeAchainandonetypeBchain.groupBcanbesynthesizedfromOsubstancebytheImmunologicabnormalities(eg,transfusionreactions,actionofaGaltransferase,employingUDP-Gal.ItisthepresenceinplasmaofwarmandcoldantibodiescrucialtoappreciatethattheproductoftheAgeneisthatlyseredbloodcells,andunusualsensitivitytocom-theGalNActransferasethataddstheterminalGalNAcplement)alsofallinthisclass,asdotoxinsreleasedbytotheOsubstance.Similarly,theproductoftheBgenevariousinfectiousagents,suchascertainbacteria(eg,istheGaltransferaseaddingtheGalresiduetotheOclostridium).Somesnakesreleasevenomsthatacttosubstance.IndividualsoftypeABpossessbothenzymeslysetheredcellmembrane(eg,viatheactionofphos-andthushavetwooligosaccharidechains(Figurepholipasesorproteinases).52–6),oneterminatedbyaGalNAcandtheotherbyaCauseswithinthemembraneincludeabnormalitiesGal.IndividualsoftypeOapparentlysynthesizeanin-ofproteins.Themostimportantconditionsareheredi-activeprotein,detectablebyimmunologicmeans;thus,taryspherocytosisandhereditaryelliptocytosis,princi-HsubstanceistheirABObloodgroupsubstance.pallycausedbyabnormalitiesintheamountorstruc-In1990,astudyusingcloningandsequencingtech-tureofspectrin(seeabove).nologydescribedthenatureofthedifferencesbetweenCausesinsidetheredbloodcellincludehemoglo-theglycosyltransferaseproductsoftheA,B,andObinopathiesandenzymopathies.Sicklecellanemiaisgenes.Adifferenceoffournucleotidesisapparentlyre-themostimportanthemoglobinopathy.AbnormalitiessponsibleforthedistinctspecificitiesoftheAandBofenzymesinthepentosephosphatepathwayandinglycosyltransferases.Ontheotherhand,theOallelehasglycolysisarethemostfrequentenzymopathiesin-asinglebase-pairmutation,causingaframeshiftmuta-volved,particularlytheformer.Deficiencyofglucose-tionresultinginaproteinlackingtransferaseactivity.6-phosphatedehydrogenaseisprevalentincertainpartsoftheworldandisafrequentcauseofhemolyticane-HEMOLYTICANEMIASARECAUSEDmia(seeabove).DeficiencyofpyruvatekinaseisnotBYABNORMALITIESOUTSIDE,frequent,butitisthesecondcommonestenzymedefi-WITHIN,ORINSIDETHEREDciencyresultinginhemolyticanemia;themechanismBLOODCELLMEMBRANEappearstobeduetoimpairmentofglycolysis,resultingindecreasedformationofATP,affectingvariousas-Causesoutsidethemembraneincludehypersplenism,apectsofmembraneintegrity.conditioninwhichthespleenisenlargedfromavarietyLaboratoryinvestigationsthataidinthediagnosisofofcausesandredbloodcellsbecomesequesteredinit.hemolyticanemiaarelistedinTable52–7.
618620/CHAPTER52Table52–7.Laboratoryinvestigationsthatassistknownasthe“acuteinflammatoryresponse.”Theyin-inthediagnosisofhemolyticanemia.clude(1)increaseofvascularpermeability,(2)entryofactivatedneutrophilsintothetissues,(3)activationofplatelets,and(4)spontaneoussubsidence(resolution)ifGeneraltestsandfindingstheinvadingmicroorganismshavebeendealtwithsuc-Increasednonconjugated(indirect)bilirubincessfully.Shortenedredcellsurvivaltimeasmeasuredbyinjectionofautologous51Cr-labeledredcellsAvarietyofmoleculesarereleasedfromcellsandReticulocytosisplasmaproteinsduringacuteinflammationwhosenetHemoglobinemiaoveralleffectistoincreasevascularpermeability,result-Lowlevelofplasmahaptoglobiningintissueedema(Table52–10).SpecifictestsandfindingsInacuteinflammation,neutrophilsarerecruitedfromHbelectrophoresis(eg,HbS)thebloodstreamintothetissuestohelpeliminatethefor-Redcellenzymes(eg,G6PDorPKdeficiency)eigninvaders.Theneutrophilsareattractedintothetis-Osmoticfragility(eg,hereditaryspherocytosis)suesbychemotacticfactors,includingcomplement1CoombstestfragmentC5a,smallpeptidesderivedfrombacteria(eg,ColdagglutininsN-formyl-methionyl-leucyl-phenylalanine),andanum-1ThedirectCoombstestdetectsthepresenceofantibodiesonberofleukotrienes.Toreachthetissues,circulatingneu-redcells,whereastheindirecttestdetectsthepresenceofcircu-trophilsmustpassthroughthecapillaries.Toachievelatingantibodiestoantigenspresentonredcells.this,theymarginatealongthevesselwallsandthenad-heretoendothelial(lining)cellsofthecapillaries.NEUTROPHILSHAVEANACTIVEIntegrinsMediateAdhesionofNeutrophilsMETABOLISM&CONTAINSEVERALtoEndothelialCellsUNIQUEENZYMES&PROTEINSAdhesionofneutrophilstoendothelialcellsemploysspecificadhesiveproteins(integrins)locatedontheirThemajorbiochemicalfeaturesofneutrophilsaresum-surfaceandalsospecificreceptorproteinsintheen-marizedinTable52–8.Prominentfeaturesareactivedothelialcells.(Seealsothediscussionofselectinsinaerobicglycolysis,activepentosephosphatepathway,Chapter47.)moderatelyactiveoxidativephosphorylation(becauseTheintegrinsareasuperfamilyofsurfaceproteinsmitochondriaarerelativelysparse),andahighcontentpresentonawidevarietyofcells.Theyareinvolvedinoflysosomalenzymes.Manyoftheenzymeslistedintheadhesionofcellstoothercellsortospecificcompo-Table52–4arealsoofimportanceintheoxidativeme-nentsoftheextracellularmatrix.Theyareheterodimers,tabolismofneutrophils(seebelow).Table52–9sum-containinganαandaβsubunitlinkednoncovalently.marizesthefunctionsofsomeproteinsthatarerela-Thesubunitscontainextracellular,transmembrane,tivelyuniquetoneutrophils.andintracellularsegments.TheextracellularsegmentsbindtoavarietyofligandssuchasspecificproteinsofNeutrophilsAreKeyPlayersintheBody’stheextracellularmatrixandofthesurfacesofotherDefenseAgainstBacterialInfectioncells.TheseligandsoftencontainArg-Gly-Asp(R-G-NeutrophilsaremotilephagocyticcellsthatplayakeyD)sequences.Theintracellulardomainsbindtovari-roleinacuteinflammation.Whenbacteriaentertissues,ousproteinsofthecytoskeleton,suchasactinandvin-anumberofphenomenaresultthatarecollectivelyculin.Theintegrinsareproteinsthatlinktheoutsidesofcellstotheirinsides,therebyhelpingtointegratere-sponsesofcells(eg,movement,phagocytosis)tochangesintheenvironment.Table52–8.SummaryofmajorbiochemicalThreesubfamiliesofintegrinswererecognizedini-featuresofneutrophils.tially.Membersofeachsubfamilyweredistinguishedbycontainingacommonβsubunit,buttheydiffered•Activeglycolysisintheirαsubunits.However,morethanthreeβsub-•Activepentosephosphatepathwayunitshavenowbeenidentified,andtheclassificationof•Moderateoxidativephosphorylationintegrinshasbecomerathercomplex.Someintegrinsof•Richinlysosomesandtheirdegradativeenzymesspecificinterestwithregardtoneutrophilsarelistedin•Containcertainuniqueenzymes(eg,myeloperoxidaseandTable52–11.NADPH-oxidase)andproteinsAdeficiencyoftheβ2subunit(alsodesignated•ContainCD11/CD18integrinsinplasmamembraneCD18)ofLFA-1andoftworelatedintegrinsfoundin
619RED&WHITEBLOODCELLS/6211Table52–9.Someimportantenzymesandproteinsofneutrophils.EnzymeorProteinReactionCatalyzedorFunctionCommentMyeloperoxidaseHO+X−(halide)+H+→HOX+HOResponsibleforthegreencolorofpus222−−(MPO)(whereX=Cl,HOX=hypochlorousGeneticdeficiencycancauserecurrentinfectionsacid)NADPH-oxidase2O+NADPH→2O−⋅+NADP+H+Keycomponentoftherespiratoryburst22DeficientinchronicgranulomatousdiseaseLysozymeHydrolyzeslinkbetweenN-acetylmura-AbundantinmacrophagesmicacidandN-acetyl-D-glucosaminefoundincertainbacterialcellwallsDefensinsBasicantibioticpeptidesof20–33aminoApparentlykillbacteriabycausingmembraneacidsdamageLactoferrinIron-bindingproteinMayinhibitgrowthofcertainbacteriabybindingironandmaybeinvolvedinregulationofprolif-erationofmyeloidcellsCD11a/CD18,CD11b/CD18,Adhesionmolecules(membersoftheDeficientinleukocyteadhesiondeficiencytypeI2CD11c/CD18integrinfamily)(MIM116920)ReceptorsforFcfragmentsofIgGsBindFcfragmentsofIgGmoleculesTargetantigen-antibodycomplexestomyeloidandlymphoidcells,elicitingphagocytosisandotherresponses1Theexpressionofmanyofthesemoleculeshasbeenstudiedduringvariousstagesofdifferentiationofnormalneutrophilsandalsoofcorrespondingleukemiccellsemployingmolecularbiologytechniques(eg,measurementsoftheirspecificmRNAs).Forthemajority,cDNAshavebeenisolatedandsequenced,aminoacidsequencesdeduced,geneshavebeenlocalizedtospecificchromosomallocations,andexonsandintronsequenceshavebeendefined.SomeimportantproteinasesofneutrophilsarelistedinTable52–12.2CD=clusterofdifferentiation.Thisreferstoauniformsystemofnomenclaturethathasbeenadoptedtonamesurfacemarkersofleuko-cytes.Aspecificsurfaceprotein(marker)thatidentifiesaparticularlineageordifferentiationstageofleukocytesandthatisrecognizedbyagroupofmonoclonalantibodiesiscalledamemberofaclusterofdifferentiation.Thesystemisparticularlyhelpfulincategorizingsub-classesoflymphocytes.ManyCDantigensareinvolvedincell-cellinteractions,adhesion,andtransmembranesignaling.neutrophilsandmacrophages,Mac-1(CD11b/CD18)toturnonmanyofthemetabolicprocessesinvolvedinandp150,95(CD11c/CD18),causestype1leukocytephagocytosisandkillingofbacteria.adhesiondeficiency,adiseasecharacterizedbyrecur-rentbacterialandfungalinfections.Amongvariousre-ActivationofNeutrophilsIsSimilarsultsofthisdeficiency,theadhesionofaffectedwhitetoActivationofPlateletsbloodcellstoendothelialcellsisdiminished,andlower&InvolvesHydrolysisofnumbersofneutrophilsthusenterthetissuestocombatPhosphatidylinositolBisphosphateinfection.OncehavingpassedthroughthewallsofsmallThemechanismsinvolvedinplateletactivationaredis-bloodvessels,theneutrophilsmigratetowardthehigh-cussedinChapter51(seeFigure51–8).Theprocessin-estconcentrationsofthechemotacticfactors,encountervolvesinteractionofthestimulus(eg,thrombin)withatheinvadingbacteria,andattempttoattackandde-receptor,activationofGproteins,stimulationofphos-stroythem.TheneutrophilsmustbeactivatedinorderpholipaseC,andliberationfromphosphatidylinositolTable52–10.Sourcesofbiomoleculeswithvasoactivepropertiesinvolvedinacuteinflammation.MastCellsandBasophilsPlateletsNeutrophilsPlasmaProteinsHistamineSerotoninPlatelet-activatingfactor(PAF)C3a,C4a,andC5afromthecomplementsystemEicosanoids(variousprostaglan-Bradykininandfibrinsplitproductsfromthecoagu-dinsandleukotrienes)lationsystem
620622/CHAPTER52Table52–11.Examplesofintegrinsthatareimportantinthefunctionofneutrophils,ofotherwhite1bloodcells,andofplatelets.IntegrinCellSubunitLigandFunctionVLA-1(CD49a)WBCs,othersα1β1Collagen,lamininCell-ECMadhesionVLA-5(CD49e)WBCs,othersα5β1FibronectinCell-ECMadhesionVLA-6(CD49f)WBCs,othersα6β1LamininCell-ECMadhesionLFA-1(CD11a)WBCsαLβ2ICAM-1AdhesionofWBCsGlycoproteinllb/lllaPlateletsαllbβ3ICAM-2PlateletadhesionandaggregationFibrinogen,fibronectin,vonWillebrandfactor1LFA-1,lymphocytefunction-associatedantigen1;VLA,verylateantigen;CD,clusterofdifferentiation;ICAM,intercellularadhesionmole-cule;ECM,extracellularmatrix.AdeficiencyofLFA-1andrelatedintegrinsisfoundintypeIleukocyteadhesiondeficiency(MIM116290).Adeficiencyofplateletglycoproteinllb/lllacomplexisfoundinGlanzmannthrombasthenia(MIM273800),aconditioncharacterizedbyahistoryofbleeding,anormalplateletcount,andabnormalclotretraction.Thesefindingsillustratehowfundamentalknowledgeofcellsurfaceadhesionproteinsissheddinglightonthecausationofanumberofdiseases.bisphosphateofinositoltriphosphateanddiacylglyc-22kDa.Whenthesystemisactivated(seebelow),twoerol.Thesetwosecondmessengersresultinaneleva-cytoplasmicpolypeptidesof47kDaand67kDaarere-2+tionofintracellularCaandactivationofproteinki-cruitedtotheplasmamembraneand,togetherwithcy-naseC.Inaddition,activationofphospholipaseA2tochromeb558,formtheNADPHoxidaseresponsibleproducesarachidonicacidthatcanbeconvertedtoafortherespiratoryburst.Thereactioncatalyzedbyvarietyofbiologicallyactiveeicosanoids.NADPHoxidase,involvingformationofsuperoxideTheprocessofactivationofneutrophilsisessentiallyanion,isshowninTable52–4(reaction2).Thissystemsimilar.Theyareactivated,viaspecificreceptors,byin-catalyzestheone-electronreductionofoxygentosuper-teractionwithbacteria,bindingofchemotacticfactors,oxideanion.TheNADPHisgeneratedmainlybytheorantibody-antigencomplexes.Theresultantriseinin-pentosephosphatecycle,whoseactivityincreasesmark-2+tracellularCaaffectsmanyprocessesinneutrophils,edlyduringphagocytosis.suchasassemblyofmicrotubulesandtheactin-myosinTheabovereactionisfollowedbythespontaneoussystem.Theseprocessesarerespectivelyinvolvedinse-production(byspontaneousdismutation)ofhydrogencretionofcontentsofgranulesandinmotility,whichperoxidefromtwomoleculesofsuperoxide:enablesneutrophilstoseekouttheinvaders.Theacti-vatedneutrophilsarenowreadytodestroytheinvadersOOHH−−⋅+⋅+→+2+OO22222bymechanismsthatincludeproductionofactivederiv-ativesofoxygen.ThesuperoxideionisdischargedtotheoutsideofTheRespiratoryBurstofPhagocyticthecellorintophagolysosomes,whereitencountersin-CellsInvolvesNADPHOxidasegestedbacteria.Killingofbacteriawithinphagolyso-somesappearstodependonthecombinedactionof&HelpsKillBacteriaelevatedpH,superoxideion,orfurtheroxygenderiva-Whenneutrophilsandotherphagocyticcellsengulf•tives(H2O2,OH,andHOCl[hypochlorousacid;seebacteria,theyexhibitarapidincreaseinoxygencon-below])andontheactionofcertainbactericidalpep-sumptionknownastherespiratoryburst.Thisphe-tides(defensins)andotherproteins(eg,cathepsinGandnomenonreflectstherapidutilizationofoxygen(fol-certaincationicproteins)presentinphagocyticcells.lowingalagof15–60seconds)andproductionfromitAnysuperoxidethatentersthecytosolofthephagocytic−⋅oflargeamountsofreactivederivatives,suchasO2,cellisconvertedtoH2O2bytheactionofsuperoxide•−H2O2,OH,andOCl(hypochloriteion).Someofdismutase,whichcatalyzesthesamereactionasthetheseproductsarepotentmicrobicidalagents.spontaneousdismutationshownabove.Inturn,H2O2isTheelectrontransportchainsystemresponsibleusedbymyeloperoxidase(seebelow)ordisposedofbyfortherespiratoryburst(namedNADPHoxidase)istheactionofglutathioneperoxidaseorcatalase.composedofseveralcomponents.OneiscytochromeNADPHoxidaseisinactiveinrestingphagocyticb558,locatedintheplasmamembrane;itisahet-cellsandisactivateduponcontactwithvariousligandserodimer,containingtwopolypeptidesof91kDaand(complementfragmentC5a,chemotacticpeptides,etc)
621RED&WHITEBLOODCELLS/623withreceptorsintheplasmamembrane.Theeventsre-MutationsingenesforthepolypeptidecomponentssultinginactivationoftheoxidasesystemhavebeenoftheNADPHoxidasesystemmuchstudiedandaresimilartothosedescribedabovefortheprocessofactivationofneutrophils.Theyin-volveGproteins,activationofphospholipaseC,andDiminishedproductionofsuperoxideiongenerationofinositol1,4,5-triphosphate(IP).Theandotheractivederivativesofoxygen3lastmediatesatransientincreaseinthelevelofcytosolic2+Ca,whichisessentialforinductionoftherespiratoryDiminishedkillingofcertainbacteriaburst.DiacylglycerolisalsogeneratedandinducesthetranslocationofproteinkinaseCintotheplasmamem-Recurrentinfectionsandformationoftissuebranefromthecytosol,whereitcatalyzesthephosphor-granulomasinordertowalloffsurvivingbacteriaylationofvariousproteins,someofwhicharecompo-nentsoftheoxidasesystem.AsecondpathwayofFigure52–7.Simplifiedschemeofthesequenceof2+activationnotinvolvingCaalsooperates.eventsinvolvedinthecausationofchronicgranuloma-tousdisease(MIM306400).MutationsinanyoftheMutationsintheGenesforComponentsgenesforthefourpolypeptidesinvolved(twoarecom-oftheNADPHOxidaseSystemCauseponentsofcytochromeb558andtwoarederivedfromChronicGranulomatousDiseasethecytoplasm)cancausethedisease.ThepolypeptideTheimportanceoftheNADPHoxidasesystemwasof91kDaisencodedbyageneintheXchromosome;clearlyshownwhenitwasobservedthattherespiratoryapproximately60%ofcasesofchronicgranulomatousburstwasdefectiveinchronicgranulomatousdisease,adiseaseareX-linked,withtheremainderbeinginher-relativelyuncommonconditioncharacterizedbyrecur-itedinanautosomalrecessivefashion.rentinfectionsandwidespreadgranulomas(chronicin-flammatorylesions)intheskin,lungs,andlymphnodes.Thegranulomasformasattemptstowalloffcauseitreactswithprimaryorsecondaryaminespre-bacteriathathavenotbeenkilled,owingtogeneticde-sentinneutrophilsandtissuestoproducevariousnitro-ficienciesintheNADPHoxidasesystem.Thedisordergen-chlorinederivatives;thesechloraminesarealsooxi-isduetomutationsinthegenesencodingthefourdants,thoughlesspowerfulthanHOCl,andactaspolypeptidesthatconstitutetheNADPHoxidasesys-microbicidalagents(eg,insterilizingwounds)withouttem.Somepatientshaverespondedtotreatmentwithcausingtissuedamage.gammainterferon,whichmayincreasetranscriptionofthe91-kDacomponentifitisaffected.TheprobableTheProteinasesofNeutrophilssequenceofeventsinvolvedinthecausationofchronicCanCauseSeriousTissueDamagegranulomatousdiseaseisshowninFigure52–7.IfTheirActionsAreNotCheckedNeutrophilsContainMyeloperoxidase,Neutrophilscontainanumberofproteinases(TableWhichCatalyzestheProduction52–12)thatcanhydrolyzeelastin,varioustypesofcol-ofChlorinatedOxidantslagens,andotherproteinspresentintheextracellularmatrix.Suchenzymaticaction,ifallowedtoproceedTheenzymemyeloperoxidase,presentinlargeamountsunopposed,canresultinseriousdamagetotissues.inneutrophilgranulesandresponsibleforthegreencolorMostoftheseproteinasesarelysosomalenzymesandofpus,canactonH2O2toproducehypohalousacids:existmainlyasinactiveprecursorsinnormalneu-trophils.SmallamountsoftheseenzymesarereleasedMYELOPEROXIDASEintonormaltissues,withtheamountsincreasingHO+X—+H+HOX+HO222markedlyduringinflammation.Theactivitiesofelas-(X—=Cl—,Br—,I—orSCN—;HOCl=hypochlorousacid)taseandotherproteinasesarenormallykeptincheckbyanumberofantiproteinases(alsolistedinTableTheH2O2usedassubstrateisgeneratedbythe52–12)presentinplasmaandtheextracellularfluid.–NADPHoxidasesystem.Clisthehalideusuallyem-Eachofthemcancombine—usuallyforminganonco-ployed,sinceitispresentinrelativelyhighconcentra-valentcomplex—withoneormorespecificproteinasestioninplasmaandbodyfluids.HOCl,theactiveingre-andthuscauseinhibition.InChapter50itwasshowndientofhouseholdliquidbleach,isapowerfuloxidantthatageneticdeficiencyof1-antiproteinaseinhi-andishighlymicrobicidal.Whenappliedtonormaltis-bitor(α1-antitrypsin)permitselastasetoactunopposedsues,itspotentialforcausingdamageisdiminishedbe-anddigestpulmonarytissue,therebyparticipatingin
622624/CHAPTER52Table52–12.Proteinasesofneutrophilsandter51)havebeengreatlyclarifiedbyinvestigationsantiproteinasesofplasmaandtissues.1usingcloningandsequencing.Thestudyofoncogenesandchromosomaltranslocationshasadvancedunder-standingoftheleukemias.Asdiscussedabove,cloningProteinasesAntiproteinasestechniqueshavemadeavailabletherapeuticamountsofElastaseα1-Antiproteinase(α1-antitrypsin)erythropoietinandothergrowthfactors.DeficiencyofCollagenaseα2-Macroglobulinadenosinedeaminase,whichaffectslymphocytespartic-GelatinaseSecretoryleukoproteinaseinhibitorularly,isthefirstdiseasetobetreatedbygenetherapy.CathepsinGα1-AntichymotrypsinLikemanyotherareasofbiologyandmedicine,hema-PlasminogenactivatorPlasminogenactivatorinhibitor–1tologyhasbeenandwillcontinuetoberevolutionizedTissueinhibitorofmetalloproteinasebythistechnology.1Thetablelistssomeoftheimportantproteinasesofneutrophilsandsomeoftheproteinsthatcaninhibittheiractions.Mostoftheproteinaseslistedexistinsideneutrophilsasprecursors.Plas-SUMMARYminogenactivatorisnotaproteinase,butitisincludedbecauseitinfluencestheactivityofplasmin,whichisaproteinase.Thepro-•Theredbloodcellissimpleintermsofitsstructureteinaseslistedcandigestmanyproteinsoftheextracellularma-andfunction,consistingprincipallyofaconcentratedtrix,causingtissuedamage.Theoverallbalanceofproteinase:an-solutionofhemoglobinsurroundedbyamembrane.tiproteinaseactioncanbealteredbyactivatingtheprecursorsof•Theproductionofredcellsisregulatedbyerythro-theproteinases,orbyinactivatingtheantiproteinases.Thelatterpoietin,whereasothergrowthfactors(eg,granulo-canbecausedbyproteolyticdegradationorchemicalmodifica-cyte-andgranulocyte-macrophagecolony-stimulat-tion,eg,Met-358ofα1-antiproteinaseinhibitorisoxidizedbycig-ingfactors)regulatetheproductionofwhitebloodarettesmoke.cells.•Theredcellcontainsabatteryofcytosolicenzymes,suchassuperoxidedismutase,catalase,andglu-thecausationofemphysema.2-Macroglobulinisatathioneperoxidase,todisposeofpowerfuloxidantsplasmaproteinthatplaysanimportantroleinthegeneratedduringitsmetabolism.body’sdefenseagainstexcessiveactionofproteases;it•Geneticallydetermineddeficiencyoftheactivityofcombineswithandthusneutralizestheactivitiesofaglucose-6-phosphatedehydrogenase,whichproducesnumberofimportantproteases(Chapter50).NADPH,isanimportantcauseofhemolyticanemia.Whenincreasedamountsofchlorinatedoxidantsare•Methemoglobinisunabletotransportoxygen;bothformedduringinflammation,theyaffectthepro-geneticandacquiredcausesofmethemoglobinemiateinase:antiproteinaseequilibrium,tiltingitinfavorofarerecognized.Considerableinformationhasaccu-theformer.Forinstance,certainoftheproteinasesmulatedconcerningtheproteinsandlipidsoftheredlistedinTable52–12areactivatedbyHOCl,whereascellmembrane.Anumberofcytoskeletalproteins,certainoftheantiproteinasesareinactivatedbythissuchasspectrin,ankyrin,andactin,interactwithspe-compound.Inaddition,thetissueinhibitorofmetallo-cificintegralmembraneproteinstohelpregulatetheproteinasesandα1-antichymotrypsincanbehydrolyzedshapeandflexibilityofthemembrane.byactivatedelastase,andα1-antiproteinaseinhibitor•Deficiencyofspectrinresultsinhereditaryspherocy-canbehydrolyzedbyactivatedcollagenaseandgelati-tosis,anotherimportantcauseofhemolyticanemia.nase.Inmostcircumstances,anappropriatebalanceofproteinasesandantiproteinasesisachieved.How-•TheABObloodgroupsubstancesintheredcellever,incertaininstances,suchasinthelungwhenmembranearecomplexglycosphingolipids;theim-α1-antiproteinaseinhibitorisdeficientorwhenlargemunodominantsugarofAsubstanceisN-acetyl-amountsofneutrophilsaccumulateintissuesbecauseofgalactosamine,whereasthatoftheBsubstanceisinadequatedrainage,considerabletissuedamagecanre-galactose.sultfromtheunopposedactionofproteinases.•Neutrophilsplayamajorroleinthebody’sdefensemechanisms.IntegrinsontheirsurfacemembranesRECOMBINANTDNATECHNOLOGYdeterminespecificinteractionswithvariouscellandtissuecomponents.HASHADAPROFOUNDIMPACT•LeukocytesareactivatedonexposuretobacteriaandONHEMATOLOGYotherstimuli;NADPHoxidaseplaysakeyroleintheRecombinantDNAtechnologyhashadamajorimpactprocessofactivation(therespiratoryburst).Muta-onmanyaspectsofhematology.Thebasesofthetha-tionsinthisenzymeandassociatedproteinscauselassemiasandofmanydisordersofcoagulation(Chap-chronicgranulomatousdisease.
623RED&WHITEBLOODCELLS/625•TheproteinasesofneutrophilscandigestmanytissueIsraelsLG,IsraelsED:MechanismsinHematology,2nded.Univproteins;normally,thisiskeptincheckbyabatteryManitobaPress,1997.(IncludesanexcellentinteractiveCD.)ofantiproteinases.However,thisdefensemechanismJaffeER,HultquistDE:Cytochromeb5reductasedeficiencyandcanbeovercomeincertaincircumstances,resultingenzymopenichereditarymethemoglobinemia.In:TheMeta-bolicandMolecularBasesofInheritedDisease,8thed.Scriverinextensivetissuedamage.CRetal(editors).McGraw-Hill,2001.•TheapplicationofrecombinantDNAtechnologyisLekstrom-HunesJA,GallinJI:Immunodeficiencydiseasescausedrevolutionizingthefieldofhematology.bydefectsingranulocytes.NEnglJMed2000;343:1703.LuzzatoLetal:Glucose-6-phosphatedehydrogenase.In:TheREFERENCESMetabolicandMolecularBasesofInheritedDisease,8thed.ScriverCRetal(editors).McGraw-Hill,2001.BorregaardN,CowlandJB:GranulesofthehumanneutrophilicRosseWFetal:NewViewsofSickleCellDiseasePathophysiologypolymorphonuclearleukocyte.Blood1997;89:3503.andTreatment.TheAmericanSocietyofHematology.DanielsG:Acenturyofhumanbloodgroups.WienKlinWochen-www.asheducationbook.orgschr2001;113:781.TseWT,LuxSE:Hereditaryspherocytosisandhereditaryellipto-GoodnoughLTetal:Erythropoietin,iron,anderythropoiesis.cytosis.In:TheMolecularBasesofInheritedDisease,8thed.Blood2000;96:823.ScriverCRetal(editors).McGraw-Hill,2001.HironoAetal:Pyruvatekinasedeficiencyandotherenzy-WeatherallDJetal:Thehemoglobinopathies.In:TheMetabolicmopathiesoftheerythrocyte.In:TheMetabolicandMolecu-andMolecularBasesofInheritedDisease,8thed.ScriverCRetlarBasesofInheritedDisease,8thed.ScriverCRetal(edi-al(editors).McGraw-Hill,2001.tors).McGraw-Hill,2001.
624MetabolismofXenobiotics53RobertK.Murray,MD,PhDBIOMEDICALIMPORTANCEInphase2,thehydroxylatedorothercompoundsproducedinphase1areconvertedbyspecificenzymesIncreasingly,humansaresubjectedtoexposuretovari-tovariouspolarmetabolitesbyconjugationwithglu-ousforeignchemicals(xenobiotics)—drugs,foodaddi-curonicacid,sulfate,acetate,glutathione,orcertaintives,pollutants,etc.Thesituationiswellsummarizedaminoacids,orbymethylation.inthefollowingquotationfromRachelCarson:“AsTheoverallpurposeofthetwophasesofmetabo-crudeaweaponasthecaveman’sclub,thechemicallismofxenobioticsistoincreasetheirwatersolubilitybarragehasbeenhurledagainstthefabricoflife.”Un-(polarity)andthusexcretionfromthebody.Veryhy-derstandinghowxenobioticsarehandledatthecellulardrophobicxenobioticswouldpersistinadiposetissuelevelisimportantinlearninghowtocopewiththealmostindefinitelyiftheywerenotconvertedtomorechemicalonslaught.polarforms.Incertaincases,phase1metabolicreac-Knowledgeofthemetabolismofxenobioticsisbasictionsconvertxenobioticsfrominactivetobiologicallytoarationalunderstandingofpharmacologyandthera-activecompounds.Intheseinstances,theoriginalpeutics,pharmacy,toxicology,managementofcancer,xenobioticsarereferredtoas“prodrugs”or“procar-anddrugaddiction.Alltheseareasinvolveadministra-cinogens.”Inothercases,additionalphase1reactionstionof,orexposureto,xenobiotics.(eg,furtherhydroxylationreactions)converttheactivecompoundstolessactiveorinactiveformspriortocon-HUMANSENCOUNTERTHOUSANDSjugation.Inyetothercases,itistheconjugationreac-OFXENOBIOTICSTHATMUSTBEtionsthemselvesthatconverttheactiveproductsofMETABOLIZEDBEFOREBEINGEXCRETEDphase1reactionstolessactiveorinactivespecies,whicharesubsequentlyexcretedintheurineorbile.InaveryAxenobiotic(Gkxenos“stranger”)isacompoundthatfewcases,conjugationmayactuallyincreasethebio-isforeigntothebody.Theprincipalclassesofxenobi-logicactivityofaxenobiotic.oticsofmedicalrelevancearedrugs,chemicalcarcino-Theterm“detoxification”issometimesusedforgens,andvariouscompoundsthathavefoundtheirwaymanyofthereactionsinvolvedinthemetabolismofintoourenvironmentbyonerouteoranother,suchasxenobiotics.However,thetermisnotalwaysappropri-polychlorinatedbiphenyls(PCBs)andcertaininsecti-atebecause,asmentionedabove,insomecasesthereac-cides.Morethan200,000manufacturedenvironmentaltionstowhichxenobioticsaresubjectactuallyincreasechemicalsexist.Mostofthesecompoundsaresubjecttotheirbiologicactivityandtoxicity.metabolism(chemicalalteration)inthehumanbody,withtheliverbeingthemainorganinvolved;occasion-ally,axenobioticmaybeexcretedunchanged.AtleastISOFORMSOFCYTOCHROME30differentenzymescatalyzereactionsinvolvedinP450HYDROXYLATEAMYRIADxenobioticmetabolism;however,thischapterwillonlyOFXENOBIOTICSINPHASE1coveraselectedgroupofthem.Itisconvenienttoconsiderthemetabolismofxeno-OFTHEIRMETABOLISMbioticsintwophases.Inphase1,themajorreactionin-Hydroxylationisthechiefreactioninvolvedinphasevolvedishydroxylation,catalyzedbymembersofa1.Theresponsibleenzymesarecalledmonooxygenasesclassofenzymesreferredtoasmonooxygenasesorcy-orcytochromeP450s;thehumangenomeencodesattochromeP450s.Hydroxylationmayterminatetheac-least14familiesoftheseenzymes.Estimatesofthetionofadrug,thoughthisisnotalwaysthecase.Inad-numberofdistinctcytochromeP450sinhumantissuesditiontohydroxylation,theseenzymescatalyzeawiderangefromapproximately35to60.Thereactioncat-rangeofreactions,includingthoseinvolvingdeamina-alyzedbyamonooxygenase(cytochromeP450)isastion,dehalogenation,desulfuration,epoxidation,per-follows:oxygenation,andreduction.Reactionsinvolvinghy-drolysis(eg,catalyzedbyesterases)andcertainothernon-P450-catalyzedreactionsalsooccurinphase1.RHO++NADPHH+→+R—OHHONADP++22626
625METABOLISMOFXENOBIOTICS/627RHabovecanrepresentaverywidevarietyofxenobi-describedaboveexceptthatitalicsareused;thus,theotics,includingdrugs,carcinogens,pesticides,petro-geneencodingCYP1A1isCYP1A1.leumproducts,andpollutants(suchasamixtureof(2)Likehemoglobin,theyarehemoproteins.PCBs).Inaddition,endogenouscompounds,suchas(3)Theyarewidelydistributedacrossspecies.Bac-certainsteroids,eicosanoids,fattyacids,andretinoids,teriapossesscytochromeP450s,andP450cam(involvedarealsosubstrates.Thesubstratesaregenerallylip-inthemetabolismofcamphor)ofPseudomonasputidaophilicandarerenderedmorehydrophilicbyhydroxy-istheonlyP450isoformwhosecrystalstructurehaslation.beenestablished.CytochromeP450isconsideredthemostversatile(4)Theyarepresentinhighestamountinliverandbiocatalystknown.Theactualreactionmechanismissmallintestinebutareprobablypresentinalltissues.complexandhasbeenbrieflydescribedpreviously(Fig-Inliverandmostothertissues,theyarepresentmainly18ure11–6).IthasbeenshownbytheuseofO2thatinthemembranesofthesmoothendoplasmicreticu-oneatomofoxygenentersR⎯OHandoneatomen-lum,whichconstitutepartofthemicrosomalfractionterswater.Thisdualfateoftheoxygenaccountsforthewhentissueissubjectedtosubcellularfractionation.Informernamingofmonooxygenasesas“mixed-hepaticmicrosomes,cytochromeP450scancomprisefunctionoxidases.”Thereactioncatalyzedbycy-asmuchas20%ofthetotalprotein.P450sarefoundtochromeP450canalsoberepresentedasfollows:inmosttissues,thoughofteninlowamountscomparedwithliver.Intheadrenal,theyarefoundinmitochon-ReducedcytochromeP450OxidizedcytochromeP450driaaswellasintheendoplasmicreticulum;thevari-RH+O2→R—OH+H2Ooushydroxylasespresentinthatorganplayanimpor-tantroleincholesterolandsteroidbiosynthesis.TheThemajormonooxygenasesintheendoplasmicreticu-mitochondrialcytochromeP450systemdiffersfromlumarecytochromeP450s—sonamedbecausetheen-themicrosomalsysteminthatitusesanNADPH-zymewasdiscoveredwhenitwasnotedthatprepara-linkedflavoprotein,adrenodoxinreductase,andationsofmicrosomesthathadbeenchemicallyreducednonhemeiron-sulfurprotein,adrenodoxin.Inaddi-andthenexposedtocarbonmonoxideexhibitedadis-tion,thespecificP450isoformsinvolvedinsteroidtinctpeakat450nm.Amongreasonsthatthisenzymebiosynthesisaregenerallymuchmorerestrictedintheirisimportantisthefactthatapproximately50%ofthesubstratespecificity.drugshumansingestaremetabolizedbyisoformsofcy-(5)AtleastsixisoformsofcytochromeP450aretochromeP450;theseenzymesalsoactonvariouscar-presentintheendoplasmicreticulumofhumanliver,cinogensandpollutants.eachwithwideandsomewhatoverlappingsubstratespecificitiesandactingonbothxenobioticsanden-IsoformsofCytochromeP450MakeUpadogenouscompounds.ThegenesformanyisoformsofSuperfamilyofHeme-ContainingEnzymesP450(frombothhumansandanimalssuchastherat)havebeenisolatedandstudiedindetailinrecentyears.Thefollowingareimportantpointsconcerningcy-(6)NADPH,notNADH,isinvolvedinthereac-tochromeP450s.tionmechanismofcytochromeP450.Theenzymethat(1)Becauseofthelargenumberofisoforms(aboutusesNADPHtoyieldthereducedcytochromeP450,150)thathavebeendiscovered,itbecameimportanttoshownattheleft-handsideoftheaboveequation,ishaveasystematicnomenclatureforisoformsofP450calledNADPH-cytochromeP450reductase.Elec-andfortheirgenes.ThisisnowavailableandinwidetronsaretransferredfromNADPHtoNADPH-useandisbasedonstructuralhomology.Theabbrevi-cytochromeP450reductaseandthentocytochromeatedrootsymbolCYPdenotesacytochromeP450.P450.Thisleadstothereductiveactivationofmolec-ThisisfollowedbyanArabicnumberdesignatingtheularoxygen,andoneatomofoxygenissubsequentlyfamily;cytochromeP450sareincludedinthesameinsertedintothesubstrate.Cytochromeb5,anotherfamilyiftheyexhibit40%ormoresequenceidentity.hemoproteinfoundinthemembranesofthesmoothTheArabicnumberisfollowedbyacapitalletterindi-endoplasmicreticulum(Chapter11),maybeinvolvedcatingthesubfamily,iftwoormoremembersexist;asanelectrondonorinsomecases.P450sareinthesamesubfamilyiftheyexhibitgreater(7)Lipidsarealsocomponentsofthecytochromethan55%sequenceidentity.TheindividualP450sP450system.Thepreferredlipidisphosphatidyl-arethenarbitrarilyassignedArabicnumerals.Thus,choline,whichisthemajorlipidfoundinmembranesCYP1A1denotesacytochromeP450thatisamemberoftheendoplasmicreticulum.offamily1andsubfamilyAandisthefirstindividual(8)MostisoformsofcytochromeP450arein-memberofthatsubfamily.Thenomenclaturefortheducible.Forinstance,theadministrationofphenobar-genesencodingcytochromeP450sisidenticaltothatbitalorofmanyotherdrugscauseshypertrophyofthe
626628/CHAPTER53smoothendoplasmicreticulumandathree-tofourfoldpotentiallyalteringthequantitiesofmetabolitesofincreaseintheamountofcytochromeP450within4–5PAHs(someofwhichcouldbeharmful)towhichthedays.Themechanismofinductionhasbeenstudiedex-fetusisexposed.tensivelyandinmostcasesinvolvesincreasedtranscrip-(10)CertaincytochromeP450sexistinpolymor-tionofmRNAforcytochromeP450.However,certainphicforms(geneticisoforms),someofwhichexhibitcasesofinductioninvolvestabilizationofmRNA,en-lowcatalyticactivity.Theseobservationsareoneim-zymestabilization,orothermechanisms(eg,aneffectportantexplanationforthevariationsindrugresponsesontranslation).notedamongmanypatients.OneP450exhibitingpolymorphismisCYP2D6,whichisinvolvedintheInductionofcytochromeP450hasimportantclini-metabolismofdebrisoquin(anantihypertensivedrug;calimplications,sinceitisabiochemicalmechanismofseeTable53–2)andsparteine(anantiarrhythmicanddruginteraction.Adruginteractionhasoccurredoxytocicdrug).CertainpolymorphismsofCYP2D6whentheeffectsofonedrugarealteredbyprior,con-causepoormetabolismoftheseandavarietyofothercurrent,orlateradministrationofanother.Asanillus-drugssothattheycanaccumulateinthebody,resultingtration,considerthesituationwhenapatientistakinginuntowardconsequences.Anotherinterestingpoly-theanticoagulantwarfarintopreventbloodclotting.morphismisthatofCYP2A6,whichisinvolvedintheThisdrugismetabolizedbyCYP2C9.Concomitantly,metabolismofnicotinetoconitine.ThreeCYP2A6al-thepatientisstartedonphenobarbital(aninducerofleleshavebeenidentified:awildtypeandtwonullorthisP450)totreatacertaintypeofepilepsy,buttheinactivealleles.Ithasbeenreportedthatindividualsdoseofwarfarinisnotchanged.After5daysorso,thewiththenullalleles,whohaveimpairedmetabolismoflevelofCYP2C9inthepatient’sliverwillbeelevatednicotine,areapparentlyprotectedagainstbecomingto-three-tofourfold.Thisinturnmeansthatwarfarinwillbacco-dependentsmokers(Table53–2).Theseindivid-bemetabolizedmuchmorequicklythanbefore,anditsualssmokeless,presumablybecausetheirbloodanddosagewillhavebecomeinadequate.Therefore,thebrainconcentrationsofnicotineremainelevatedlongerdosemustbeincreasedifwarfarinistobetherapeuti-thanthoseofindividualswiththewild-typeallele.Itcallyeffective.Topursuethisexamplefurther,aprob-hasbeenspeculatedthatinhibitingCYP2A6maybealemcouldariselateronifthephenobarbitalisdiscon-novelwaytohelppreventandtotreatsmoking.tinuedbuttheincreaseddosageofwarfarinstaysthesame.Thepatientwillbeatriskofbleeding,sincetheTable53–1summarizessomeprincipalfeaturesofhighdoseofwarfarinwillbeevenmoreactivethanbe-cytochromeP450s.fore,becausethelevelofCYP2C9willdeclineoncephenobarbitalhasbeenstopped.AnotherexampleofenzymeinductioninvolvesCONJUGATIONREACTIONSPREPARECYP2E1,whichisinducedbyconsumptionofXENOBIOTICSFOREXCRETIONINethanol.Thisisamatterforconcern,becausethisPHASE2OFTHEIRMETABOLISMP450metabolizescertainwidelyusedsolventsandalsocomponentsfoundintobaccosmoke,manyofwhichInphase1reactions,xenobioticsaregenerallycon-areestablishedcarcinogens.Thus,iftheactivityofvertedtomorepolar,hydroxylatedderivatives.InphaseCYP2E1iselevatedbyinduction,thismayincreasethe2reactions,thesederivativesareconjugatedwithmole-riskofcarcinogenicitydevelopingfromexposuretoculessuchasglucuronicacid,sulfate,orglutathione.suchcompounds.Thisrendersthemevenmorewater-soluble,andtheyareeventuallyexcretedintheurineorbile.(9)CertainisoformsofcytochromeP450(eg,CYP1A1)areparticularlyinvolvedinthemetabolismofpolycyclicaromatichydrocarbons(PAHs)andrelatedFiveTypesofPhase2Reactionsmolecules;forthisreasontheywereformerlycalledaro-AreDescribedHerematichydrocarbonhydroxylases(AHHs).ThisenzymeisimportantinthemetabolismofPAHsandincar-A.GLUCURONIDATIONcinogenesisproducedbytheseagents.Forexample,inTheglucuronidationofbilirubinisdiscussedinChap-thelungitmaybeinvolvedintheconversionofinac-ter32;thereactionswherebyxenobioticsareglu-tivePAHs(procarcinogens),inhaledbysmoking,toac-curonidatedareessentiallysimilar.UDP-glucuronictivecarcinogensbyhydroxylationreactions.Smokersacidistheglucuronyldonor,andavarietyofglu-havehigherlevelsofthisenzymeinsomeoftheircellscuronosyltransferases,presentinboththeendoplasmicandtissuesthandononsmokers.Somereportshavein-reticulumandcytosol,arethecatalysts.Moleculessuchdicatedthattheactivityofthisenzymemaybeelevatedas2-acetylaminofluorene(acarcinogen),aniline,ben-(induced)intheplacentaofawomanwhosmokes,thuszoicacid,meprobamate(atranquilizer),phenol,and
627METABOLISMOFXENOBIOTICS/629Table53–1.SomepropertiesofhumanGSH(becauseofthesulfhydrylgroupofitscysteine,cytochromeP450s.whichisthebusinesspartofthemolecule).Anumberofpotentiallytoxicelectrophilicxenobiotics(suchascertaincarcinogens)areconjugatedtothenucleophilic•InvolvedinphaseIofthemetabolismofinnumerableGSHinreactionsthatcanberepresentedasfollows:xenobiotics,includingperhaps50%ofthedrugsadminis-teredtohumansRGSH+→R——SG•Involvedinthemetabolismofmanyendogenouscom-pounds(eg,steroids)whereR=anelectrophilicxenobiotic.Theenzymes•AllarehemoproteinscatalyzingthesereactionsarecalledglutathioneS-•Oftenexhibitbroadsubstratespecificity,thusactingontransferasesandarepresentinhighamountsinlivermanycompounds;consequently,differentP450smaycat-cytosolandinloweramountsinothertissues.AvarietyalyzeformationofthesameproductofglutathioneS-transferasesarepresentinhumantis-•Extremelyversatilecatalysts,perhapscatalyzingabout60sue.Theyexhibitdifferentsubstratespecificitiesandtypesofreactionscanbeseparatedbyelectrophoreticandothertech-•However,basicallytheycatalyzereactionsinvolvingintro-niques.IfthepotentiallytoxicxenobioticswerenotductionofoneatomofoxygenintothesubstrateandoneconjugatedtoGSH,theywouldbefreetocombineco-intowatervalentlywithDNA,RNA,orcellproteinandcould•Theirhydroxylatedproductsaremorewater-solublethanthusleadtoseriouscelldamage.GSHisthereforeantheirgenerallylipophilicsubstrates,facilitatingexcretion•Livercontainshighestamounts,butfoundinmostifnotimportantdefensemechanismagainstcertaintoxicalltissues,includingsmallintestine,brain,andlungcompounds,suchassomedrugsandcarcinogens.Ifthe•Locatedinthesmoothendoplasmicreticulumorinmito-levelsofGSHinatissuesuchasliverarelowered(aschondria(steroidogenichormones)canbeachievedbytheadministrationtoratsofcertain•Insomecases,theirproductsaremutagenicorcarcino-compoundsthatreactwithGSH),thenthattissuecangenicbeshowntobemoresusceptibletoinjurybyvarious•Manyhaveamolecularmassofabout55kDachemicalsthatwouldnormallybeconjugatedtoGSH.•Manyareinducible,resultinginonecauseofdruginterac-Glutathioneconjugatesaresubjectedtofurthermetab-tionsolismbeforeexcretion.Theglutamylandglycinyl•Manyareinhibitedbyvariousdrugsortheirmetabolicgroupsbelongingtoglutathioneareremovedbyspe-products,providinganothercauseofdruginteractionscificenzymes,andanacetylgroup(donatedbyacetyl-•Someexhibitgeneticpolymorphisms,whichcanresultinCoA)isaddedtotheaminogroupoftheremainingatypicaldrugmetabolismcysteinylmoiety.Theresultingcompoundisamercap-•Theiractivitiesmaybealteredindiseasedtissues(eg,cir-turicacid,aconjugateofL-acetylcysteine,whichisrhosis),affectingdrugmetabolismthenexcretedintheurine.•GenotypingtheP450profileofpatients(eg,todetectGlutathionehasotherimportantfunctionsinpolymorphisms)mayinthefuturepermitindividualizationhumancellsapartfromitsroleinxenobioticmetabo-ofdrugtherapylism.1.Itparticipatesinthedecompositionofpotentiallytoxichydrogenperoxideinthereactioncat-manysteroidsareexcretedasglucuronides.Theglu-alyzedbyglutathioneperoxidase(Chapter20).curonidemaybeattachedtooxygen,nitrogen,orsulfurgroupsofthesubstrates.Glucuronidationisprobably2.Itisanimportantintracellularreductant,help-themostfrequentconjugationreaction.ingtomaintainessentialSHgroupsofenzymesintheirreducedstate.ThisroleisdiscussedinChap-B.SULFATIONter20,anditsinvolvementinthehemolyticane-Somealcohols,arylamines,andphenolsaresulfated.miacausedbydeficiencyofglucose-6-phosphateThesulfatedonorintheseandotherbiologicsulfationdehydrogenaseisdiscussedinChapters20andreactions(eg,sulfationofsteroids,glycosaminoglycans,52.glycolipids,andglycoproteins)isadenosine3-phos-3.AmetaboliccycleinvolvingGSHasacarrierhasphate-5-phosphosulfate(PAPS)(Chapter24);thisbeenimplicatedinthetransportofcertaincompoundiscalled“activesulfate.”aminoacidsacrossmembranesinthekidney.Thefirstreactionofthecycleisshownbelow.C.CONJUGATIONWITHGLUTATHIONEGlutathione(γ-glutamyl-cysteinylglycine)isatripep-Aminoacid+GSH→γ-Glutamylaminoacid+tideconsistingofglutamicacid,cysteine,andglycine(Figure3–3).GlutathioneiscommonlyabbreviatedCysteinylglycine
628630/CHAPTER53ThisreactionhelpstransfercertainaminoacidsacrossAgain,thiscanaffectthedosesofcertaindrugsthataretheplasmamembrane,theaminoacidbeingsubse-administeredtopatients.Variousdiseases(eg,cirrhosisquentlyhydrolyzedfromitscomplexwithGSHandoftheliver)canaffecttheactivitiesofdrug-metaboliz-theGSHbeingresynthesizedfromcysteinylglycine.ingenzymes,sometimesnecessitatingadjustmentofTheenzymecatalyzingtheabovereactionis-glu-dosagesofvariousdrugsforpatientswiththesedisor-tamyltransferase(GGT).Itispresentintheplasmaders.membraneofrenaltubularcellsandbileductulecells,andintheendoplasmicreticulumofhepatocytes.TheenzymehasdiagnosticvaluebecauseitisreleasedintoRESPONSESTOXENOBIOTICSthebloodfromhepaticcellsinvarioushepatobiliaryINCLUDEPHARMACOLOGIC,diseases.TOXIC,IMMUNOLOGIC,D.OTHERREACTIONS&CARCINOGENICEFFECTSThetwomostimportantotherreactionsareacetylationXenobioticsaremetabolizedinthebodybythereac-andmethylation.tionsdescribedabove.Whenthexenobioticisadrug,1.Acetylation—Acetylationisrepresentedbyphase1reactionsmayproduceitsactiveformormayXAc+→etylCoA--AcetylX+CoAdiminishorterminateitsactionifitispharmacologi-callyactiveinthebodywithoutpriormetabolism.ThewhereXrepresentsaxenobiotic.Asforotheracetyla-diverseeffectsproducedbydrugscomprisetheareaoftionreactions,acetyl-CoA(activeacetate)istheacetylstudyofpharmacology;hereitisimportanttoappreci-donor.Thesereactionsarecatalyzedbyacetyltrans-atethatdrugsactprimarilythroughbiochemicalmech-ferasespresentinthecytosolofvarioustissues,particu-anisms.Table53–2summarizesfourimportantreac-larlyliver.Thedrugisoniazid,usedinthetreatmentoftionstodrugsthatreflectgeneticallydeterminedtuberculosis,issubjecttoacetylation.Polymorphicdifferencesinenzymeandproteinstructureamongin-typesofacetyltransferasesexist,resultinginindividualsdividuals—partofthefieldofstudyknownaspharma-whoareclassifiedassloworfastacetylators,andinflu-cogenetics(seebelow).encetherateofclearanceofdrugssuchasisoniazidfromblood.SlowacetylatorsaremoresubjecttocertaintoxiceffectsofisoniazidbecausethedrugpersistsTable53–2.Someimportantdrugreactionsduelongerintheseindividuals.tomutantorpolymorphicformsofenzymesor2.Methylation—Afewxenobioticsaresubjectto1methylationbymethyltransferases,employingS-adeno-proteins.sylmethionine(Figure30–17)asthemethyldonor.EnzymeorProteinTHEACTIVITIESOFXENOBIOTIC-AffectedReactionorConsequenceMETABOLIZINGENZYMESAREGlucose-6-phosphateHemolyticanemiafollowingin-AFFECTEDBYAGE,SEX,dehydrogenase(G6PD)gestionofdrugssuchasprim-&OTHERFACTORS[mutations](MIM305900)aquine2+Careleasechannel(ryan-Malignanthyperthermia(MIMVariousfactorsaffecttheactivitiesoftheenzymesme-odinereceptor)inthe145600)followingadministra-tabolizingxenobiotics.Theactivitiesoftheseenzymessarcoplasmicreticulumtionofcertainanesthetics(eg,maydiffersubstantiallyamongspecies.Thus,forexam-[mutations](MIM180901)halothane)ple,thepossibletoxicityorcarcinogenicityofxenobi-oticscannotbeextrapolatedfreelyfromonespeciestoCYP2D6[polymorphisms]Slowmetabolismofcertainanother.Therearesignificantdifferencesinenzymeac-(MIM124030)drugs(eg,debrisoquin),result-tivitiesamongindividuals,manyofwhichappeartobeingintheiraccumulationduetogeneticfactors.TheactivitiesofsomeoftheseCYP2A6[polymorphisms]Impairedmetabolismofnico-enzymesvaryaccordingtoageandsex.(MIM122720)tine,resultinginprotectionIntakeofvariousxenobioticssuchasphenobarbital,againstbecomingatobacco-PCBs,orcertainhydrocarbonscancauseenzymein-dependentsmokerduction.Itisthusimportanttoknowwhetherornot1G6PDdeficiencyisdiscussedinChapters20and52andmalig-anindividualhasbeenexposedtotheseinducingagentsnanthyperthermiainChapter49.Atleastonegeneotherthaninevaluatingbiochemicalresponsestoxenobiotics.thatencodingtheryanodinereceptorisinvolvedincertaincasesMetabolitesofcertainxenobioticscaninhibitorstimu-ofmalignanthypertension.Manyotherexamplesofdrugreac-latetheactivitiesofxenobiotic-metabolizingenzymes.tionsbasedonpolymorphismormutationareavailable.
629METABOLISMOFXENOBIOTICS/631Certainxenobioticsareverytoxicevenatlowlevelsintheendoplasmicreticulumtobecomecarcinogenic(eg,cyanide).Ontheotherhand,therearefewxenobi-(theyarethuscalledindirectcarcinogens).Theactivi-otics,includingdrugs,thatdonotexertsometoxicef-tiesofthemonooxygenasesandofotherxenobiotic-fectsifsufficientamountsareadministered.Thetoxicmetabolizingenzymespresentintheendoplasmicretic-effectsofxenobioticscoverawidespectrum,buttheulumthushelptodeterminewhethersuchcompoundsmajoreffectscanbeconsideredunderthreegeneralbecomecarcinogenicorare“detoxified.”Otherchemi-headings(Figure53–1).cals(eg,variousalkylatingagents)canreactdirectly(di-Thefirstiscellinjury(cytotoxicity),whichcanberectcarcinogens)withDNAwithoutundergoingintra-severeenoughtoresultincelldeath.Therearemanycellularchemicalactivation.mechanismsbywhichxenobioticsinjurecells.TheoneTheenzymeepoxidehydrolaseisofinterestbe-consideredhereiscovalentbindingtocellmacromol-causeitcanexertaprotectiveeffectagainstcertaincar-eculesofreactivespeciesofxenobioticsproducedbycinogens.Theproductsoftheactionofcertainmetabolism.ThesemacromoleculartargetsincludemonooxygenasesonsomeprocarcinogensubstratesareDNA,RNA,andprotein.Ifthemacromoleculetoepoxides.Epoxidesarehighlyreactiveandmutagenicwhichthereactivexenobioticbindsisessentialfororcarcinogenicorboth.Epoxidehydrolase—likecy-short-termcellsurvival,eg,aproteinorenzymein-tochromeP450,alsopresentinthemembranesofthevolvedinsomecriticalcellularfunctionsuchasoxida-endoplasmicreticulum—actsonthesecompounds,tivephosphorylationorregulationofthepermeabilityconvertingthemintomuchlessreactivedihydrodiols.oftheplasmamembrane,thensevereeffectsoncellularThereactioncatalyzedbyepoxidehydrolasecanberep-functioncouldbecomeevidentquiterapidly.resentedasfollows:Second,thereactivespeciesofaxenobioticmaybindtoaprotein,alteringitsantigenicity.Thexenobi-CCH+2OCCoticissaidtoactasahapten,ie,asmallmoleculethatbyitselfdoesnotstimulateantibodysynthesisbutwillOHOOHcombinewithantibodyonceformed.Theresultingan-EpoxideDihydrodioltibodiescanthendamagethecellbyseveralimmuno-logicmechanismsthatgrosslyperturbnormalcellularPHARMACOGENOMICSWILLDRIVETHEbiochemicalprocesses.DEVELOPMENTOFNEW&SAFERDRUGSThird,reactionsofactivatedspeciesofchemicalcar-cinogenswithDNAarethoughttobeofgreatimpor-Asindicatedabove,pharmacogeneticsisthestudyoftanceinchemicalcarcinogenesis.Somechemicals(eg,therolesofgeneticvariationsintheresponsestodrugs.benzo[α]pyrene)requireactivationbymonooxygenasesAsaresultoftheprogressmadeinsequencingtheGSHS-transferaseorCytochromeP450epoxidehydrolaseXenobioticReactivemetaboliteNontoxicmetaboliteCovalentbindingtomacromoleculesCellinjuryHaptenMutationAntibodyproductionCancerCellinjuryFigure53–1.Simplifiedschemeshowinghowmetabolismofaxenobioticcanresultincellinjury,immuno-logicdamage,orcancer.Inthisinstance,theconversionofthexenobiotictoareactivemetaboliteiscatalyzedbyacytochromeP450,andtheconversionofthereactivemetabolite(eg,anepoxide)toanontoxicmetaboliteiscatalyzedeitherbyaGSHS-transferaseorbyepoxidehydrolase.
630632/CHAPTER53humangenome,anewfieldofstudy—pharmacoge-•CytochromeP450saregenerallylocatedintheendo-nomics—hasdevelopedrecently.Itincludespharmaco-plasmicreticulumofcellsandareparticularlyen-geneticsbutcoversamuchwidersphereofactivity.In-richedinliver.formationfromgenomics,proteomics,bioinformatics,•ManycytochromeP450sareinducible.Thishasim-andotherdisciplinessuchasbiochemistryandtoxicol-portantimplicationsinphenomenasuchasdrugin-ogywillbeintegratedtomakepossiblethesynthesisofteraction.newerandsaferdrugs.Asthesequencesofallourgenes•MitochondrialcytochromeP450salsoexistandareandtheirencodedproteinsaredetermined,thiswillre-involvedincholesterolandsteroidbiosynthesis.vealmanynewtargetsfordrugactions.Itwillalsore-Theyuseanonhemeiron-containingsulfurprotein,vealpolymorphisms(thistermisbrieflydiscussedinadrenodoxin,notrequiredbymicrosomalisoforms.Chapter50)ofenzymesandproteinsrelatedtodrug•CytochromeP450s,becauseoftheircatalyticactivi-metabolism,action,andtoxicity.DNAprobescapableties,playmajorrolesinthereactionsofcellstoofdetectingthemwillbesynthesized,permittingchemicalcompoundsandinchemicalcarcinogenesis.screeningofindividualsforpotentiallyharmfulpoly-morphismspriortothestartofdrugtherapy.Asthe•Phase2reactionsarecatalyzedbyenzymessuchasstructuresofrelevantproteinsandtheirpolymorphismsglucuronosyltransferases,sulfotransferases,andglu-arerevealed,modelbuildingandothertechniqueswilltathioneS-transferases,usingUDP-glucuronicacid,permitthedesignofdrugsthattakeintoaccountbothPAPS(activesulfate),andglutathione,respectively,normalproteintargetsandtheirpolymorphisms.Atasdonors.leasttosomeextent,drugswillbetailor-madeforindi-•Glutathionenotonlyplaysanimportantroleinvidualsbasedontheirgeneticprofiles.Aneweraofra-phase2reactionsbutisalsoanintracellularreducingtionaldrugdesignbuiltoninformationderivedfromagentandisinvolvedinthetransportofcertaingenomicsandproteomicshasalreadycommenced.aminoacidsintocells.•Xenobioticscanproduceavarietyofbiologiceffects,SUMMARYincludingpharmacologicresponses,toxicity,immuno-•Xenobioticsarechemicalcompoundsforeigntothelogicreactions,andcancer.body,suchasdrugs,foodadditives,andenvironmen-•Catalyzedbytheprogressmadeinsequencingthetalpollutants;morethan200,000havebeenidenti-humangenome,thenewfieldofpharmacogenomicsfied.offersthepromiseofbeingabletomakeavailablea•Xenobioticsaremetabolizedintwophases.Thehostofnewrationallydesigned,saferdrugs.majorreactionofphase1ishydroxylationcatalyzedbyavarietyofmonooxygenases,alsoknownastheREFERENCEScytochromeP450s.Inphase2,thehydroxylatedspeciesareconjugatedwithavarietyofhydrophilicEvansWE,JohnsonJA:Pharmacogenomics:theinheritedbasisforcompoundssuchasglucuronicacid,sulfate,orglu-interindividualdifferencesindrugresponse.AnnuRevGe-tathione.ThecombinedoperationofthesetwonomicsHumGenet2001;2:9.phasesrenderslipophiliccompoundsintowater-GuengerichFP:CommonanduncommoncytochromeP450reac-solublecompoundsthatcanbeeliminatedfromthetionsrelatedtometabolismandchemicaltoxicity.ChemResToxicol2001;14:611.body.HonkakakoskiP,NegishiM:RegulationofcytochromeP450•CytochromeP450scatalyzereactionsthatintroduce(CYP)genesbynuclearreceptors.BiochemJ2000;347:321.oneatomofoxygenderivedfrommolecularoxygenKalowW,GrantDM:Pharmacogenetics.In:TheMetabolicandintothesubstrate,yieldingahydroxylatedproduct.MolecularBasesofInheritedDisease,8thed.ScriverCRetalNADPHandNADPH-cytochromeP450reductase(editors).McGraw-Hill,2001.areinvolvedinthecomplexreactionmechanism.KatzungBG(editor):Basic&ClinicalPharmacology,8thed.Mc-•AllcytochromeP450sarehemoproteinsandgener-Graw-Hill,2001.allyhaveawidesubstratespecificity,actingonmanyMcLeodHL,EvansWE:Pharmacogenomics:unlockingthehumangenomeforbetterdrugtherapy.AnnuRevPharmacolexogenousandendogenoussubstrates.Theyrepre-Toxicol2001;41:101.sentthemostversatilebiocatalystknown.NelsonDRetal:P450superfamily:updateonnewsequences,gene•Membersofatleast11familiesofcytochromeP450mapping,accessionnumbersandnomenclature.Pharmacoge-arefoundinhumantissue.netics1996;6:1.
631TheHumanGenomeProject54RobertK.Murray,MD,PhDBIOMEDICALSIGNIFICANCErizesthedifferencesbetweenageneticmap,acytoge-neticmap,andaphysicalmapofachromosome.TheseTheinformationderivingfromdeterminationofthese-andotherinitialgoalswereachievedandsurpassedbyquencesofthehumangenomeandthoseofotheror-themid-nineties.In1998,newgoalsfortheUnitedganismswillchangebiologyandmedicineforalltime.StateswingoftheHGPwereannounced.Thesein-Forexample,withreferencetothehumangenome,newcludedtheaimofcompletingtheentiresequencebyinformationonourorigins,ondiseasegenes,ondiag-theendof2003orsooner.Otherspecificobjectivesnosis,andpossibleapproachestotherapyarealreadyconcernedsequencingtechnology,comparativege-floodingin.Progressinfieldssuchasgenomics,pro-nomics,bioinformatics,ethicalconsiderations,andteomics,bioinformatics,biotechnology,andpharma-otherissues.Bythefallof1998,about6%ofthecogenomicsisacceleratingrapidly.humangenomesequencehadbeencompletedandtheTheaimsofthischapteraretobrieflysummarizefoundationsforfutureworklaid.FurtherprogresswasthemajorfindingsoftheHumanGenomeProjectcatalyzedbytheannouncementthatasecondgroup,(HGP)andindicatetheirimplicationsforbiologyandtheprivatecompanyCeleraGenomics,ledbyCraigmedicine.Venter,hadundertakentheobjectiveofsequencingthehumangenome.VenterandcolleagueshadpublishedTHEHUMANGENOMEPROJECTin1995theentiregenomesequencesofHaemophilusHASAVARIETYOFGOALSinfluenzaeandMycoplasmagenitalium,thefirstofmanyspeciestohavetheirgenomicsequencesdetermined.AnTheHGP,whichstartedin1990,isaninternationalimportantfactorinthesuccessoftheseworkerswastheeffortwhoseprincipalgoalsweretosequencetheentireuseofashotgunapproach,ie,sonicatingtheDNA,se-humangenomeandthegenomesofseveralothermodelquencingthefragments,andreassemblingthese-organismsthathavebeenbasictothestudyofgeneticsquence,basedonoverlaps.Forcomparison,avarietyof(eg,Escherichiacoli,Saccharomycescerevisiae[ayeast],approachesthathavebeenusedatdifferenttimestoDrosophilamelanogaster[thefruitfly],CaenorhabditisstudynormalanddiseasegenesarelistedinTable54–1.elegans[theroundworm],andMusmusculus[thecom-monhousemouse]).Mostofthesegoalshavebeenac-ADraftSequenceoftheHumanGenomecomplished.IntheUnitedStates,theNationalCenterWasAnnouncedinJune2000forHumanGenomeResearch(NCHGR)wasestab-lishedin1989,initiallydirectedbyJamesD.WatsonInJune2000,leadersoftheIHGSCandthepersonnelandsubsequentlybyFrancisCollins.TheNCHGRatCeleraGenomicsannouncedcompletionofworkingplayedaleadingroleindirectingtheUnitedStatesef-draftsofthesequenceofthehumangenome,coveringfortintheHGP.In1997,itbecametheNationalmorethan90%ofit.TheprincipalfindingsofthetwoHumanGenomeResearchInstitute(NHGRI).Thein-groupswerepublishedseparatelyinFebruary2001internationalcollaboration—involvinggroupsfromthespecialissuesofNature(theIHGSC)andScience(Cel-USA,UK,Japan,France,Germany,andChina—cameera).ThedraftpublishedbytheConsortiumwasthetobeknownastheInternationalHumanGenomeSe-productofatleast10yearsofworkinvolving20se-quencingConsortium(IHGSC).Initially,anumberofquencingcenterslocatedinsixcountries.Thatpub-short-termgoalswereestablishedfortheUnitedStateslishedbyCeleraandassociateswastheproductofsomeeffort—eg,producingahumangeneticmapwithmark-3yearsorlessofwork;itreliedinpartondataobtaineders2–5centimorgans(cM)apartandconstructingabytheIHGSC.Thecombinedachievementhasbeenphysicalmapofall24humanchromosomes(22auto-hailed,amongotherdescriptions,asprovidingaLibrarysomalplusXandY)withmarkersspacedatapproxi-ofLife,supplyingaPeriodicTableofLife,andfindingmately100,000basepairs(bp).Figure54–1summa-theHolyGrailofHumanGenetics.633
632634/CHAPTER54Geneticmap2030302025cMCytogeneticmapPhysicalmap255075100125150MbEcoRIHindIIINotIRestrictionmapSTSmapContigmapFigure54–1.Principalmethodsusedtoidentifyandisolatenormalanddiseasegenes.Forthegeneticmap,thepositionsofseveralhypo-theticalgeneticmarkersareshown,alongwiththegeneticdistancesincentimorgansbetweenthem.Thecircleshowsthepositionofthecen-tromere.Forthecytogeneticmap,theclassicbandingpatternofahypo-theticalchromosomeisshown.Forthephysicalmap,theapproximatephysicalpositionsoftheabovegeneticmarkersareshown,alongwiththerelativephysicaldistancesinmegabasepairs.Examplesofarestric-tionmap,acontigmark,andanSTSmaparealsoshown.(Reproduced,withpermission,fromGreenED,WaterstonRH:TheHumanGenomeProject:Prospectsandimplicationsforclinicalmedicine.JAMA1991;266:1966.Copyright©1991bytheAmericanMedicalAssociation.)DifferentApproachesWereUsed(STSs),whoselocationshadbeenalreadydetermined.bytheTwoGroupsSTSsareshort(usually<500bp),uniquegenomiclociforwhichaPCRassayisavailable.ClonesoftheBACsWeshallsummarizethemajorfindingsreportedinthewerethenbrokenintosmallfragments(shotgunning).twodraftsandcommentontheirimplications.WhileEachfragmentwasthensequenced,andcomputeralgo-therearedifferencesbetweenthedrafts,theywillnotberithmswereusedthatrecognizedmatchingsequencedweltonhere,astheareasofgeneralagreementareinformationfromoverlappingfragmentstopieceto-muchmoreextensive.Itisworthwhile,however,togetherthecompletesequence.summarizethedifferentapproachesusedbythetwoCelerausedthewholegenomeshotgunapproach,groups.Basically,theIHGSCemployedamapfirst,ineffectbypassingthemappingstep.Shotgunfrag-sequencelaterapproach.Inpart,thiswasbecausese-mentswereassembledbyalgorithmsontolargescaf-quencingwasaslowprocesswhenthepublicprojectfolds,andthecorrectpositionofthesescaffoldsinthestarted,andthestrategyoftheConsortiumevolvedgenomewasdeterminedusingSTSs.Ascaffoldisase-overtimeasadvancesweremadeinsequencingandriesof“contigs”thatareintherightorderbutnotnec-othertechniques.Theoverallapproach,referredtoasessarilyconnectedinonecontinuoussequence.Contigshierarchicalshotgunsequencing,consistedoffrag-arecontiguoussequencesofDNAmadebyassemblingmentingtheentiregenomeintopiecesofapproxi-overlappingsequencedfragmentsofanaturalchromo-mately100–200kbandinsertingthemintobacterialsomeoraBAC.Theavailabilityofhigh-throughputartificialchromosomes(BACs).TheBACswerethensequenators,powerfulcomputerprograms,theele-positionedonindividualchromosomesbylookingformentofcompetition,andotherfactorsaccountedforthemarkersequencesknownassequence-taggedsitesrapidprogressmadebybothgroupsfrom1998onward.
633THEHUMANGENOMEPROJECT/635Table54–1.Principalmethodsusedtoidentifyandisolatenormalanddiseasegenes.ProcedureCommentsDetectionofspecificcytogeneticForinstance,asmalldeletionofbandXp21.2wasimportantincloningthegeneinvolvedinabnormalitiesDuchennemusculardystrophy.ExtensivelinkagestudiesLargefamilieswithdefinedpedigreesaredesirable.Dominantgenesareeasiertorecognizethanrecessives.1UseofprobestodefinemarkerProbesidentifySTSs,RFLPs,SNPs,etc;thousands,coveringallthechromosomes,arenowlociavailable.Itisdesirabletoflankthegeneonbothsides,clearlydelineatingit.2RadiationhybridmappingNowthemostrapidmethodoflocalizingageneorDNAfragmenttoasubregionofahumanchromosomeandconstructingaphysicalmap.UseofrodentorhumansomaticPermitsassignmentofagenetoonespecificchromosomebutnottoasubregion.cellhybridsFluorescenceinsituhybridizationPermitslocalizationofagenetoonechromosomalband.Useofpulsed-fieldgelPermitsisolationoflargeDNAfragmentsobtainedbyuseofrestrictionendonucleases(rareelectrophoresis(PFGE)tocutters)thatresultinverylimitedcuttingofDNA.separatelargeDNAfragmentsChromosomewalkingInvolvesrepeatedcloningofoverlappingDNAsegments;theprocedureislaboriousandcanusuallycoveronly100–200kb.ChromosomejumpingBycuttingDNAintorelativelylargefragmentsandcircularizingit,onecanmovemorequicklyandcovergreaterlengthsofDNAthanwithchromosomalwalking.CloningviaYACs,BACs,cosmids,Permitsisolationoffragmentsofvaryinglengths.phages,andplasmidsDetectionofexpressionofThemRNAshouldbeexpressedinaffectedtissues.mRNAsintissuesbyNorthernblottingusingoneormorefragmentsofthegeneasaprobePCRCanbeusedtoamplifyfragmentsofthegene;alsomanyotherapplications.DNAsequencingEstablishesthehighestresolutionphysicalmap.Identifiesopenreadingframe.Facilitieswithmanyhighthroughputinstrumentscouldsequencemillionsofbasepairsperday.DatabasesComparisonofDNAandproteinsequencesobtainedfromunknowngenewithknownse-quencesindatabasescanfacilitategeneidentification.Abbreviations:STS,sequencetaggedsite;RFLP,restrictionfragmentlinkedpolymorphism;SNP,singlenucleotidepolymorphism;YAC,yeastartificialchromosome;BAC,bacterialartificialchromosome;PCR,polymerasechainreaction.1Manysinglenucleotidepolymorphisms(SNPs)arebeingdetectedandcatalogued.Thesearestableandfrequent,andtheirdetectioncanbeautomated.Itisanticipatedthattheywillbeparticularlyusefulformappingcomplextraitssuchasdiabetesmellitus.2Radiationhybridmapping(consulthttp://compgen.rutgers.edu/rhmap/foradetailedbibliographyofthistechnique)makesuseofapanelofsomaticcellhybrids,witheachcelllinecontainingarandomsetofirradiatedhumangenomicDNAinahamsterbackground.Briefly,theradiationfragmentstheDNAintosmallpiecesofvariablelength;ifageneislocatedclosetoanotherknowngene,itislikelythatthetwowillremainlinked(comparegeneticlinkage)onthesamefragment.AnSTSmarkeristypedagainstaradiationhybridpanelbyusingitstwooligonucleotideprimerstoperformaPCRassayagainsttheDNAfromeachhybridcelllineofthepanel.Ifenoughmark-ersaretypedononepanel,continuouslinkagecanbeestablishedalongeacharmofachromosome,andthemarkerscanbeassembledintothemapasasinglelinkagegroup.
634636/CHAPTER54DETERMINATIONOFTHESEQUENCEOFfly(13,061).ThefiguressuggestthatthecomplexityofTHEHUMANGENOMEHASPRODUCEDAhumanscomparedwiththatofthetwosimplerorgan-WEALTHOFNEWFINDINGSismsmusthaveexplanationsotherthanstrictlygenenumber.Onlyasmallfractionofthefindingscanbecoveredhere.Theinterestedreaderisreferredtotheoriginalar-Only1.1–1.5%oftheHumanticles.Table54–2summarizesanumberofthehigh-lights,whichcannowbedescribed.GenomeEncodesProteinsAnalysesoftheavailabledatarevealthat1.1–1.5%ofMostoftheHumanGenomethegenomeconsistsofexons.About24%consistsofHasBeenSequencedintrons,and75%ofsequenceslyingbetweengenes(in-tergenic).Comparisonswiththedataontheround-Over90%ofthehumangenomehadbeensequencedwormandfruitflyhaveshownthatexonsizeacrossthebyJuly2000.Thisisbyfarthelargestgenomese-threespeciesisrelativelyconstant(meansizeof145bpquenced,withanestimatedsizeofapproximately3.2inhumans).However,intronsizeinhumansismuchgigabases(Gb).Priortothehumangenome,thatofthemorevariable(meansizeofover3300bp),resultinginfruitflyhadbeenthelargest(~180Mb)sequenced.greatvariationingenesize.Gapsstillexist,smallandlarge,andthequalityofsomeofthesequencingdatawillberefinedsincesomeofthefindingsareprobablynotexactlyright.TheLandscapeofHumanChromosomesVariesWidelyTheHumanGenomeIsEstimatedtoTherearemarkeddifferencesamongindividualchro-EncodeAbout30,000–40,000Proteinsmosomesinmanyfeatures,suchasgenenumberperThegreatestsurpriseprovidedbytheresultstodatehasmegabase,densityofsinglenucleotidepolymorphismsbeentheapparentlylownumberofgenesencodingpro-(SNPs),GCcontent,numberoftransposableelementsteins,estimatedtoliebetween30,000and40,000.TheandCpGislands,andrecombinationrate.Totakeonehighernumbercouldincreaseasnewdataareobtained.example,chromosome19hastherichestgenecontentThisnumberisapproximatelytwicethatfoundinthe(23genespermegabase),whereaschromosome13androundworm(19,099)andthreetimesthatofthefruittheYchromosomehavethesparsestcontent(5genespermegabase).Explanationsforthesevariationsarenotapparentatthistime.Table54–2.Majorfindingsreportedintheroughdraftsofthehumangenome.HumanGenesDoMoreWorkThanThoseofSimplerOrganisms•Morethan90%ofthegenomehasbeensequenced;gaps,Alternativesplicingappearstobemoreprevalentinhu-largeandsmall,remaintobefilledin.mans,involvingatleast35%oftheirgenes.Dataindi-•Estimatednumberofprotein-codinggenesrangesfromcatethattheaveragenumberofdistincttranscriptsper30,000to40,000.geneforchromosomes22and19were2.6and3.2,re-•Only1.1–1.5%ofthegenomecodesforproteins.spectively.Thesefiguresarehigherthanfortheround-•Therearewidevariationsinfeaturesofindividualchromo-worm,whereonly12.2%ofgenesappeartobealterna-somes(eg,ingenenumberperMb,SNPdensity,GCcon-tivelysplicedandonly1.34splicevariantspergenetent,numbersoftransposableelementsandCpGislands,werenoted.recombinationrate).•Humangenesdomoreworkthanthoseoftheroundwormorfruitfly(eg,alternativesplicingisusedmorefrequently).TheHumanProteomeIsMoreComplex•ThehumanproteomeismorecomplexthanthatfoundinThanThatofInvertebratesinvertebrates.•Repeatsequencesprobablyconstitutemorethan50%ofRelativelyfewnewproteindomainsappeartohavethegenome.emergedamongvertebrates.However,thenumberof•Approximately100codingregionshavebeencopiedanddistinctdomainarchitectures(~1800)inhumanpro-movedbyRNA-basedtransposons.teinsis1.8timesthatoftheroundwormorfruitfly.•Approximately200genesmaybederivedfrombacteriabyAbout90vertebrate-specificfamiliesofproteinshavelateraltransfer.beenidentified,andthesehavebeenfoundtobeen-•Morethan3millionSNPshavebeenidentified.richedinproteinsoftheimmuneandnervoussystems.
635THEHUMANGENOMEPROJECT/637TheresultsofthetwodraftsarerichininformationSegmentalduplicationshavebeenfoundtobemuchaboutproteinfamiliesandclasses.Oneexampleismorecommonthanintheroundwormorfruitfly.ItisshowninTable54–3,inwhichthemajorclassesofpossiblethatthesestructuresmaybeinvolvedinexonproteinsencodedbyhumangenesarelisted.Ascanbeshufflingandtheincreaseddiversityofproteinsfoundseen,thelargestclassis“unknown.”Identificationofinhumans.theseunknownproteinswillbeamajorfocusofactivityformanylaboratories.OtherFindingsofInterestRepeatSequencesProbablyConstituteThelastthreemajorpointsofinterestlistedinTable54–2willbebrieflydescribedtogether.MoreThan50%oftheHumanGenomeApproximately100codingregionsareestimatedtoRepeatsequencesprobablyaccountforatleasthalfofhavebeencopiedandmovedbyRNA-basedtrans-thegenome.Theyfallintofiveclasses:(1)transposon-posons(retrotransposons).Itispossiblethatsomeofderivedrepeats(interspersedrepeats);(2)processedthesegenesmayadoptnewrolesinthecourseoftime.pseudogenes;(3)simplesequencerepeats;(4)segmentalAsurprisingfindingisthatover200genesmaybede-duplications,madeupof10–300kbthathavebeenrivedfrombacteriabylateraltransfer.Noneofthesecopiedfromoneregionofthegenomeintoanother;genesarepresentinnonvertebrateeukaryotes.Moreand(5)blocksoftandemlyrepeatedsequences,foundthan3millionSNPshavebeenidentified.Itislikelyatcentromeres,telomeres,andotherareas.Consider-thattheywillproveinvaluableforcertainaspectsofableinformationonmostoftheaboveclassesofrepeatgenemapping.sequences—ofgreatvalueinunderstandingthearchi-Itisstressedthatthefindingslistedhereareonlyatectureanddevelopmentofthehumangenome—isre-fewofthosereportedinthedrafts,andthereaderisportedinthedrafts.Onlytwopointsofinterestwillbeurgedtoconsulttheoriginalreports(seeReferences,mentionedhere.ItisspeculatedthatAluelements,thebelow).mostprominentmembers(about10%ofthetotalgenome)oftheshortinterspersedelements(SINEs),FURTHERWORKISPLANNEDONTHEmaybepresentinGC-richareasbecauseofpositivese-HUMAN&OTHERGENOMESlection,implyingthattheyareofbenefittothehost.TheIHGSChasindicatedthatitwilldeterminethecompletesequence,itishoped,by2003.Thetaskin-Table54–3.Majorclassesofproteinsencodedbyvolvesfillinginthegapsandidentifyingnewgenes,humangenes.1theirlocations,andfunctions.Regulatoryregionswillbeidentified,andthesequencesofotherlargegenomes2(eg,ofthehousemouse;ofRattusnorvegicus,theNor-ClassofProteinNumber(%)wayrat;ofDaniorerio,thezebrafish;ofFugurubripes,Unknown12,809(41%)thetigerpufferfish;andofoneormoreprimates)willNucleicacidenzymes2,308(7.5%)beobtained;indeed,adraftversionofthegenomeofthetigerpufferfishwaspublishedin2002.AdditionalTranscriptionfactors1,850(6%)SNPswillbeidentified;acompletecatalogoftheseReceptors1,543(5%)variantsisexpectedtobeofgreatvalueinmappinggenesassociatedwithcomplextraitsandforotherusesHydrolases1,227(4.0%)aswell.Alongwiththeabove,existingdatabaseswillbeSelectregulatorymolecules(eg,988(3.2%)addedtoasnewinformationflowsin,andnewdata-Gproteins,cellcycleregulators)baseswillprobablybeestablishedtoservespecificpur-poses.Avarietyofstudiesinfunctionalgenomics(ie,Protooncogenes902(2.9%)thestudyofgenomestodeterminethefunctionsofallCytoskeletalstructuralproteins876(2.8%)theirgenesandtheirproducts)willalsobeundertaken.Kinases868(2.8%)1DatafromVenterJCetal:Thesequenceofthehumangenome.IMPLICATIONSFORPROTEOMICS,Science2001;291:1304.BIOTECHNOLOGY,&BIOINFORMATICS2Thepercentagesarederivedfromatotalof26,383genesre-portedintheroughdraftbyCeleraGenomics.ClassescontainingManyfieldswillbeinfluencedbyknowledgeofthemorethan2.5%ofthetotalproteinsencodedbythegenesiden-humangenome.Onlyafewarebrieflydiscussedhere.tifiedwhenthisroughdraftwaswrittenarearbitrarilylistedasProteomics(seeChapter4)initsbroadestsenseismajor.thestudyofalltheproteinsencodedinanorganism(ie,
636638/CHAPTER54theproteome),includingtheirstructures,modifica-effectsonhealthservicesandthediagnosisandtreat-tions,functions,andinteractions.Inanarrowersense,mentofdisease.itinvolvestheidentificationandstudyofmultiplepro-teinslinkedthroughcellularactions—butnotnecessar-SUMMARYilytheentireproteome.Withregardtohumans,manyindividualproteinswillbeidentifiedandcharacterized;•Determinationofthecompletesequenceofthetheirinteractionsandlevelswillbedeterminedinphys-humangenome,nowalmostcompleted,isoneoftheiologicandpathologicstates,andtheresultinginforma-mostsignificantscientificachievementsofalltime.tionwillbeenteredintoappropriatedatabases.Tech-•Manyimportantfindingshavealreadyemerged.Theniquessuchastwo-dimensionalelectrophoresis,aonetodatethathasgeneratedthemostdiscussionisvarietyofmodesofmassspectrometry,andantibodythatthenumberofhumangenesmaybeonlytwotoarrayswillbecentraltoexpansionofthisrapidlygrow-threetimesthatestimatedfortheroundwormandingfield.Overall,proteomicswillgreatlyadvanceourthefruitfly.knowledgeofproteinsatthebasiclevelandwillalso•InformationflowingfromtheHumanGenomeProj-nourishbiotechnologyasnewproteinsthatarelikelyectishavingmajorinfluencesinfieldssuchaspro-tohavediagnostic,therapeutic,andotherusesaredis-teomics,bioinformatics,biotechnology,andphar-coveredandmethodsfortheireconomicproductionaremacogenomicsaswellasallareasofbiologyanddeveloped.Specialistsinbioinformaticswillbeinde-medicine.mand,asthisfieldrapidlygearsuptomanage,analyze,•Itishopedthattheknowledgederivedwillbeusedandutilizethefloodofdatafromgenomicandpro-wiselyandfairlyandthatthebenefitsthatwillensueteomicstudies.regardinghealth,disease,andothermatterswillbemadeavailabletoallpeopleeverywhere.IMPLICATIONSFORMEDICINEREFERENCESPracticallyeveryareaofmedicinewillbeaffectedbytheCollinsFS,McKusickVA:ImplicationsoftheHumanGenomenewinformationaccruingfromknowledgeoftheProjectformedicalscience.JAMA2001;285:540.(TheFeb-humangenome.Thetrackingofdiseasegeneswillberuary7,2001,issuedescribesopportunitiesformedicalre-enormouslyfacilitated.Asmentionedabove,SNPmapssearchinthe21stcentury.Manyarticlesofinterest.)shouldgreatlyassistdeterminationofgenesinvolvedinHedgesSB,KumarS:Vertebrategenomescompared.Sciencecomplexdiseases.Probesforanygenewillbeavailable2002;297:1283.(Thesameissue—No.5585,August23—ifneeded,leadingtoimproveddiagnostictestingforcontainsadraftversionofthegenomeofthetigerpufferfish.)diseasesusceptibilitygenesandforgenesdirectlyin-volvedinthecausationofspecificdiseases.ThefieldofMcKusickVA:Theanatomyofthehumangenome:aneo-Vesalianbasisformedicineinthe21stcentury.JAMA2001;286:pharmacogenomics(seeChapter53)isalreadyex-2289.(TheNovember14,2001,issuecontainsanumberofpandinggreatly,anditispossiblethatinthefutureotherexcellentarticles—eg,onclinicalproteomics,pharma-drugswillbetailoredtoaccommodatethevariationsincogenomics—relatingtotheHumanGenomeProjectanditsenzymesandotherproteinsinvolvedindrugactionandimpactonmedicine.)metabolismfoundamongindividuals.StudiesofgenesNature2001;409(6822)(February15),andScience2001;291involvedinbehaviormayleadtonewinsightsintothe(5507)(February16).(Thesetwoissuespresenttheroughcausationandpossibletreatmentofpsychiatricdisor-draftspreparedbytheIHGSCandCelera,respectively,alongwithmanyotherarticlesanalyzingthemeaningandsignifi-ders.Manyethicalissues—eg,privacyconcernsandcanceofthefindings.)theuseofgenomicinformationforcommercialpur-Science2001;294(5540)(October5).(Thisissuecontainsanum-poses—willhavetobeaddressed.Itwillalsobeimpor-berofarticlesunderthetitleGenome:UnlockingBiology’stantthatmedicalandeconomicbenefitsaccruetoindi-Storehouse.Theydescribenewideas,approaches,andre-vidualsinThirdWorldcountriesfromtheanticipatedsearchrelatedtogenomeinformation.)
637APPENDIXSELECTEDWORLDWIDEWEBSITES(MaintainstheEMBLNucleotideandSWISS-PROTdatabasesaswellasotherdatabases.)ThefollowingisalistofWebsitesthatreadersmayGeneCards:http://bioinformatics.weizmann.ac.il/cards/finduseful.Thesiteshavebeenvisitedatvarioustimes(Adatabaseofhumangenes,theirproducts,andtheirinvolvementsbyoneoftheauthors(RKM).Mostarelocatedintheindiseases.FromtheWeizmannInstituteofScience.)USA,butmanyprovideextensivelinkstointernationalGeneTestsGeneClinics:http://www.geneclinics.org/sitesandtodatabases(eg,forproteinandnucleicacid(Amedicalgeneticsinformationresourcewithcomprehensivearti-sequences)andonlinejournals.RKMwouldbegratefulclesonmanygeneticdiseases.)ifreaderswhofindotherusefulsiteswouldnotifyhimGenesandDisease:http://www.ncbi.nlm.nih.gov/disease/oftheirURLsbye-mail(rmurray6745@rogers.com)so(Coverageofthegeneticbasisofmanydifferenttypesofdiseases.)thattheymaybeconsideredforinclusioninfutureedi-TheGlycoscienceNetwork(TGN):http://www.vei.co.uk/TGN/tionsofthistext.tgn_side.htmReadersshouldnotethatURLsmaychangeorcease(TGNisaninformalworldwidegroupingofscientistswhoshareantoexist.interestincarbohydrates.Thesitecontainsconsiderablein-formationoncarbohydratesandanextensivelistoflinkstoothersitesdealingwithsugar-containingmolecules).HowardHughesMedicalInstitute:http://www.hhmi.org/ACCESSTOTHEBIOMEDICAL(Anexcellentsiteforfollowingcurrentbiomedicalresearch.Con-tainsacomprehensiveResearchNewsArchive.)LITERATURETheHumanGeneMutationDatabase:http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.htmlHighWirePress:http://highwire.stanford.edu(AnextensivetabulationofmutationsinhumangenesfromtheIn-stituteofMedicalGeneticsinCardiff,Wales.)(Extensivelistsofvariousclassesofjournals—biology,medicine,etc—andoffersalsothemostextensivelistofjournalswithHumanGenomeProjectInformation:http://www.ornl.gov/hgmis/freeonlineaccess.)(FromtheHumanGenomeProgramoftheUnitedStatesDepart-NationalLibraryofMedicine:http://www.nlm.nih.gov/mentofEnergy.)(FreeaccesstoMedlineviaPubMed.)TheInstituteforGeneticResearch:http://www.tigr.org/(Sequencesofvariousbacterialgenomesandotherinformation.)GENERALRESOURCESITESKarolinskaInstituteNutritionalandMetabolicDiseases:http://www.mic.ki.se/Diseases/c18.htmlTheBiologyProject(fromtheUniversityofArizona):http://(Accesstoinformationonmanynutritionalandmetabolicdisor-www.biology.arizona.edu/default.htmlders.)HarvardDepartmentofMolecular&CellularBiologyLinks:MITOMAP:http://www.mitomap.org/http://mcb.harvard.edu/BioLinks.html(Ahumanmitochondrialgenomedatabase.)NationalCenterforBiotechnologyInformation:http://ncbi.nlm.SITESONSPECIFICTOPICSnih.gov/AmericanHeartAssociation:http://www.americanheart.org(Informationonmolecularbiologyandhowmolecularprocessesaf-fecthumanhealthanddisease.)(Usefulinformationonnutrition,ontheroleofvariousbiomole-cules—eg,cholesterol,lipoproteins—inheartdisease,andonNationalHumanGenomeResearchInstitute:http://www.genome.themajorcardiovasculardiseases.)gov/CancerGenomeAnatomyProject(CGAP):http://www.ncbi.nlm.(ExtensiveinformationabouttheHumanGenomeProject.)nih.gov/ncicgapNationalInstitutesofHealth(NIH):http://www.nih.gov/(Aninterdisciplinaryprogramtogeneratetheinformationand(IncludeslinkstotheseparateInstitutesandCentersthatconstitutetechnicaltoolsneededtodecipherthemolecularanatomyofNIH,coveringawiderangeofbiomedicalresearch.)thecancercell.)Neuroscience(Biosciences):http://neuro.med.cornell.edu/VL/EuropeanBioinformaticsInstitute:http://www.ebi.ac.uk/ebi_(Acomprehensivelistofneuroscienceresources;partoftheWorld-home.htmlWideWebVirtualLibrary.)639
638640/APPENDIXOfficeofRareDiseases:http://rarediseases.info.nih.gov/index_http://www.mc.vanderbilt.edu/peds/pidl/genetic/ehlers.htmmain.html(Accesstoinformationonmorethan7000rarediseases,includingChapter6currentresearch.)OMIMHomePage—OnlineMendelianInheritanceinMan:http://sickle.bwh.harvard.edu/http://www.ncbi.nlm.nih.gov/omim/http://globin.cse.psu.edu/(Anextensivecatalogofhumangeneticdisorders,updateddaily.Listsaccesstovariousalliedresources.)ProteinDataBank(PDB):http://www.rcsb.org/pdb/Chapter7(Aworldwiderepositoryfortheprocessinganddistributionofhttp://s02.middlebury.edu/CH441A/EnzymeTutorials.htmlthree-dimensionalbiologicmacromolecularstructuredata.)http://www.i-a-s.de/IAS/botanik/e18/18.htmTheProteinKinaseResource:http://pkr.sdsc.edu/html/index.shtml(Informationontheproteinkinasefamilyofenzymes.)Chapter8TheProteinSociety:http://www.faseb.org/protein/index.html(AnextensivelistofWebresourcesforproteinscientists.)http://www.indstate.edu/thcme/mwking/enzyme-kinetics.htmlSignalingUpdate:http://www.signaling-update.org/http://ntri.tamuk.edu/cell/kinetics.html(Aone-stopoverviewforthespecialistornonspecialistofwhatishappeningincellsignaling.)SocietyforEndocrinology:http://www.endocrinology.orgChapter9(Aimstopromoteadvancementofpubliceducationinendocrinol-http://users.wmin.ac.uk/~mellerj/physiology/bernard.htmogy.Containsanumberofarticlesonendocrinologyandalistoflinkstootherrelevantsites.)http://www.cm.utexas.edu/academic/courses/Fall2001/CH369/LEC06/Lec6.htmtbase—theTransgenic/TargetedMutationDatabaseattheJacksonLaboratory,BarHarbor,Maine:http://tbase.jax.org/http://www.cellularsignaling.org(Anattempttoorganizeinformationontransgenicanimalsandtar-http://arethusa.unh.edu/bchm752/ppthtml/Jan27/sld015.htmgetedmutationsgeneratedandanalyzedworldwide.)TheWellcomeTrustSangerInstitute:http://www.sanger.ac.ukChapter22(Agenomeresearchcenterwhosepurposeitistoincreaseknowl-edgeofgenomes,particularlythroughlarge-scalesequencinghttp://www.auhs.edu/netbiochem/NetWelco.htmandanalysis,)WhiteheadInstitute/MITCenterforGenomeResearch:http://www.genome.wi.mit.edu/Chapter28(Accesstovariousdatabasesandarticlesentitled“What’sNewinGenomeResearch.”)http://www.people.virginia.edu/~rjh9u/scurvy.htmlhttp://www.mc.vanderbilt.edu/biolib/hc/journeys/scurvy.htmlChapter2http://opbs.okstate.edu/~melcher/MG/MGW2/MG2411.htmlhttp://www.geocities.com/bioelectrochemistry/sorensen.htmChapter29Chapter3http://www.nucdf.org/whatis.htmhttp://www.bio.cmu.edu/Courses/03231http://www.bcbp.gu.se/~orjan/bmstruct/Chapter30http://www.pkunetwork.org/Chapter4http://www.rarediseases.org/search/rdblist.htmlhttp://www.lundberg.bcbp.gu.se/~orjan/bmstruct/http://www.msud-support.org/overv.htmChapter5Chapter34http://www.rarediseases.org/search/rdblist.htmlhttp://www.umass.edu/microbio/chime/explorer/http://www.rheumatology.org/patients/factsheet/gout.htmlhttp://molvis.sdsc.edu/protexpl/index.htmhttp://www.merck.com/pubs/mmanual/section5/chapter55/55a.htmhttp://www.umass.edu/microbio/rasmol/http://www.nlm.nih.gov/medlineplus/goutandpseudogout.htmlhttp://www.cryst.bbk.ac.uk/PPS2/course/section10/membrane.htmlhttp://www.amg.gda.pl/~essppmm/ppd/ppd_py_umps.htmlhttp://molbio.info.nih.gov/cgi-bin/pdb/
639APPENDIX/641BIOCHEMICALJOURNALSANDREVIEWSBiochimicaetBiophysicaActa(BiochimBiophysActa)ThefollowingisapartiallistofbiochemistryjournalsBiochimie(Biochimie)andreviewseriesandofsomebiomedicaljournalsthatcontainbiochemicalarticles.BiochemistryandbiologyEuropeanJournalofBiochemistry(EurJBiochem)journalsnowusuallyhaveWebsites,oftenwithusefulIndianJournalofBiochemistryandBiophysics(In-links,andsomejournalsarefullyaccessiblewithoutdianJBiochemBiophys)charge.ThereadercanobtaintheURLsforthefollow-JournalofBiochemistry(Tokyo)(JBiochemingbyusingasearchengine.[Tokyo])JournalofBiologicalChemistry(JBiolChem)AnnualReviewsofBiochemistry,CellandDevelop-JournalofClinicalInvestigation(JClinInvest)mentalBiology,Genetics,GenomicsandHumanJournalofLipidResearch(JLipidRes)GeneticsNature(Nature)ArchivesofBiochemistryandBiophysics(ArchBiochemBiophys)NatureGenetics(NatGenet)BiochemicalandBiophysicalResearchCommunica-ProceedingsoftheNationalAcademyofSciencestions(BiochemBiophysResCommun)USA(ProcNatlAcadSciUSA)BiochemicalJournal(BiochemJ)Science(Science)Biochemistry(Biochemistry)TrendsinBiochemicalSciences(TrendsBiochemSci)Biochemistry(Moscow)(Biochemistry[Mosc])
640IndexNote:Pagenumbersinboldfacetypeindicateamajordiscussion.Atfollowingapagenumberindicatestabularma-terialandanffollowingapagenumberindicatesafigure.Abands,556,557f,558fformationof,254f,255–259,255f,256f,molecularstructureaffectingstrengthof,Abloodgroupsubstance,618,619f257f,258f,259f12,12tAcyclins,333,334f,335tlipogenesisand,173–177,174f,175fpolyfunctional,nucleotidesas,290Agene,GalNActransferaseencodedby,asfattyacidbuildingblock,176–177asprotondonors,9618–619pyruvatedehydrogenaseregulatedby,strong,9A(aminoacyl/acceptor)site,aminoacyl-141–142,142f,178weak.SeeWeakacidstRNAbindingto,inproteinpyruvateoxidationto,134,135f,Aciduriasynthesis,368,368f140–142,141f,142f,143tdicarboxylic,188ABC-1.SeeATP-bindingcassettexenobioticmetabolismand,630methylmalonic,155transporter-1Acetyl-CoAcarboxylase,156t,179orotic,300,301Abetalipoproteinemia,207,228tinlipogenesisregulation,156t,173,urocanic,250ABObloodgroupsystem,biochemicalbasis174f,178,178f,179Aconitase(aconitatehydratase),130of,617–619,619fN-Acetylgalactosamine(GalNAc),inACP.SeeAcylcarrierproteinAbsorption,474–480glycoproteins,515,516tAcrosomalreaction,glycoproteinsin,528Absorptionchromatography,forN-Acetylglucosamine(GlcNAc),inACTH.SeeAdrenocorticotropichormoneprotein/peptidepurification,22glycoproteins,516tActin,557,559,560Absorptionspectra,ofporphyrins,N-Acetylglucosaminephosphotransferasedecorationof,561,561f273–274,277f(GlcNAcphosphotransferase)fibronectinreceptorinteractingwith,Absorptivepinocytosis,430inI-celldisease,532540,541fACAT(acyl-CoA:cholesterolacyltrans-inpseudo-Hurlerpolydystrophy,532inmusclecontraction,557–559,558f,ferase),223N-Acetylglutamate,inureabiosynthesis,561–562,561f,562fAccelerator(Ac-)globulin(factorV),600t,245,246fregulationofstriatedmuscleand,601,602fAcetylhexosamines,inglycoproteins,109t562–563Acceptor(A/aminoacyl)site,aminoacyl-N-Acetyllactosamines,onN-linkedglycaninnonmusclecells,576–577tRNAbindingto,inproteinchains,521inredcellmembranes,615f,616f,616t,synthesis,368,368fAcetyl(acyl)-malonylenzyme,173,175f617Acceptorarm,oftRNA,310,312f,360,N-Acetylneuraminicacid,169,171finstriatedvs.smoothmuscle,572t361fingangliosides,201,203fβ-Actin,577Aceruloplasminemia,589inglycoproteins,169,171f,515,516tF-Actin,559,559f,561ACEs.SeeAngiotensin-convertingenzymeinmucins,519f,520innonmusclecells,576,577inhibitorsAcetyltransacylase,173,174f,175fG-Actin,559,559fAcetallinks,105–106Acetyltransferases,xenobioticmetabolisminnonmusclecells,576Aceticacid,112tand,630γ-Actin,577pK/pKavalueof,12tAcholuricjaundice,282Actin-filamentcappingprotein,540,541fAcetoacetate,183–184,184fAchondroplasia,432t,551t,553–554,554fActin(thin)filaments,557,558f,559fintyrosinecatabolism,254f,255Acidanhydridebonds,287α-Actinin,540,566tAcetoacetyl-CoAsynthetase,inmevalonateAcidanhydrides,grouptransferpotentialActivatedproteinC,inbloodcoagulation,synthesis,219,220ffor,289–290,289f,290f,290t603Acetone,183Acid-basebalance,ammoniametabolismin,Activationenergy,61,63Acetonebodies.SeeKetonebodies245Activationenergybarrier,enzymesaffecting,AcetylationAcid-basecatalysis,51–5263incovalentmodification,massincreasesHIVproteasein,52,53fActivationfunction1,460and,27tα1-Acidglycoprotein(orosomucoid),583tActivationfunction2,460ofxenobiotics,630Acidhydrolysis,forpolypeptidecleavage,Activationreaction,456,458fAcetyl-CoA,122,122f26tActivator-recruitedcofactor(ARC),472t,carbohydratemetabolismand,122,122f,Acidphosphatase,diagnosticsignificance473123fof,57tActivatorscatabolismof,130–135,131f,132f.SeeAcidemia,isovaleric,259,259–262inregulationofgeneexpression,374,alsoCitricacidcycleAcidosis376.SeealsoEnhancerscholesterolsynthesisand,219–220,220f,lactic.SeeLacticacidosistranscription,351,351t221f,222fmetabolic,ammoniain,245Activechromatin,316–318,318f,383fattyacidoxidationto,123–124,123f,AcidsActivesite,51,51f.SeealsoCatalyticsite181–183,181f,182fconjugate,10ΔGFand,63643
641644/INDEXActivesulfate(adenosine3′-phosphate-metabolismin,214–215,214f,235tAlanine(alanine-pyruvate)aminotransferase5′-phosphosulfate),289,289f,629controlof,216–217(ALT/SGPT)Activetransport,423,423t,424f,426–427,ADP,287fdiagnosticsignificanceof,57t427–428,428ffreeenergyofhydrolysisof,82tinureasynthesis,243–244,244finbilirubinsecretion,280,281fmitochondrialrespiratoryrateand,β-Alanyldipeptides,264,265fActivity(physical),energyexpenditureand,94–95,97t,98fAlbumin,580,581f,583–584,583t478myosin,musclecontractionand,561,conjugatedbilirubinbindingto,283Actomyosin,560561ffreefattyacidsincombinationwith,180,ACTRcoactivator,472,472tinplateletactivation,606f,607206,206t,584Acutefattyliverofpregnancy,188inrespiratorycontrol,94–95,97,97t,glomerularmembranepermeabilityto,Acuteinflammatoryresponse,neutrophils98f,134–135540–541in,620structureof,83fAlbuminuria,542Acutephaseproteins,583,583tADP/ATPtransporter,95,98fAlcohol,ethyl.SeeEthanolnegative,vitaminAas,483–484ADPase,607,607tAlcoholdehydrogenase,infattyliver,212Acylcarnitine,180–181,181fADP-chaperonecomplex,508.SeealsoAlcoholismAcylcarrierprotein(ACP),173,174fChaperonescirrhosisand,212synthesisof,frompantothenicacid,173,ADP-ribose,NADassourceof,490fattyliverand,212–214495ADP-ribosylation,490Aldehydedehydrogenase,87Acyl-CoA:cholesterolacyltransferaseAdrenalcorticalhormones.Seealsospecificinfattyliver,212(ACAT),223hormoneandGlucocorticoids;AldolaseA,deficiencyof,143Acyl-CoAdehydrogenase,87,181,182fMineralocorticoidsAldolaseB,167,168fmedium-chain,deficiencyof,188synthesisof,438–442,440f,441fdeficiencyof,171Acyl-CoAsynthetase(thiokinase)Adrenalgland,cytochromeP450isoformsAldolases,inglycolysis,137,138finfattyacidactivation,180,181fin,627Aldose-ketoseisomerism,103f,104,104fintriacylglycerolsynthesis,199,214f,215Adrenalmedulla,catecholaminesproducedAldosereductase,167,169f,172Acylglycerols,197in,445Aldoses,102,102t,104tmetabolismof,197–201Adrenergicreceptors,inglycogenolysis,148ringstructureof,104catabolism,197Adrenocorticotropichormone(ACTH),Aldosteroneclinicalaspectsof,202437,438,439f,453,453fbindingof,455synthesis,197–201,197f,198fAdrenodoxin,627synthesisof,438–440,441finendoplasmicreticulum,126,127fAdrenodoxinreductase,627angiotensinaffecting,451,452Adapterproteins,inabsorptivepinocytosis,Adrenogenitalsyndrome,442Aldosteronesynthase(18-hydroxylase),in430Adrenoleukodystrophy,neonatal,503,503tsteroidsynthesis,440,441fAdenine,288f,288tAerobicglycolysis,139AlkalinephosphataseAdenosine,287f,288tasmuscleATPsource,575,575f,575tinbonemineralization,550basepairingofinDNA,303,304,305fAerobicrespirationisozymesof,diagnosticsignificanceof,inuricacidformation,299,299fcitricacidcycleand,13057tAdenosinedeaminaseAF-1.SeeActivationfunction1inrecombinantDNAtechnology,400tdeficiencyof,300AF-2.SeeActivationfunction2Alkalosis,metabolic,ammoniain,245localizationofgenefor,407tAF-2domain,470Alkaptonuria,255Adenosinediphosphate.SeeADPAffinitychromatographyAllergicreactions,peptideabsorptionAdenosinemonophosphate.SeeAMPforprotein/peptidepurification,23causing,474Adenosine3′-phosphate-5′-phosphosulfate,inrecombinantfusionproteinAllopurinol,290,297289,289f,629purification,58,59fAllostericactivators,157Adenosinetriphosphate.SeeATPAFP.SeeAlpha-fetoproteinAllostericeffectors/modifiers,129S-Adenosylmethionine,258f,259,264,Agammaglobulinemia,595ingluconeogenesisregulation,157266f,289,290f,290tAge,xenobiotic-metabolizingenzymesnegative,74.SeealsoFeedbackinhibitionAdenylicacid,assecondmessenger,457affectedby,630secondmessengersas,76AdenylylcyclaseAggrecan,542,551t,553,553fAllostericenzymes,75,129incAMP-dependentsignaltransduction,Aggregates,formationofafterdenaturation,aspartatetranscarbamoylaseasmodelof,458–459,460t3675cAMPderivedfrom,147Aging,glycosaminoglycansand,549Allostericpropertiesofhemoglobin,42–46inlipolysis,215,216fAglycone,105,106Allostericregulation,ofenzymaticcatalysis,Adenylylkinase(myokinase),84AHG.SeeAntihemophilicfactorA/globulin74,74–76,75f,128f,129deficienciesof,151–152AIB1coactivator,472,472tgluconeogenesisregulationand,157ingluconeogenesisregulation,157ALA.SeeAminolevulinateAllostericsite,74,75assourceofATPinmuscle,573,575fAlanine,15tAlpha-adrenergicreceptors,inAdhesionmolecules,529,529t.SeealsoCellpIof,17glycogenolysis,148adhesioninpyruvateformation,250,252fAlpha-aminoacids.SeealsoAminoacidsAdiposetissue,111,214–215,214fsynthesisof,237,238fgeneticcodespecifying,14,15–16tbrown,217,217fβ-Alanine,264,300,301finproteins,14
642INDEX/645Alpha-aminonitrogen.SeeAminoacidabsorptionof,477Aminolevulinatedehydratase,270,273fnitrogenanalysis/identificationof,20inporphyria,274,277tAlphaanomers,104bloodglucoseand,159Aminolevulinatesynthase,270,272–273,Alpha1antiproteinase/antitrypsin.Seebranchedchain,catabolismof,259,273f,276fα1-Antiproteinase260f,261f,262finporphyria,274,277f,278Alpha-(α)carotene,482disordersof,259–262Aminopeptidases,477Alpha-fetoprotein,583tincatalysis,conservationof,54,55tAminophospholipids,membraneAlpha-globingene,localizationof,407tchemicalreactionsof,functionalgroupsasymmetryand,420Alpha(α)helix,31–32,32f,33fdictating,18–20Aminoproteinase,procollagen,537amphipathic,31–32deaminationof.SeeDeaminationAminosugars(hexosamines),106,106finmyoglobin,40,41fdeficiencyof,237,480glucoseasprecursorof,169,171fAlpha(α)ketoglutarate.Seeexcitatory.SeealsoAspartate;Glutamateinglycosaminoglycans,109,169,171fα-Ketoglutarateglucogenic,231–232inglycosphingolipids,169,171fAlpha-lipoproteins,205.SeealsoHigh-ingluconeogenesis,133–134,134finterrelationshipsinmetabolismof,densitylipoproteinsinterconvertabilityof,231–232171ffamilialdeficiencyof,228tketoacidreplacementofindiet,240Aminotransferases(transaminases),Alpha-Rgroups,aminoacidpropertiesketogenic,232133–134,134faffectedby,18meltingpointof,18diagnosticsignificanceof,57tAlpha(α)thalassemia,47metabolismof,122f,124,124f,125f.Seeinureabiosynthesis,243–244,244fAlpha-tocopherol.SeeTocopherolalsoAminoacidcarbonskeletons,AmmoniaAlportsyndrome,538,538tcatabolismof;Aminoacidinacid-basebalance,245ALT.SeeAlanineaminotransferasenitrogen,catabolismofdetoxificationof,242Alteplase(tissueplasminogenactivator/t-PA),pyridoxalphosphatein,491excessof,247604–605,605f,606t,607tnetchargeof,16–17,17fglutaminesynthasefixing,245,245fAlternativepathway,ofcomplementnutritionallyessential,124nitrogenremovedas,244,244factivation,596nutritionallynonessential,124Ammoniaintoxication,244Altitude,high,adaptationto,46synthesisof,237–241Ammoniumion,pK/pKavalueof,12tAlufamily,321–322inpeptides,14,19,19fAmobarbital,oxidativephosphorylation/Alzheimerdisease,amyloidin,37,590pK/pKavaluesof,15–16t,17,17frespiratorychainaffectedby,92,α-Amanitin,RNApolymerasesaffectedby,environmentaffecting,18,18t95,96f343productsderivedfrom,264–269.SeealsoAMP,287f,288f,288t,297fAmbiguity,geneticcodeand,359specificproductcoenzymederivativesof,290tAminoacidcarbonskeletons,catabolismof,propertiesof,14–18cyclic.SeeCyclicAMP249–263α-Rgroupdetermining,18freeenergyofhydrolysisof,82tacetyl-CoAformationand,254f,proteindegradationand,242,243fIMPconversionto,293,296f255–259,255f,256f,257f,258f,inproteins,14feedback-regulationof,294,296f259fremovalofammoniafrom,244,244fPRPPglutamylamidotransferasebranched-chain,259,260f,261f,262frequirementsfor,480regulatedby,294disordersof,259–262sequenceof,primarystructurestructureof,83f,288fpyruvateformationand,250–255,252f,determinedby,18–19Ampresistancegenes,402,403f253fsolubilitypointof,18Amphibolicpathways/processes,122transaminationininitiationof,249–250,substitutionsof,missensemutationscitricacidcycleand,133249f,250f,251fcausedby,362–363,362fAmphipathicα-helix,31–32Aminoacidnitrogensynthesisof,237–241Amphipathiclipids,119–121,120fcatabolismof,242–248incarbohydratemetabolism,123inlipoproteins,205,207finaminoacidcarbonskeletoncitricacidcyclein,133,134finmembranes,119,120f,417–418,417fcatabolism,249,249ftransaminationof.SeeTransaminationAmphipathicmolecules,foldingand,6endproductsof,242–243transporter/carriersystemsfor,99Ampicillinresistancegenes,402,403fureaas,245–247,246fglutathioneand,629–630Amplification,gene,ingeneexpressionL-glutamatedehydrogenasein,hormonesaffecting,427regulation,392–393,393f244–245,244f,245fAminoacylresidues,18–19Amylasestransaminationof,243–244,243fpeptidestructureand,19diagnosticsignificanceof,57tL-Aminoacidoxidase,86–87Aminoacyl(A/acceptor)site,aminoacyl-inhydrolysisofstarch,474innitrogenmetabolism,244,244ftRNAbindingto,inproteinβ-Amyloid,inAlzheimerdisease,37,590Aminoacidsequences.SeealsoProteinsynthesis,368,368fAmyloid-associatedprotein,590sequencingAminoacyl-tRNA,inproteinsynthesis,368Amyloidprecursorproteins,590determinationof,forglycoproteins,515tAminoacyl-tRNAsynthetases,360,360finAlzheimerdisease,37,590primarystructuredeterminedby,18–19γ-Aminobutyrate,267,268fAmyloidosis,590–591repeating,inmucins,519,520fβ-Aminoisobutyrate,300,301fAmylopectin,107,108fAminoacids,2,14–20,15–16t.SeealsoAminolevulinate(ALA),270,273fAmylopectinosis,152tPeptidesinporphyria,278Amylose,107,108f
643646/INDEXAnabolicpathways/anabolism,81,122.SeeAnticonformers,287,287fApoA-IV,206,206talsoEndergonicreaction;AntibioticsApoB-48,206,206tMetabolismaminosugarsin,106ApoB-100,206,206tAnaerobicglycolysis,136,137f,139bacterialproteinsynthesisaffectedby,inLDLmetabolism,209,210fasmuscleATPsource,574–576,575f,371–372regulationof,223575tfolateinhibitorsas,494ApoC-I,206,206tAnalbuminemia,584Antibodies,580,581.SeealsoApoC-II,206,206tAnaphylaxis,slow-reactingsubstanceof,Immunoglobulinsinlipoproteinlipaseactivity,207–208196monoclonal,hybridomasinproductionApoC-III,206,206tAnapleroticreactions,incitricacidcycle,of,595–596,596flipoproteinlipaseaffectedby,208133inxenobioticcellinjury,631,631fApoD,206,206tAnchorin,incartilage,551tAntibodydiversity,591ApoE,206,206tAndersen’sdisease,152tDNA/generearrangementand,593–594receptorsforAndrogenresponseelement,459tAntichymotrypsin,583tinchylomicronremnantuptake,AndrogensAnticoagulants,coumarin,604208–209,209festrogensproducedfrom,442–445,444fAnticodonregion,oftRNA,359,360,360finLDLmetabolism,209,210freceptorsfor,471Antifolatedrugs,purinenucleotideApoferritin,586synthesisof,440–442,441f,443fsynthesisaffectedby,293Apolipoproteins/apoproteins,205,AndrostenedioneAntigenicdeterminant(epitope),33,591205–206estroneproducedfrom,444f,445Antigenicity,xenobioticsaltering,cellinjurydistributionof,205–206,206tsynthesisof,442,443fand,631,631fhemoglobin;oxygenationaffecting,42Anemias,47Antigens,591Apomyoglobin,hinderedenvironmentforFanconi’s,338AntihemophilicfactorA/globulin(factorhemeironand,41,41fhemolytic,136,143,609,619,620tVIII),599f,600,600tApoproteins.SeeApolipoproteins/glucose-6-phosphatedehydrogenasedeficiencyof,604apoproteinsdeficiencycausing,163,AntihemophilicfactorB(factorIX),599f,Apoptosis,201169–170,613,614f,619600,600tp53and,339haptoglobinlevelsin,584coumarindrugsaffecting,604Apo-transketolase,activationof,inthiaminhyperbilirubinemia/jaundicein,282,deficiencyof,604nutritionalstatusassessment,489284,284tAntimalarialdrugs,folateinhibitorsas,APP.SeeAmyloidprecursorproteinpentosephosphatepathway/494Apurinicendonuclease,inbaseexcision-glutathioneperoxidaseand,166,AntimycinA,respiratorychainaffectedby,repair,337167f,169–17095,96fApyrimidinicendonuclease,inbaseprimaquine-sensitive,613Antioxidants,91,119,611–613,613texcision-repair,337redcellmembraneabnormalitiesretinoidsandcarotenoidsas,119,482tAquaporins,424–426causing,619vitaminCas,119D-Arabinose,104f,105tirondeficiency,478,497,586,610tvitaminEas,91,119,486,487fArabinosylcytosine(cytarabine),290megaloblasticAntiparallelloops,mRNAandtRNA,360Arachidonicacid/arachidonate,113t,190,folatedeficiencycausing,482t,492,Antiparallelβsheet,32,33f190f494Antiparallelstrands,DNA,303eicosanoidformationand,192,193f,vitaminB12deficiencycausing,482t,Antiportsystems,426,426f194,194f,195f492,610tfornucleotidesugars,517foressentialfattyaciddeficiency,pernicious,482t,492α1-Antiproteinase(α1-antitrypsin),583t,191–192recombinanterythropoietinfor,526,610589ARC,472t,473sicklecell.SeeSicklecelldiseasedeficiencyof,589–590,589f,590f,ARE.SeeAndrogenresponseelementAngiotensinII,437,451,452f623Argentaffinoma(carcinoid),serotoninin,synthesisof,451–452,452fasthrombininhibitor,603266–267AngiotensinIII,452,452fAntiproteinases,623,624tArginaseAngiotensin-convertingenzyme,451–452,Antithrombin/antithrombinIII,583t,inperiodichyperlysinemia,258452f603–604inureasynthesis,246f,247Angiotensin-convertingenzymeinhibitors,heparinbindingto,547,603–604Arginine,16t,265,266f451–452α1-Antitrypsin(α1-antiproteinase),583t,catabolismof,250,251fAngiotensinogen,451,452f589inureasynthesis,246f,247Anionexchangeprotein,615,615f,616tdeficiencyof,589–590,589f,590f,ArginosuccinaseAnkyrin,615f,616f,616t,617623–624deficiencyof,248Anomericcarbonatom,104asthrombininhibitor,603inureasynthesis,246f,247Anomers,αandβ,104APC.SeeActivatedproteinCArginosuccinate,inureasynthesis,245,Anserine,264,265,265fApoA-I,206,206t,224246f,247Antennae(oligosaccharidebranches),521deficienciesof,228tArginosuccinatesynthase,246f,247Anteriorpituitaryglandhormones,bloodApoA-II,206tdeficiencyof,247glucoseaffectedby,161lipoproteinlipaseaffectedby,207–208Arginosuccinicaciduria,248
644INDEX/647Aromataseenzymecomplex,442,444f,445AsymmetryATPsynthase,membrane-located,96,97f,ARS(autonomouslyreplicatingsequences),importinbindingand,50198f326,413lipidandprotein,membraneassemblyAtractyloside,respiratorychainaffectedby,Arsenate,oxidationandphosphorylationand,511,512f95affectedby,137,142inmembranes,416,419–420Attachmentproteins,540,541fArterialwall,intimaof,proteoglycansin,Ataxia-telangiectasia,338Autoantibodies,inmyastheniagravis,431548ATCase.SeeAspartatetranscarbamoylaseAutonomouslyreplicatingsequences(ARS),ArthritisAtherosclerosis,205,607326,413gouty,299cholesteroland,117,219,227Auto-oxidation.SeePeroxidationproteoglycansin,548HDLand,210–211Autoradiography,definitionof,413rheumatoid,glycosylationalterationsin,hyperhomocysteinemiaand,folicacidAutotrophicorganisms,82533supplementsinpreventionof,Avidin,biotindeficiencycausedby,494Artificialmembranes,421–422494Axialratios,30Ascorbate,167,168fLDLreceptordeficiencyin,209Axonemaldyneins,577Ascorbicacid(vitaminC),163,482t,lysophosphatidylcholine(lysolecithin)5-or6-Azacytidine,290495–496,496fand,1168-Azaguanine,290,291fasantioxidant,119Atorvastatin,229Azathioprine,290incollagensynthesis,38,496,535ATP,82,82–85,287f,2895-or6-Azauridine,290,291fdeficiencyof,482t,496inactivetransport,427–428,428fcollagenaffectedin,38–39,496,incoupling,82,84538–539fattyacidoxidationproducing,182Bbloodgroupsubstance,618,619fironabsorptionand,478,496freeenergyofhydrolysisof,82–83,82tB(β)cells,pancreatic,insulinproducedby,supplemental,496infreeenergytransferfromexergonicto160Asialoglycoproteinreceptorsendergonicprocesses,82–83,82fBcyclins,333,334f,335tincotranslationalinsertion,506,506fhydrolysisofBgene,Galtransferaseencodedby,inglycoproteinclearance,517inmusclecontraction,561–562,561f618–619Asn-GlcNAclinkagebyNSF,509,510fBlymphocytes,591inglycoproteins,521inorganicpyrophosphateproductioninhybridomaproduction,595–596,596finglycosaminoglycans,543and,85Bvitamins.SeeVitaminBcomplexAsparaginase,inaminoacidnitrogeninmitochondrialproteinsynthesisandBACvector.SeeBacterialartificialcatabolism,245,245fimport,499chromosome(BAC)vectorAsparagine,15tinmuscle/musclecontraction,556,Bacteriainaminoacidnitrogencatabolism,245561–562,561fintestinal,inbilirubindeconjugation,281catabolismof,249,250fdecreaseinavailabilityof,564transcriptioncyclein,342–343,342fsynthesisof,237–238,238fsourcesof,573–574,574–576,575f,Bacterialartificialchromosome(BAC)Asparaginesynthetase,238,238f575tvector,401–402,402tAspartateinpurinesynthesis,293–294,295fforcloningingeneisolation,635tcatabolismof,249,250frespiratorycontrolinmaintenanceofinHumanGenomeproject,634synthesisof,237–238,238fsupplyof,94–95,97,97t,98f,Bacterialgyrase,306inureasynthesis,246f,247134–135Bacterialpromoters,intranscription,Aspartate102,incovalentcatalysis,53–54,structureof,83f345–346,345f54fsynthesisofBacteriophage,definitionof,413Aspartateaminotransferase(AST/SGOT),ATPsynthasein,96,97f,98fBacteriophagelambda(λ),378–383,379f,diagnosticsignificanceof,57tincitricacidcycle,131f,133,142,380f,381f,382fAspartatetranscarbamoylase,75143tBAL.SeeDimercaprolinpyrimidinesynthesis,298f,299glucoseoxidationyielding,142,143tBAL31nuclease,inrecombinantDNAAsparticacid,15trespiratorychainin,93–95,98ftechnology,400tpIof,17ATP/ADPcycle,83,84fBalancedchemicalequations,60Asparticproteasefamily,inacid-baseATPaseBamHI,398,399tcatalysis,52,53finactivetransport,427–428,428fBarbiturates,respiratorychainaffectedby,Aspartylglycosaminuria,532–533,533tchaperonesexhibitingactivityof,50895,96fAspirincopper-bindingP-type,mutationsinBasallamina,lamininascomponentof,antiplateletactionsof,607–608genefor540–542cyclooxygenaseaffectedby,193Menkesdiseasescausedby,588Basalmetabolicrate,478prostaglandinsaffectedby,190Wilsondiseasecausedby,588–589Baseexcision-repairofDNA,336t,337,Assemblyparticles,inabsorptiveATP-bindingcassettetransporter-1,210,337fpinocytosis,430211fBasepairinginDNA,7,303,304,305fAST.SeeAspartateaminotransferaseATP-chaperonecomplex,508.Seealsomatchingofforrenaturation,305–306Asthma,leukotrienesin,112ChaperonesrecombinantDNAtechnologyand,Asymmetricsubstitution,inporphyrins,ATP-citratelyase,134,135f,156t,157396–397270,271facetyl-CoAforlipogenesisand,177replication/synthesisand,328–330,330f
645648/INDEXBasesubstitution,mutationsoccurringby,BilirubinBloodcells,609–625.SeealsoErythrocytes;361,361f,362accumulationof(hyperbilirubinemia),Neutrophils;PlateletsBasementmembranes,collagenin,537281–284,284tBloodclotting.SeeCoagulationBasesconjugatedBloodglucoseconjugate,10bindingtoalbuminand,283normal,145asprotonacceptors,9reductionoftourobilinogen,281,regulationofstrong,9282fclinicalaspectsof,161–162,161fweak,9conjugationof,280,280f,281fdiet/gluconeogenesis/glycogenolysisin,BenceJonesprotein,595fecal,injaundice,284t158–161,159f,160fBends(proteinconformation),32–33,hemecatabolismproducing,278–280,glucagonin,160–16134f279fglucokinasein,159–160,160fBeriberi,482t,489liveruptakeof,280–281,280f,281f,glycogenin,145Beta-alanine.Seeβ-Alanine282finsulinin,160Betaanomers,104normalvaluesfor,284tlimitsof,158Beta-(β)carotene,482,482t,483,483f.secretionofintobile,280,281fmetabolicandhormonalmechanismsSeealsoVitaminAunconjugated,disordersoccurringin,in,159,160t,161asantioxidant,119,482t282–283Bloodgroupsubstances,618,619fBeta-endorphins,453,453furine,injaundice,284,284tglycoproteinsas,514,618Beta-globingeneBiliverdin,278,279fBloodgroupsystems,617–619,619flocalizationof,407tBiliverdinreductase,278Bloodplasma.SeePlasmarecombinantDNAtechnologyinBimolecularmembranelayer,418–419.SeeBloodtype,617–618detectionofvariationsin,alsoLipidbilayerBloodvessels,nitricoxideaffecting,407–408,408f,409tBindingconstant,Michaelisconstant(Km)571–573,573f,574tBeta-lipoproteins,205.Seealsolowdensityapproximating,66Blottransfertechniques,403,404flipoproteinsBindingproteins,454–455,454t,455t,583tBluntendligation/blunt-endedDNA,398,Beta-oxidationoffattyacids,181–183,Biochemistry399–400,400f,413181f,182fasbasisofhealth/disease,2–4,3tBMR.SeeBasalmetabolicrateketogenesisregulationand,186–187,definitionof,1Bodymassindex,478187f,188fHumanGenomeProjectand,3–4Bodywater.SeeWatermodified,183,183fmethodsandpreparationsusedin,1,2tBohreffect,44,45fBeta(β)sheet,32,33frelationshipoftomedicine,1–4,3finhemoglobinM,46Betasubunitsofhemoglobin,myoglobinBiocytin,494,495fBone,549–550,549f,550fand,42Bioenergetics,80–85.SeealsoATPmetabolicandgeneticdisordersBeta(β)thalassemia,47Bioinformatics,412,638involving,551–552,551tBeta(β)turn,32,34fproteinfunctionand,28–29proteinsin,548t,549BFU-E.SeeBurst-formingunit-erythroidBiologicoxidation.SeealsoOxidationBoneGlaprotein,548tBgIII,399tBiomolecules.SeealsospecifictypeBonemarrow,hemesynthesisin,272BHA.SeeButylatedhydroxyanisolestabilizationof,7BonematrixGlaprotein,488BHT.SeeButylatedhydroxytoluenewateraffectingstructureof,6–7,6tBonemorphogeneticproteins,548tBi-Bireactions,69–70,69f,70fBiotechnology,HumanGenomeProjectBonesialoprotein,548tMichaelis-Mentenkineticsand,70,70faffecting,638BoneSPARCprotein,548tBicarbonate,inextracellularandBiotin,482t,494–495,495fBotulinumBtoxin,511intracellularfluid,416tdeficiencyof,482t,494Bovinespongiformencephalopathy,37Biglycaninmalonyl-CoAsynthesis,173,174fBPG.See1,3-Bisphosphoglycerate;inbone,548tasprostheticgroup,502,3-Bisphosphoglycerateincartilage,551tBiP.SeeImmunoglobulinheavychainBradykinin,ininflammation,621Bilayers,lipid,418–419,418f,419fbindingproteinBrain,metabolismin,235tmembraneproteinsand,4191,3-Bisphosphoglycerate(BPG),freeenergyglucoseasnecessityfor,232Bile,bilirubinsecretioninto,280,281fofhydrolysisof,82tBranchedchainaminoacids,catabolismof,Bileacids(salts),225–2272,3-Bisphosphoglycerate(BPG),Tstructure259,260f,261f,262fenterohepaticcirculationof,227ofhemoglobinstabilizedby,45,disordersof,259–262inlipiddigestionandabsorption,475,45fBranched-chainα-ketoaciddehydrogenase,476fBisphosphoglyceratemutase,inglycolysisin259secondary,226f,227erythrocytes,140,140fBranchedchainketonuria(maplesyrupsynthesisof,225–227,226f2,3-Bisphosphoglyceratephosphatase,inurinedisease),259regulationof,226,226f,227erythrocytes,140,140fBranchingenzymesBilepigments,278–284,282f.SeealsoBlindness,vitaminAdeficiencycausing,483absenceof,152tBilirubinBloodinglycogenbiosynthesis,145,147fBiliaryobstruction,coagulationof,598–608.SeealsoBrefeldinA,510–511hyperbilirubinemia/jaundiceCoagulation;CoagulationfactorsBrittlebones(osteogenesisimperfecta),551tcausedby,283,284,284tfunctionsof,580,581tBroadbetadisease,228t
646INDEX/649Bronzediabetes,587inplateletactivation,606f,607inureasynthesis,245,246–247,246f,Brownadiposetissue,217,217fassecondmessenger,436–437,437t,247Brushborderenzymes,475457,463–465,463tCarbamoylphosphatesynthaseIBSE.SeeBovinespongiformencephalopathyphosphatidylinositidemetabolismdeficiencyof,247Buffersaffecting,464–465,464f,465finureasynthesis,245–246,246fHenderson-HasselbalchequationvitaminDmetabolismaffectedby,CarbamoylphosphatesynthaseII,inpyrim-describingbehaviorof,11,12f485–486idinesynthesis,296,298f,299weakacidsandtheirsaltsas,11–12,12fCalciumATPase,463,568Carbohydrates,102–110.Seealsospecific“Bulkflow,”ofmembraneproteins,507Calcium-bindingproteins,vitaminKandtypeandGlucose;SugarsBurst-formingunit-erythroid,610,611fglutamatecarboxylationandincellmembranes,110Butylatedhydroxyanisole(BHA),aspostsyntheticmodificationcellsurface,glycolipidsand,116antioxidant/foodpreservative,119and,487–488,488fclassificationof,102,102tButylatedhydroxytoluene(BHT),assynthesisand,488,604complex(glycoconjugate),glycoproteinsantioxidant/foodpreservative,119Calcium/calmodulin,463as,514Butyricacid,112tCalcium/calmodulin-sensitivephosphorylasedigestionandabsorptionof,474–475,kinase,inglycogenolysis,148475fCalciumchannels,463.SeealsoCalciuminterconvertibilityof,231C1–9(complementproteins),596releasechannelisomerismof,102–104,103fC-peptide,449,450fincardiacmuscle,566–567inlipoproteins,110C20polyunsaturatedacids,eicosanoidsCalcium-inducedcalciumrelease,incardiacmetabolismof,122–123,122f,123f,formedfrom,190,192,193f,muscle,567124–125,125f194fCalciumpump,463,568diseasesassociatedwith,102C-reactiveprotein,583,583tCalciumreleasechannelvitaminB1(thiamin)in,488–489,Cregions/segments.SeeConstantdihydropyridinereceptorand,563–564,489fregions/segments563finproteoglycans,542,543,543fCa2+ATPase,463mutationsingenefor,malignantCarbondioxideCa2+-Na+exchanger,463,567–568hyperthermiacausedby,citricacidcycleinproductionof,Cachexia,cancer,136,479564–565,565f,630t130–133,132fCaffeine,289,289fCalciumreleasechannel,563,564ftransportof,byhemoglobin,44,45fhormonalregulationoflipolysisand,215Calcium-sodiumexchanger,463CarbonmonoxideCalbindin,477Caldesmon,571hemecatabolismproducing,278Calcidiol(25-hydroxycholecalciferol),inCalmodulin,463,463t,562oxidativephosphorylation/respiratoryvitaminDmetabolism,484,485fmusclephosphorylaseand,148,149fchainaffectedby,92,95,96fCalciferol.SeeVitaminDCalmodulin-4Ca2+,insmoothmuscleCarbonskeleton,aminoacid.SeeAminoCalcineurin,566tcontraction,570–571,571facidcarbonskeletonsCalcinosis,486Calnexin,508,526Carbonicacid,pK/pKavalueof,12tCalcitonin,437Calreticulin,508,526Carbonicanhydrase,inosteopetrosis,552Calcitriol(1,25(OH)2-D3),437,439f,485Calsequestrin,563,563fCarboxybiotin,494,495fcalciumconcentrationregulatedby,485cAMP.SeeCyclicAMPγ-Carboxyglutamate,vitaminKinsynthesisstorage/secretionof,453,454tCancer/cancercells.Seealsoof,487,488fsynthesisof,445,446f,484,485fCarcinogenesis/carcinogensCarboxylterminalrepeatdomain,350Calcium,496tcyclinsand,334Carboxylaseenzymes,biotinascoenzymeabsorptionof,477glycoproteinsand,514,526,530t,531of,494–495vitaminDmetabolismand,477,484,hormone-dependent,vitaminB6Carboxypeptidases,477484–485deficiencyand,491Carboxyproteinase,procollagen,537inbloodcoagulation,599f,600,600t,membraneabnormalitiesand,432tCarcinogenesis/carcinogens,631601mucinsproducedby,520chemical,631inbone,549Cancercachexia,136,479cytochromeP450inductionand,628inextracellularfluid,416,416tCancerchemotherapyindirect,631inintracellularfluid,416,416tfolateinhibitorsin,494Carcinoid(argentaffinoma),serotoninin,ironabsorptionaffectedby,478neutropeniacausedby,610266–267inmalignanthyperthermia,564–565,syntheticnucleotideanalogsin,Carcinoidsyndrome,490565f290–291,291fCardiacdevelopmentaldefects,570metabolismof,463Cancerphototherapy,porphyrinsin,Cardiacglycosides,106vitaminDmetabolismand,484–485273Cardiacmuscle,556,566–570,568t,569tinmusclecontraction,562CAP.SeeCatabolitegeneactivatorproteinCardiolipin,115,115fincardiacmuscle,566–568Caproicacid,112tsynthesisof,197,197f,199,199fphosphorylaseactivationand,148Carbamates,hemoglobin,44Cardiomyopathies,556,569–570,569tsarcoplasmicreticulumand,563–564,CarbamoylphosphateCargoproteins/molecules,510563f,564fexcess,301inexport,503insmoothmuscle,570,571freeenergyofhydrolysisof,82tinimport,501,502f
647650/INDEXCarnitinefactorsaffectingratesof,61–63,62f,CDK-cyclininhibitor/CDKI,DNA/-deficiencyof,180,18763–64,64fchromosomeintegrityand,339infattyacidtransport,180–181,181ffreeenergychangesand,60–61CDKs.SeeCyclin-dependentproteinCarnitine-acylcarnitinetranslocase,180,initialvelocityand,64kinases181fmultiplesubstratesand,69–70,69f,cDNA,413Carnitinepalmitoyltransferase,deficiency70fsequencing,inglycoproteinanalysis,515tof,180substrateconcentrationand,64,64f,cDNAlibrary,402,413Carnitinepalmitoyltransferase-I,180,65fCDRs.SeeComplementarity-determining181fmodelsof,65–67,66f,67fregionsdeficiencyof,187transitionstatesand,61Celiacdisease,474inketogenesisregulation,186–187,187f,mechanismsof,51–52,52fCell,1188fprostheticgroups/cofactors/coenzymesinjurytoCarnitinepalmitoyltransferase-II,181,181fin,50–51,51foxygenspeciescausing,611–613,deficiencyof,187–188site-directedmutagenesisinstudyof,613tCarnosinasedeficiency,26458xenobioticscausing,631,631fCarnosine,264,265,265foxaloacetateand,130lysisof,complementin,596Carnosinuria,264ping-pong,69–70,69finmacromoleculetransport,428–431,β-Carotene,482,482t,483,483f.Seealsoprostheticgroupsin,50–51,51f429f,430fVitaminAbyproximity,51Celladhesionasantioxidant,119,482tregulationof,72–79,128f,129fibronectinin,540,541fCarotenedioxygenase,482–483,483factiveandpassiveprocessesin,72,73fglycosphingolipidsin,202Carotenoids,482–484,483f,484f.Seealsoallosteric,74,74–76,75f,128f,129integrinsin,620–621,622tVitaminAcompartmentationin,72–73selectinsin,528–529,529t,530fCarrierproteins/systems,426,426fcovalent,74,76,77–78,78fCellbiology,1fornucleotidesugars,517enzymequantityand,73–74Cell-cellinteractions,415Cartilage,543,551t,552–553,553ffeedbackinhibitionand,74–76,75f,mucinsin,520chondrodysplasiaaffecting,553–55476,129Cellcycle,Sphaseof,DNAsynthesisCatabolicpathways/catabolism,81,122.feedbackregulationand,76,129during,333–335,334f,335tSeealsoExergonicreaction;metaboliteflowand,72,73fCelldeath,201MetabolismMichaelisconstant(Km)in,72,73fCell-freesystems,vesiclesstudiedin,509Catabolitegeneactivatorprotein(cyclicphosphorylation-dephosphorylationCellfusion,595AMPregulatoryprotein),376,in,78–79,78tCell-mediatedimmunity,591378proteolysisin,76–77,77fCellmembrane.SeePlasmamembraneCataboliteregulatoryprotein,460secondmessengersin,76Cellmigration,fibronectinin,540Catalase,88–89RNAand,356Cellrecognition,glycosphingolipidsin,202asantioxidant,119,611–613,613tsequential(single)displacement,69,69fCellsap.SeeCytosolinnitrogenmetabolism,244,244fspecificityof,49,50fCellsurfacecarbohydrates,glycolipidsand,Catalysis/catalyticreactions(enzymatic).Seebystrain,52116alsoMetabolismsubstrateconcentrationaffectingrateof,Cellsurfaces,heparansulfateon,545acid-base,51–5264,64f,65fCellulose,109HIVproteasein,52,53fHillmodelof,66–67,67fCelluloseacetatezoneelectrophoresis,580,atactivesite,51,51fMichaelis-Mentenmodelof,65–66,582fBi-Bi,69–70,69f,70f66fCentralcoredisease,565,569tMichaelis-Mentenkineticsand,70,Catalyticresidues,conserved,54,55tCentralnervoussystem,glucoseas70fCatalyticsite,75.SeealsoActivesitemetabolicnecessityfor,232coenzymes/cofactorsin,50–51,51fCataracts,diabetic,172Centromere,318,319fconservationofresiduesand,54,55tCatecholamines.SeealsospecifictypeCephalin(phosphatidylethanolamine),115,covalent,52,52f,63receptorsfor,436115fchymotrypsinin,52–54,54f,63storage/secretionof,453,454tmembraneasymmetryand,420fructose-2,6-bisphosphatasein,54,55fsynthesisof,445–447,447fsynthesisof,197,197fdoubledisplacement,69–70,69fCathepsins,inacid-basecatalysis,52Ceramide,116,116f,201–202,202f,203fenzymedetectionfacilitatedby,55–56,Caveolae,422synthesisof,201–202,202f56fCaveolin-1,422Cerebrohepatorenal(Zellweger)syndrome,equilibriumconstantand,63CBG.SeeCorticosteroid-bindingglobulin188,503,503tisozymesand,54–55CBP/CBP/p300(CREB-bindingprotein),Cerebrosides,201kineticsof,63–70461,468,469,469f,471–472,Ceruloplasmin,583t,587,588activationenergyand,61,63472tdeficiencyof,589balancedequationsand,60CD11a-c/CD18,inneutrophils,621,621tdiagnosticsignificanceof,57t,587competitiveversusnoncompetitiveCD18,620–621Cervonicacid,113tinhibitionand,67–69,67f,CD49a/e/f,622tCFTR.SeeCysticfibrosistransmembrane68f,69fCD59,531regulator
648INDEX/651CFU-E.SeeColony-formingunit-erythroidinlipoprotein,205,207freconstitutionof,inDNAreplication,Chainelongation.SeealsoElongationinmembranes,417333byDNApolymerase,328fluidmosaicmodeland,422remodelingofingeneexpression,inglycosaminoglycansynthesis,543metabolismof,123–124,123f383–384intranscriptioncycle,342,342fclinicalaspectsof,227–229,228tChromatography.SeealsospecifictypeChaininitiation.SeealsoInitiationdiurnalvariationsin,220affinityintranscriptioncycle,342,342fhigh-densitylipoproteinsin,forprotein/peptidepurification,23Chaintermination.SeealsoTermination209–211,211fforrecombinantfusionproteininglycosaminoglycansynthesis,543plasmalevelsofpurification,58,59fintranscriptioncycle,342,342fatherosclerosisandcoronaryheartforprotein/peptidepurification,21–24Channeling,incitricacidcycle,130diseaseand,227Sepharose-lectincolumn,forChannelopathies,568,569tdietarychangesaffecting,227glycoproteinanalysis,515tChaperones,36–37,507–508,508tdrugtherapyaffecting,229Chromium,496tATPaseactivityof,508lifestylechangesaffecting,227–229Chromosomalintegration,324,324fATP-dependentproteinbindingto,499,normal,223Chromosomalrecombination,323–324,508synthesisof,219–220,220f,221f,222f323f,324fhistone,315acetyl-CoAin,123f,124,219–220,Chromosomaltransposition,324–325inproteinsorting,499,508t220f,221f,222fChromosomejumping,635tChaperonins,36–37carbohydratemetabolismand,123Chromosomewalking,411,411f,635tCharging,inproteinsynthesis,360,360fHMG-CoAreductaseinregulationof,Chromosomes,318–319,319f,319t,320f,Checkpointcontrols,339220,223f321fChédiak-Higashisyndrome,512tintissues,118,119fintegrityof,monitoring,339Chemicalcarcinogenesis/carcinogens,631factorsaffectingbalanceof,220–223,interphase,chromatinfibersin,316Chemiosmotictheory,92,95–97,97f224fmetaphase,317f,318,319texperimentalfindingsinsupportof,96transportof,223–224,225fpolytene,318,318frespiratorycontrolanduncouplersand,reverse,210,211f,219,224variationsin,63697Cholesterylesterhydrolase,223Chronicgranulomatousdisease,623,623fChemotacticfactors,620Cholesterylestertransferprotein,224,225fChyle,207Chemotherapy,cancerCholesterylesters,118,205,224Chylomicronremnants,206t,208,209ffolateinhibitorsin,494inlipoproteincore,205,207fliveruptakeof,208–209neutropeniacausedby,610Cholestyramineresins,forChylomicrons,125,205,206tsyntheticnucleotideanalogsin,hypercholesterolemia,229apolipoproteinsof,206,206t290–291,291fCholicacid,225metabolismof,125,126f,207–209,Chenodeoxycholicacid,225,226fCholine,114–115,115f209fChenodeoxycholylCoA,226,226fdeficiencyof,fattyliverand,212intriacylglyceroltransport,207,208f,Chimericgeneapproach,385–386,387f,inglycinesynthesis,238,239f209f388fmembraneasymmetryand,420Chymotrypsin,477Chimericmolecules,397–406,413Cholinesterase.SeeAcetylcholinesteraseconservedresiduesand,55trestrictionenzymesandDNAligaseinCholuricjaundice,282incovalentcatalysis,52–54,54fpreparationof,399–400,401fCholylCoA,inbileacidsynthesis,226,indigestion,477Chips,genearray,proteinexpressionand,226fforpolypeptidecleavage,26t28Chondrodysplasias,551t,553–554,554fChymotrypsinogen,477Chitin,109,109fChondroitinsulfates,109,109f,538,cIrepressorprotein/cIrepressorgene,Chloride543–545,544f,544t379–383,380f,381f,382finextracellularandintracellularfluid,functionsof,547CICR.SeeCalcium-inducedcalciumrelease416,416tChondronectin,551t,553Cirrhosisofliver,130,212permeabilitycoefficientof,419fChorionicgonadotropin,human(hCG),inα1-antitrypsindeficiency,590Chlorophyll,270438Cistron,375–376Cholecalciferol(vitaminD3)Christmasfactor(factorIX),599f,600,600tCitratesynthesisofinskin,445,446f,484,485fcoumarindrugsaffecting,604incitricacidcycle,130,131finvitaminDmetabolism,484,485fdeficiencyof,604inlipogenesisregulation,178Cholestaticjaundice,283ChromatidsCitratesynthase,130,132fCholesterol,117,118,119f,205,219–230nucleoproteinpackingin,318,319t,Citricacid,pK/pKavalueof,12tinbileacidsynthesis,225–227,226f320fCitricacidcycle(Krebs/tricarboxylicacidincalcitriol(1,25(OH)2-D3)synthesis,sister,318,319fcycle),83,130–135,131f,132f445,446fexchangesbetween,325,325fATPgeneratedby,131f,133,142,143tdietary,219Chromatin,314–316,315f,315tcarbondioxideliberatedby,130–133,excessof.SeeHypercholesterolemiaactivevs.inactiveregionsof,316–318,132fexcretionof,225–227,226f318fdeaminationand,133–134inhormonesynthesis,438,438–445,higherorderstructure/compactionof,gluconeogenesisand,133,134f,439t,440f316,317f153–155,154f
649652/INDEXCitricacidcycle(cont.)vitaminKin,486–488,488fsecretionof,537inmetabolism,122,122f,123f,124f,coumarinanticoagulantsaffecting,604triplehelixstructureof,38,38f,126,127f,130,133–135,134fCoagulationfactors,600t.Seealsospecific535–539,536faminoacid,122f,124ftypeunderFactortypesof,535,536tcarbohydrate,122–123,122f,123f,vitaminKinsynthesisof,486–488,488fCollision-induceddissociation,inmass133–134,134fCoatproteins,recruitmentof,509,510fspectrometry,27lipid/fattyacid,122f,123,123f,134,Coatedpits,inabsorptivepinocytosis,429f,Collision(kinetic)theory,61135f430Coloncancer.SeeColorectalcanceratsubcellularlevel,126,127fCoating,vesicle,509,510fColony-formingunit-erythroid,610,611finmitochondria,126,127fbrefeldinAaffecting,510–511Colonyhybridization,403–404.Seealsoreducingequivalentsliberatedby,Cobalamin(vitaminB12),482t,491–492,Hybridization130–133,132f492fColorectalcancer,mismatchrepairgenesin,regulationof,134–135absorptionof,491–492336respiratorychainsubstratesprovidedby,intrinsicfactorin,477,491–492Coltranslationalglycosylation,504130,131fdeficiencyof,482t,492Columnchromatography,forprotein/transaminationand,133–134,134ffunctionalfolatedeficiencyand,492,peptidepurification,21,22fvitaminsin,133494Combinatorialdiversity,592Citrulline,inureasynthesis,245,246–247,inmethylmalonicaciduria,155Compartmentation,72–73246f,247Cobalophilin,492Competitiveinhibition,noncompetitiveCitrullinemia,247Cobalt,496tinhibitiondifferentiatedfrom,CJD.SeeCreutzfeldt-JakobdiseaseinvitaminB1267–69,67f,68f,69fCl.SeeChlorideCobamide,coenzymesderivedfrom,51Complement,583t,596–597ClassBscavengerreceptorB1,210,211fCodingregions,319,321f,637ininflammation,596,621tClass(isotype)switching,594inrecombinantDNAtechnology,397,ComplementarityClassicpathway,ofcomplementactivation,398fofDNA,306,307f596Codingstrand,304recombinantDNAtechnologyand,Clathrin,429f,430inRNAsynthesis,341396–397Clathrin-coatedvesicles,510Codonusagetables,359–360ofRNA,306,309fCleavage,inproteinsequencing,25,26tCodons,358,359tComplementarity-determiningregions,CLIP,453,453faminoacidsequenceofencodedprotein591–592,594fClofibrate,229specifiedby,358ComplementaryDNA(cDNA),413Clonesnonsense,359ComplementaryDNA(cDNA)library,definitionof,413CoenzymeA,synthesisoffrompantothenic402,413libraryof,402,413acid,495,495fComplex(glycoconjugate)carbohydrates,inmonoclonalantibodyproduction,596CoenzymeQ(Q/ubiquinone),92,95fglycoproteinsas,514Cloning,400–402,401f,402t,403fCoenzymes,50Complexoligosaccharidechains,521,522fingeneisolation,635tincatalysis,50–51,51fformationof,521,524Cloningvectors,400–402,401f,402t,nucleotidederivatives,290,290tConcanavalinA(ConA),110,518t403f,414Cofactors,50Conformationaldiseases,590Closedcomplex,345inbloodcoagulation,600,600t,603CongenitaldisordersofglycosylationClottingfactors,600t.Seealsospecifictypeincatalysis,50–51,51f(CDG),530t,531underFactorincitricacidcycleregulation,134–135CongenitallongQTsyndrome,432tvitaminKinsynthesisof,486–488,488fColipase,475Congenitalnonhemolyticjaundice(typeICMP,288tCollagen,37–39,371,535–539,536tCrigler-Najjarsyndrome),283CMP-NeuAc,516t,517inbone,548t,549Conjugateacid,10CNBr.SeeCyanogenbromideincartilage,551t,552,553fConjugatebase,10CO.SeeCarbonmonoxideclassificationof,535,536tConjugationCO2.SeeCarbondioxideelastindifferentiatedfrom,539tofbilirubin,280,280f,281fCoA.SeeCoenzymeAfibrilformationby,535–539,536f,537tofxenobiotics,626,628–630Coactivators,transcription,351,351tgenesfor,535,536tConnectivetissue,535Coagulation(blood),598–608diseasescausedbymutationsin,39,boneas,549–550endothelialcellproductsin,607,607t538–539,538tkeratansulfateIin,545extrinsicpathwayof,598,599f,601chondrodysplasias,551t,553Connexin,431fibrinformationin,598–601,599fosteogenesisimperfecta,551Consensussequences,353,353ffinalcommonpathwayin,598,599,maturation/synthesisof,38–39Kozak,365601,602fascorbicacid(vitaminC)in,38,496Conservationofenergy,83intrinsicpathwayof,598,599f,600–601disordersof,38–39Conservedresidues,54,55tlaboratorytestsinevaluationof,608O-glycosidiclinkagein,518Constantregions/segments,593prostaglandinsin,190inplateletactivation,605,606f,607genefor,593proteinsinvolvedin,599–600,600t.Seeposttranslationalmodificationof,DNArearrangementand,325–326,alsoCoagulationfactors537–538,537t393,593–594
650INDEX/653immunoglobulinheavychain,591,592fCorticotropin.SeeAdrenocorticotropicbindingoftoDNA,byhelix-turn-heliximmunoglobulinlightchain,325–326,hormonemotif,389–390,389f393,591,592fCortisol,439f,440fCross-bridges,557,557–559,558f,562fConstitutiveenzymes,75bindingof,454,455,455tCross-links,covalentincollagen,537Constitutivegeneexpression,376,378synthesisof,440,441fCrossing-over,inchromosomalrecombina-Constitutiveheterochromatin,316Cossites,401tion,323–324,323f,324fConstitutivemutation,376Cosmids,401,402t,413Crouzonsyndrome,551tConstitutivesecretion,498forcloningingeneisolation,635tCRP.SeeC-reactiveprotein;CataboliteContigmap,634fCothromboplastin(factorVII),599f,600t,regulatoryprotein;CyclicAMPContractility/contraction.SeeMuscle601regulatoryproteincontractioncoumarindrugsaffecting,604Cryoprecipitates,recombinantDNAtech-CooperativebindingCotranslationalinsertion,504,505–506nologyinproductionof,604hemoglobin,42Cotransportsystems,426,426fCryptoxanthin,482Bohreffectand,44,45fCoulomb’slaw,5Crystallography,x-ray,proteinstructureHillequationdescribing,66–67,67fCoumarin,604demonstratedby,35COPIvesicles,510Coupling,81–82,81f,82fCS-PGI/II/III,inbone,548tCOPIIvesicles,510ATPin,82,84CT.SeeCalcitoninCoplanaratoms,partialdouble-bondhormonereceptor-effector,435–436CTD.SeeCarboxylterminalrepeatdomaincharacterand,19,20fCouplingdomains,onhormonereceptors,CTP,290Copper,496t435–436inphosphorylation,85ceruloplasmininbindingof,587,588CovalentbondsCY282Ymutation,inhemochromatosis,587ascofactor,588,588tbiologicmoleculesstabilizedby,6,6tCyanide,oxidativephosphorylation/enzymescontaining,588tmembranelipid-proteininteractionand,respiratorychainaffectedby,92,inMenkesdisease,58841995,96fmetallothioneinsinregulationof,588xenobioticcellinjuryand,631,631fCyanogenbromide,forpolypeptidecleav-inoxidases,86Covalentcatalysis,52,52f,63age,25,26ttestsfordisordersofmetabolismof,588,chymotrypsinin,52–54,54f,63CyclicAMP,147,148f,289,289f,290t,589tfructose-2,6-bisphosphatasein,54,55f458–462,460t,462finWilsondisease,587,588–589Covalentcross-links,collagen,537adenylylcyclaseaffecting,147,458–459,Copper-bindingP-typeATPase,mutationsCovalentmodification460tingeneformassspectrometryindetectionof,27,incardiacmuscleregulation,566Menkesdiseasescausedby,58827f,27tingluconeogenesis,158,158fWilsondiseasecausedby,588–589inproteinmaturation,37–39inglycogenmetabolismregulation,Coppertoxicosis,588.SeealsoWilsoninregulationofenzymaticcatalysis,74,147–150,148f,149f,150fdisease76,77–78,78f.Seealsoassecondmessenger,147,436,437t,CoproporphyrinogenI,271,275fPhosphorylation;Proteolysis457,458–462,460t,462fCoproporphyrinogenIII,271,275fgluconeogenesisregulationand,157smoothmusclecontractionaffectedby,Coproporphyrinogenoxidase,271,275f,irreversible,76–77,77f571276fmetaboliteflowand,79CyclicAMP-dependentproteinkinase.Seeinporphyria,277treversible,77–79,78f,78tProteinkinasesCoproporphyrins,272fCPT-I.SeeCarnitinepalmitoylCyclicAMPregulatoryprotein(catabolitespectrophotometryindetectionof,transferase-Igeneactivatorprotein),376,378273–274CRE.SeeCyclicAMPresponseelementCyclicAMPresponseelement,459t,461Coprostanol(coprosterol),225Creatine,267,268fCyclicAMPresponseelementbindingCoreproteins,542,543fCreatinekinase,diagnosticsignificanceof,protein,461inglycosaminoglycansynthesis,542–54357tCyclicGMP,289f,290Coregulatorproteins,469,471–473,472fCreatinephosphate,267,268fassecondmessenger,290,436,437t,Corepressors,472t,473freeenergyofhydrolysisof,82t457,462–463Coricycle,159,159finmuscle,573–574,574–576,575f,roleinsmoothmuscle,573fCori’sdisease,152t575tCyclic3′,5′-nucleotidephosphodiesterase,Cornea,keratansulfateIin,545,546,547Creatinephosphateshuttle,100,101finlipolysis,215Coronary(ischemic)heartdisease.SeealsoCreatinine,267,268fCyclin-dependentproteinkinases,333,AtherosclerosisCREB,461334f,335tcholesteroland,227CREB-bindingprotein,461,469,469f,471inhibitionof,DNA/chromosomeCorrinoids,491.SeealsoCobalaminCreutzfeldt-Jakobdisease,37integrityand,339Corticosteroid-bindingglobulin(CBG/Crigler-NajjarsyndromeCyclins,333–335,334f,335ttranscortin),454–455,455t,583ttypeI(congenitalnonhemolyticCycloheximide,372cyclooxygenasesaffectedby,193jaundice),283Cyclooxygenase,192CorticosteronetypeII,283as“suicideenzyme,”194bindingof,454,455tCroprotein/crogene,379–383,380f,381f,Cyclooxygenasepathway,192,192–194,synthesisof,438,440,441f382f193f,194f
651654/INDEXCYPnomenclature,forcytochromeP450Cytoskeleton/cytoskeletalproteins,556,Delta4(Δ4)(progesterone)pathway,442,isoforms,627576–578443fCYP2A6,polymorphismof,628,630tredcell,615f,616–617,616f,616tDelta5(Δ5)(dehydroepiandrosterone)CYP2C9,inwarfarin–phenobarbitalCytosolpathway,442,443finteraction,628ALAsynthesisin,270,273fDelta9(Δ9)desaturaseCYP2D6,polymorphismof,628,630tglycolysisin,126,127f,136inmonounsaturatedfattyacidsynthesis,CYP2E1,enzymeinductionand,628lipogenesisin,173–177,174f,175f191,191fCysteine,15t,265pentosephosphatepathwayreactionsin,inpolyunsaturatedfattyacidsynthesis,metabolismof,250,252f,253f163191,191fabnormalitiesof,250–255,253fpyrimidinesynthesisin,296,298fDenaturationinpyruvateformation,250,252fCytosolicbranch,forproteinsorting,498,DNAstructureanalysisand,304–305requirementsfor,480499fproteinrefoldingand,36synthesisof,238–239,239fCytosolicdynein,577temperatureand,63Cysticfibrosis,431–432,432t,474,569tCytosolicproteins,O-glycosidiclinkagesin,Deoxoynojirimycin,527,527tCysticfibrosistransmembraneregulator518Deoxyadenylate,303(CFTR),431,431f,432tCytotoxicity,xenobiotic,631,631fDeoxycholicacid,synthesisof,226Cystinereductase,250,252fDeoxycorticosteroneCystinosis(cystinestoragedisease),bindingof,454–455250–255D-aminoacids,free,14synthesisof,438,441fCystinuria(cystine-lysinuria),250Darm,oftRNA,310,312f,360,361f11-Deoxycortisol,synthesisof,440,441fCytarabine(arabinosylcytosine),290Dcyclins,333,334f,335tDeoxycytidineresidues,methylationof,Cytidine,287f,288tcancerand,334geneexpressionaffectedby,383Cytidinemonophosphate(CMP),288tDisomerism,102–104,103fDeoxycytidylate,303CytidinemonophosphateDAF.SeeDecayacceleratingfactorDeoxyguanylate,303N-acetylneuraminicaciddAMP,288fDeoxyhemoglobin,protonbindingby,44,(CMP-NeuAc),516t,517Dantrolene,formalignanthyperthermia,45fCytidinetriphosphate(CTP),290564DeoxyhemoglobinA,“stickypatch”recep-inphosphorylation,85DBD.SeeDNA-bindingdomaintoron,46Cytochromeb5,89,627DBH.SeeDopamine-β-hydroxylaseDeoxyhemoglobinS,“stickypatch”Cytochromeb558,622Deamination,124,124freceptoron,46Cytochromec,93citricacidcyclein,133–134Deoxynucleotides,303–304,304f,305fCytochromeoxidase/cytochromeaa3,86,liverin,125Deoxyribonucleases(DNase)/DNaseI,31293Debranchingenzymesactivechromatinand,316CytochromeP450-dependentmicrosomalabsenceof,152tDeoxyribonucleicacid.SeeDNAethanoloxidizingsystem,inglycogenolysis,146–147,148fDeoxyribonucleosidediphosphates212–214Debrisoquin,CYP2D6inmetabolismof,(dNDPs),reductionofNDPsto,CytochromeP450sidechaincleavageen-628294,297fzyme(P450scc),438,440f,442Decayacceleratingfactor,531Deoxyribonucleosides,286CytochromeP450system,86,89–90,90f,Decorininpyrimidinesynthesis,296626inbone,548tDeoxyribose,102,106,106fALAsynthaseaffectedby,272,278incartilage,551tDeoxysugars,106,106fenzymeinductionand,272–273,Defensins,621t3-Deoxyuridine,290627–628Degeneracy,ofgeneticcode,359Dephosphorylation.Seealsogenesencoding,nomenclaturefor,627Degradation,rateof(kdeg),74Phosphorylationisoformsof,627–628Dehydrocholesterol,invitaminDincovalentmodification,78–79,78tinmetabolismofxenobiotics,626–628,metabolism,484,485fDepolarization,innerveimpulse629tDehydroepiandrosterone(DHEA),transmission,428membraneinsertion,504synthesisof,440,441fDepurination,DNA,baseexcision-repairmitochondrial,89–90Dehydroepiandrosterone(Δ5)pathway,442,and,337nomenclaturesystemfor,627443fDermatansulfate,544f,544t,545inxenobioticcellinjury,631,631fDehydrogenases,86,87–88,88ffunctionsof,547Cytochromes,asdehydrogenases,88inenzymedetection,56,56fΔ9DesaturaseCytogeneticabnormalities,detectionof,nicotinamidecoenzyme-dependent,87,inmonounsaturatedfattyacidsynthesis,635t89f191,191fCytogeneticmap,633,634finrespiratorychain,87inpolyunsaturatedfattyacidsynthesis,Cytokines,α2-macroglobulinbindingof,riboflavin-dependent,87191,191f590Deletions,DNA,recombinantDNAtech-Desmin,566t,577tCytosine,288tnologyindetectionof,409,Desmosines,539basepairingofinDNA,303,304,305f409tDesmosterol,incholesterolsynthesis,220,545,4deoxyribonucleosidesof,inpyrimidineDelta,(Δ)isomerase,438,441f,442,222fsynthesis,296–297,298f443fDetergents,417–418
652INDEX/655Detoxification,626Dihydrobiopterin,defectinsynthesisof,255DiversitycytochromeP450systemin,89–90,90f,Dihydrobiopterinreductase,defectin,255antibody,592,593–594626–628,629tDihydrofolate/dihydrofolatereductase,combinatorial,592Dextrinosis,limit,152tmethotrexateaffecting,296–297,ingeneexpression,387,388fDextrins,109494junctional,593–594Dextrose,104Dihydrolipoyldehydrogenase,140,141fDiversitysegment,DNArearrangementDHA.SeeDocosahexaenoicacidDihydrolipoyltransacetylase,140,141fand,593–594DHEA.SeeDehydroepiandrosteroneDihydropyridinereceptor,563–564,563f,DNA,303,303–306,314–340DHPR.SeeDihydropyridinereceptor564fbaseexcision-repairof,336t,337,337fDHT.SeeDihydrotestosteroneDihydrotestosterone,442,444fbasepairingin,7,303,304,305fDiabetesmellitus,102,161–162bindingof,455tmatchingofforrenaturation,305–306fattyliverand,212Dihydroxyacetone,106frecombinantDNAtechnologyand,freefattyacidlevelsin,206Dihydroxyacetonephosphate,inglycolysis,396–397hemochromatosisand,587197,197f,198freplication/synthesisand,328–330,insulinresistanceand,6111,25-DihydroxyvitaminD3.SeeCalcitriol330fketosis/ketoacidosisin,18824,25-DihydroxyvitaminD3(24-hydroxy-bindingoftoregulatoryproteins,motifslipidtransportandstoragedisordersand,calcidiol),invitaminDfor,387–390,388t,389f,390f,205metabolism,484,485f391flipogenesisin,173Diiodotyrosine(DIT),447,448f,449blunt-ended,398,399–400,400f,413asmetabolicdisease,122,231Dilatedcardiomyopathy,570inchromatin,314–318,315f,315t,starvationand,236Dimercaprol(BAL),respiratorychain317f,318fDiabeticcataract,172affectedby,95,96fchromosomal,318–319,319f,319t,Diacylglycerol,115,475,476fDimericproteins,34320f,321fincalcium-dependentsignalDimersrelationshipoftomRNA,321ftransduction,464,465fCroprotein,380,381fcodingregionsof,319,321f,637formationof,197f,198fhistone,315complementarityof,306,307finplateletactivation,606,606flambdarepressor(cI)protein,380,381frecombinantDNAtechnologyand,inrespiratoryburst,623Dimethylallyldiphosphate,incholesterol396–397Diacylglycerolacyltransferase,198f,199synthesis,219,221fdamageto,335,335tDiagnosticenzymology,57,57tDimethylaminoadenine,289frepairof,335–339,335t,336tDicarboxylateanions,transportersystemsDinitrophenol,respiratorychainaffectedADP-ribosylationfor,490for,98–99by,95,96fdeletionsin,recombinantDNAtechnol-Dicarboxylicaciduria,188Dinucleotide,291ogyindetectionof,409,409tDicumarol(4-hydroxydicoumarin),486Dioxygenases,89depurinationof,baseexcision-repairand,Dielectricconstant,ofwater,5Dipalmitoyllecithin,115337Diet.SeealsoNutritionDipeptidases,477double-strandbreakrepairof,336t,bloodglucoseregulationand,159–161Diphosphates,nucleoside,287,287f337–338,338fcholesterollevelsaffectedby,227Diphosphatidylglycerol.SeeCardiolipindouble-stranded,304hepaticVLDLsecretionand,211–212,Diphtheriatoxin,372flankingsequence,397213fDipoles,waterforming,5,6fgeneticinformationcontainedin,high-fat,fattyliverand,212Disaccharidases,102,475303–306Diet-inducedthermogenesis,217,478Disaccharides,106–107,107f,107t.Seegroovesin,305f,306Diethylenetriaminepentaacetate(DTPA),alsospecifictypeinsertionsin,recombinantDNAaspreventiveantioxidant,119Diseasetechnologyindetectionof,409Diffusionbiochemicalbasisof,2,3tintegrityof,monitoring,339facilitated,423,423t,424f,426–427,HumanGenomeProjectand,3–4“jumping,”325427,427fmajorcausesof,3tmismatchrepairof,36f,336,336f,ofbilirubin,280Displacementreactions336tofglucose.SeealsoGlucosedouble,69–70,69fmitochondrial,322–323,322f,323ttransporterssequential(single),69,69fmutationsin,314,323–326,323f,324f,insulinaffecting,427Dissociation,ofwater,8–9325f.SeealsoMutationsinredcellmembrane,611Dissociationconstant,8–9innucleosomes,315–316,316fhormonesinregulationof,427Michaelisconstant(Km)and,66nucleotideexcision-repairof,336,337,“Ping-Pong”modelof,427,427finpHcalculation,10338fnet,423,424fofweakacids,10–11,12rearrangementsofpassive,423,423t,424fDistalhistidine(histidineE7),inoxygeninantibodydiversity,325–326,393,simple,423,423t,424fbinding,40,41f593–594Digestion,474–480Disulfidebonds,proteinfoldingand,37recombinantDNAtechnologyinDigitalisDIT.SeeDiiodotyrosinedetectionof,409,409tCa2+-Na+exchangerinactionof,568Diurnalrhythm,incholesterolsynthesis,recombinant.SeeRecombinantDNA/Na+-K+ATPaseaffectedby,428,568220recombinantDNAtechnology
653656/INDEXDNA(cont.)DNA-PK.SeeDNA-dependentproteinDopamine,446,447f.SeealsoCate-relaxedformof,306kinasecholaminesrenaturationof,basepairmatchingand,DNApolymerases,326,327–328,327f,synthesisof,267,267f,445–447,447f305–306328,328tDopamine-β-hydroxylase,447repairof,335–339,335t,336tinrecombinantDNAtechnology,400tvitaminCascoenzymefor,495–496repetitive-sequence,320–322DNAprimase,327,327f,328tDopamineβ-oxidase,267,267freplication/synthesisof,306,307f,DNAprobes,402,414Doubledisplacementreactions,69–70,69f326–339,326t,327f,328tforgeneisolation,635tDoublehelix,ofDNAstructure,7,303,DNApolymerasecomplexin,328,librarysearchedwith,402304,305f328tinporphyriadiagnosis,274recombinantDNAtechnologyand,396,DNAprimerin,328,329f,330fDNA-proteininteractions,bacteriophage397initiationof,328–330,329f,330f,lambdaasparadigmfor,Doublereciprocalplot331f378–383,379f,380f,381f,382finhibitorevaluationand,68,68f,69foriginof,326DNAsequencesKmandVmaxestimatedfrom,66,66fpolarityof,330–331amplificationofbyPCR,405–406,Double-strandbreakrepairofDNA,336t,proteinsinvolvedin,328t406f337–338,338freconstitutionofchromatinstructuredeterminationof,404,405fDouble-strandedDNA,304,314and,333ingeneisolation,635tunwindingrepairduring,335–339,335t,336tproteinsequencingand,25–26forreplication,326,326–327replicationbubbleformationand,DNAtopoisomerases,306,328t,332,RNAsynthesisand,344331–333,331f,332f,333f332fDownstreampromoterelement,346–348,replicationforkformationand,DNAtransfection,identificationof347f327–328,327fenhancers/regulatoryelementsDPE.SeeDownstreampromoterelementribonucleosidediphosphatereductionand,386DRIPs,472t,473and,294,297fDNAunwindingelement,326Drugdetoxification/interactions,inSphaseofcellcycle,333–335,DNase(deoxyribonuclease)/DNaseI,312cytochromesP450and,334f,335tactivechromatinand,31689–90,90f,628semiconservativenatureof,306,307finrecombinantDNAtechnology,400tDrugdevelopment,pharmacogeneticsand,semidiscontinuous,327f,331,331fdNDPs.SeeDeoxyribonucleoside631–632unwindingand,326,326–327diphosphatesDrugresistance,geneamplificationin,393inRNAsynthesis,341–343,342f,343tDOC.See11-DeoxycorticosteroneDS-PGI/DS-PGII,incartilage,551tstabilizationof,7Docking,innuclearimport,501,502fdsDNA.SeeDouble-strandedDNAstructureof,303–306,304f,305fDockingprotein,504DTPA,aspreventiveantioxidant,119denaturationinanalysisof,304–305Docosahexaenoicacid,191–192Dubin-Johnsonsyndrome,283double-helical,7,303,304,305fDolichol,118,119f,522,523fDuchennemusculardystrophy,556,recombinantDNAtechnologyand,incholesterolsynthesis,220,221f565–566,566f396,397inN-glycosylation,522DUE.SeeDNAunwindingelementsupercoiled,306,332,333fDolicholkinase,522Dwarfism,551t,553–554transcriptionof,306Dolichol-P-P-GlcNAc,522–523Dynamin,inabsorptivepinocytosis,430,transpositionof,325Dolichol-P-P-oligosaccharide(dolichol-577unique-sequence(nonrepetitive),320,pyrophosphate-oligosaccharide),Dyneins,577320–321521,524fDysbetalipoproteinemia,familial,228tunwindingof,326,326–327inN-glycosylation,521–524,523fDyslipoproteinemias,228t,229RNAsynthesisand,344Dolicholphosphate,522Dystrophin,556,565–566,566t,567fxenobioticcellinjuryand,631Domains.Seealsospecifictypemutationingenefor,inmuscularDNAbindingdomains,390–391,392f,470albumin,584dystrophy,565–566,566fDNAbindingmotifs,387–390,388t,389f,carboxylterminalrepeat,350390f,391fchromatin,316,318,319fDNA-dependentproteinkinase,incoupling,onhormonereceptors,435–436E0.SeeRedox(oxidation-reduction)double-strandbreakrepair,338DNAbinding,390–391,392f,470potentialDNA-dependentRNApolymerases,fibronectin,540,541fEact.SeeActivationenergy342–343,342f,343tprotein,33–34Ecoli,lactosemetabolismin,operonDNAelements,geneexpressionaffectedby,Srchomology2(SH2)hypothesisand,376–378,384–385,384f,385t,386fininsulinsignaltransmission,465,376f,377fdiversityand,387,388f466f,467EcolibacteriophageP1-based(PAC)vector,DNAfingerprinting,413inJak/STATpathway,467,467f401–402,402t,413DNAfootprinting,413trans-activation,ofregulatoryproteins,Ecyclins,333,334f,335tDNAhelicase,326–327,327f,328,328t390–391,392fE-selectin,529tDNAligase,328t,330transcription,387E(exit)site,inproteinsynthesis,38f,368inrecombinantDNAtechnology,L-Dopa,446,447fECF.SeeExtracellularfluid399–400,400t,401fDopadecarboxylase,267,267f,446,447fECM.SeeExtracellularmatrix
654INDEX/657EcoRI,398,399t,401finglycosaminoglycansynthesis,543neutrophilinteractionandEcoRII,399tinproteinsynthesis,367–370,368fintegrinsin,529t,620–621,622tEdemainRNAsynthesis,342,342f,344selectinsin,528–529,529t,530finkwashiorkor,479Elongationarrest,504Endothelium-derivedrelaxingfactor,572,plasmaproteinconcentrationand,580Elongationfactor2,inproteinsynthesis,607t.SeealsoNitricoxideinthiamindeficiency,489368,368fEnergyEdmanreaction,forpeptide/proteinElongationfactorEF1A,inproteinactivation,61,63sequencing,25,26fsynthesis,368,368fconservationof,83Edmanreagent(phenylisothiocyanate),inElongationfactors,inproteinsynthesis,free.SeeFreeenergyproteinsequencing,25,26f367,368,368finmuscle,creatinephosphateasreserveEDRF.SeeEndothelium-derivedrelaxingEmelin,539for,573–574,575ffactorEmphysema,α1-antiproteinasedeficiencynutritionalrequirementfor,478EDTA,aspreventiveantioxidant,119and,589,589f,623–624transductionofEFA.SeeEssentialfattyacidsEmulsions,amphipathiclipidsforming,membranesin,415EFs.SeeElongationfactors120f,121inmuscle,556–559EGF.SeeEpidermalgrowthfactorEncephalopathiesEnergybalance,478–479Eggwhite,uncooked,biotindeficiencyhyperbilirubinemiacausing(kernicterus),Energycapture,82–83,82f,83causedby,494282,283Energyexpenditure,478Ehlers-Danlossyndrome,538,538tmitochondrial,withlacticacidosisandEnergytransfer,82–83,82fEicosanoids,112,190,192,193f,194f,stroke(MELAS),100–101Enhanceosome,385,386f621tspongiform(priondiseases),37Enhancers/enhancerelements,348Eicosapentaenoicacid,190fWernicke’s,489ingeneexpression,384–385,384f,385teIF-4Ecomplex,inproteinsynthesis,367,Endergonicreaction,80,81tissue-specificexpressionand,385367fcouplingand,81–82,81f,82frecombinantDNAtechnologyand,397eIFs,inproteinsynthesis,365ATPin,82,84reportergenesindefinitionof,385–386,80Sinitiationcomplex,inproteinsynthesis,Endocrinesystem,434–455.Seealso387f,388f366f,367HormonesEnolase,inglycolysis,137,138fElaidicacid,112,113,113t,114fdiversityof,437–438Δ2Enoyl-CoAhydratase,181,182fElastase,indigestion,477Endocytosis,428,429–430,429fΔ3cis-Enoyl-CoAisomerase,183Elastin,539,539treceptor-mediated,429fΔ2trans-Enoyl-CoAisomerase,183Electrochemicalpotentialdifference,inEndoglycosidaseF,517Entactin,inbasallamina,540oxidativephosphorylation,96EndoglycosidaseH,517Enterohepaticcirculation,227Electroncarriers,flavincoenzymesas,490Endoglycosidases,inglycoproteinanalysis,lipidabsorptionand,475Electronmovement,inactivetransport,427515t,517,517tEnterohepaticurobilinogencycle,281Electron-transferringflavoprotein,87,181Endonucleases,312,413Enteropeptidase,477Electrontransportchainsystem,622.Seeapurinicandapyrimidinic,inbaseEnthalpy,80alsoRespiratorychainexcision-repair,337Entropy,80Electrophiles,7restriction,312,397–399,399t,400f,Enzymeinduction,630Electrophoresis414cytochromeP450and,272–273,forplasmaproteinanalysis,580inrecombinantDNAtechnology,627–628polyacrylamide,forprotein/peptide399–400,399t,400f,400t,401fingluconeogenesisregulation,155–157,purification,24,24f,25fEndopeptidases,477156tpulsed-fieldgel,forgeneisolation,635tEndoplasmicreticulum,370Enzyme-linkedimmunoassays(ELISAs),55two-dimensional,proteinexpressionand,acylglycerolsynthesisand,126,127fEnzymes,7–828coreproteinsynthesisin,543activesitesof,51,51fElectrospraydispersion,inmassfattyacidchainelongationin,177,177fassayof,55–56,56fspectrometry,27roughbranching,inglycogenbiosynthesis,145,Electrostaticbonds/interactions,7.Seealsoglycosylationin,524–525,525f147fSalt(electrostatic)bondsinproteinsorting,498,499f,500fcatalyticactivityof,49,50f.Seealsooxygenbindingrupturing,Bohreffectproteinsynthesisand,370Catalysisprotonsand,44–45,45froutesofproteininsertioninto,detectionfacilitatedby,55–56,56fELISAs.SeeEnzyme-linkedimmunoassays505–507,506fkineticsof,63–70.SeealsoKineticsElliptocytosis,hereditary,617signalhypothesisofpolyribosome(enzyme)Elongase,177,177fbindingto,503–505,504t,505fregulationof,72–79,128f,129inpolyunsaturatedfattyacidsynthesis,smooth,cytochromeP450isoformsin,RNAand,356191,191f,192f627specificityof,49,50fElongationEndoproteinase,forpolypeptidecleavage,classificationof,49–50chain26tconstitutive,75infattyacidsynthesis,177,177fEndorphins,453,453fdebranchingintranscriptioncycle,342,342fEndothelialcellsabsenceof,152tinDNAsynthesis,328inclottingandthrombosis,607,607tinglycogenolysis,146–147,148f
655658/INDEXEnzymes(cont.)Erythrocytes,609–610,610–619ironabsorptionand,478degradationof,controlof,742,3-bisphosphoglyceratepathwayin,thiamindeficiencyand,489indiseasediagnosis/prognosis,56–57,140,140fEthylenediaminetetraacetate(EDTA),as57,57t,58f,580disordersaffecting,609,610tpreventiveantioxidant,119inDNArepair,335,336t,338–339erythropoietininregulationof,609–610,Euchromatin,316hydrolysisrateaffectedby,7–8611fEukaryoticgeneexpression,383–387,irreversibleinhibition(“poisoning”)of,glucose-6-phosphatedehydrogenasedefi-391–395,392t.SeealsoGene69ciencyaffecting,613,614f,619expressionisozymesand,54–55glucoseasmetabolicnecessityfor,232chromaticremodelingin,383–384kineticsof,60–71.SeealsoKineticsglycolysisin,140,140fdiversityof,387,388f(enzyme)hemoglobinS“stickypatch”affecting,46DNAelementsaffecting,384–385,384f,mechanismsofactionof,49–59hemolysisof,pentosephosphate385t,386fmembranesinlocalizationof,415pathway/glutathioneperoxidaseDNA-proteininteractionsin,bacterio-metal-activated,50and,166,167f,169–170phagelambdaasparadigmfor,asmitochondrialcompartmentmarkers,lifespanof,609378–383,379f,380f,381f,382f92membranesof,614–617,615f,615t,locuscontrolregionsandinsulatorsin,plasma,diagnosticsignificanceof,57,57t616f,616t387quantityof,catalyticcapacityaffectedby,glucosetransporterof,611,612tprokaryoticgeneexpressioncompared73–74hemolyticanemiasand,619,620twith,391–395,392trateofdegradationof(kdeg),controlof,metabolismof,235t,610–614,612treportergenesand,385–386,387f,388f74oxidantsproducedduring,611–613,tissue-specific,385rateofsynthesisof(ks),controlof,74613tEukaryoticpromoters,intranscription,recombinantDNAtechnologyinstudyrecombinantDNAtechnologyinstudy346–349,347f,348f,349fof,58,59fof,624Eukaryotictranscriptioncomplex,regulatory,126–129,128fstructureandfunctionof,609–610350–352,351trestriction.SeeRestrictionendonucleasesErythroidALAsynthase(ALAS2),272,273Exchangetransporters,98–100,98f,99fspecificityof,49,50finporphyria,274,277tExcitation-responsecoupling,membranessubstratesaffectingconformationof,52,Erythropoiesis,609–610,611fin,41553fErythropoietin/recombinanterythropoietinExergonicreaction,80,81Enzymopathies,619(epoitinalfa/EPO),526,583t,couplingand,81–82,81f,82fEpidermalgrowthfactor(EGF),receptor609–610,611fATPin,82,84for,436D-Erythrose,104fExinuclease,inDNArepair,337,337fEpidermolysisbullosa,538,538tEscherichiacoli,lactosemetabolismin,Exit(E)site,inproteinsynthesis,368,Epimerasesoperonhypothesisand,376–378,368fingalactosemetabolism,167,170f376f,377fExocytosis,429,430–431,430finglycosaminoglycansynthesis,543EscherichiacolibacteriophageP1-basedininsulinsynthesis,430–431inpentosephosphatepathway,163,165f(PAC)vector,401–402,402t,Exocytotic(secretory)pathway,498Epimers,104,104f413Exoglycosidases,inglycoproteinanalysis,Epinephrine,439f,447,447f.SeealsoEssentialaminoacids.SeeNutritionally515t,517,517tCatecholaminesessentialaminoacidsExons,319,358,413bloodglucoseaffectedby,161Essentialfattyacids,190,190f,193interruptionsin.SeeIntronsingluconeogenesisregulation,157abnormalmetabolismof,195–196inrecombinantDNAtechnology,397,inlipogenesisregulation,178deficiencyof,191–192,194–195398fsynthesisof,267,267f,445–447,447fprostaglandinproductionand,190splicing,352–354,414Epitope(antigenicdeterminant),33,591Essentialfructosuria,163,171–172alternative,inregulationofgeneEpoxidehydrolase,631Essentialpentosuria,163,170expression,354,354f,Epoxides,631Estradiol/17β-Estradiol,439f,440f393–394,636Equilibriumconstant(Keq),62–63bindingof,455,455trecombinantDNAtechnologyand,inenzymaticcatalysis,63synthesisof,442–445,444f397,398ffreeenergychangesand,60–61Estriol,synthesisof,442,444fExonucleases,312,413ER.SeeEstrogens,receptorsforEstrogenresponseelement,459tinrecombinantDNAtechnology,400tErcalcitriol,484EstrogensExopeptidases,477ERE.SeeEstrogenresponseelementbindingof,455,455tExportins,503eRF.SeeReleasingfactorsreceptorsfor,471Expressionvector,402Ergocalciferol(vitaminD2),484synthesisof,442–445,444f,445fExtraarm,oftRNA,310,312fErgosterol,118,119fEstroneExtracellularenvironment,membranesinErythrocyteaminotransferases,invitaminbindingof,455tmaintenanceof,415–416,416tB6statusassessment,491synthesisof,442,444fExtracellularfluid(ECF),415–416,416,Erythrocytetransketolaseactivation,inEthanol416tthiaminnutritionalstatusCYP2E1inductionand,628Extracellularmatrix,535–555.Seealsospe-assessment,489fattyliverand,212–214cificcomponentandunderMatrix
656INDEX/659Extrinsicpathwayofbloodcoagulation,Farber’sdisease,203ttrans,113–114,192598,599f,601FarnesoidXreceptor,inbileacidsynthesistransportof,carnitinein,180–181,181fEye,fructoseandsorbitolin,diabeticregulation,227triacylglycerols(triglycerides)asstoragecataractand,172Farnesyldiphosphate,incholesterol/formof,114,115fpolyisoprenoidsynthesis,unesterified(free).SeeFreefattyacids219,220,221funsaturated.SeeUnsaturatedfattyacidsF0,inATPsynthesis,96,97f,98fFastacetylators,630FattyliverF1,inATPsynthesis,96,97f,98fFast(white)twitchfibers,574–576,575talcoholismand,212–214Fabregion,591,592fFattissue.SeeAdiposetissueofpregnancy,188Fabry’sdisease,203tFatalinfantilemitochondrialmyopathyandtriacylglycerolmetabolismimbalanceFacilitateddiffusion/transportsystem,423,renaldysfunction,oxidoreductaseand,212423t,424f,426–427,427,427fdeficiencycausing,100Favism,170forbilirubin,280Fatigue(muscle),136Fcfragment,591,592fforglucose.SeealsoGlucosetransportersFats,111.SeealsoLipidsreceptorsfor,inneutrophils,621tinsulinaffecting,427dietshighin,fattyliverand,212Fe.SeeIroninredcellmembrane,611metabolismof,122f,123–124,123f,Fedstate,metabolicfuelreservesand,232,hormonesinregulationof,427125–126,126f234t“Ping-Pong”modelof,427,427fFattyacid-bindingprotein,180,207Feedbackinhibition,inallostericregulation,FactorI(fibrinogen),580,600,600tFattyacidchains,elongationof,177,177f74–76,75f,76,129conversionoftofibrin,601–602Fattyacidelongasesystem,177,177fFeedbackregulationFactorII(prothrombin),600t,601inpolyunsaturatedfattyacidsynthesis,inallostericregulation,76,129coumarindrugsaffecting,487,604191,191f,192fthrombinlevelscontrolledby,602vitaminKinsynthesisof,487Fattyacidoxidase,181,182fFentonreaction,612FactorIII(tissuefactor),599f,600t,601Fattyacidsynthase,156t,173Ferriciron,278FactorIV.SeeCalciumFattyacidsynthasecomplex,173–176,inmethemoglobinemia,46FactorV(proaccelerin/labilefactor/accelera-174f,175f,179Ferrireductase,585torglobulin),600t,601,602fFattyacid-transportprotein,membrane,Ferritin,478,585,586FactorVLeiden,603–604207proteinsynthesisaffectedby,370FactorVII(proconvertin/serumFattyacids,2,111–114Ferritinreceptor,586prothrombinconversionaccelera-activationof,180–181,181fFerrochelatase(hemesynthase),271,272ftor/cothromboplastin),599f,calciumabsorptionaffectedby,477inporphyria,277t600t,601eicosanoidsformedfrom,190,192,Ferrousironcoumarindrugsaffecting,604193f,194fincorporationofintoprotoporphyrin,FactorVIII(antihemophilicfactoressential,190,190f,193271–272,272fA/globulin),599f,600,600tabnormalmetabolismof,195–196inoxygentransport,40–41deficiencyof,604deficiencyof,191–192,194–195Fertilization,glycoproteinsin,528FactorVIIIconcentrates,recombinantprostaglandinproductionand,190FeS.SeeIronsulfurproteincomplexDNAtechnologyinproductionfree.SeeFreefattyacidsFetalhemoglobin,P50of,42of,604interconvertibilityof,231Fetalwarfarinsyndrome,488FactorIX(antihemophilicfactorB/Christ-inmembranes,417,418fα-Fetoprotein,583tmasfactor/plasmathromboplas-metabolismof,123–124,123fFFA.SeeFreefattyacidstincomponent),599f,600,600tnomenclatureof,111–112,112fFGFs.SeeFibroblastgrowthfactorscoumarindrugsaffecting,604oxidationof,180–189.SeealsoFibrillin,535,539deficiencyof,604KetogenesisMarfansyndromecausedbymutationsinFactorX(Stuart-Prowerfactor),599f,600,acetyl-CoAreleaseand,123–124,genefor,539–540,540f600t123f,181–183,181f,182fFibrils,collagen,535–539,536f,537tactivationof,599f,600–601β,181–183,181f,182fFibrincoumarindrugsaffecting,604ketogenesisregulationand,dissolutionofbyplasmin,604–605,FactorXI(plasmathromboplastin186–187,187f,188f604fantecedent),599f,600,600tmodified,183,183fformationof,598–601,599fdeficiencyof,601clinicalaspectsof,187–189thrombinin,601–602,603fFactorXII(Hagemanfactor),599f,600,hypoglycemiacausedbyimpairmentinthrombi,598600tof,187–188Fibrindeposit,598FactorXIII(fibrinstabilizingfactor/inmitochondria,180–181,181fFibrinmesh,formationof,598fibrinoligase),600tphysical/physiologicpropertiesof,114Fibrinsplitproducts,ininflammation,621tFacultativeheterochromatin,316saturated,111,112,112tFibrinstabilizingfactor(factorXIII),600tFAD.SeeFlavinadeninedinucleotidesynthesisof,173–179,174f,175f.SeeFibrinogen(factorI),580,600,600tFADH2,fattyacidoxidationyielding,181alsoLipogenesisconversionoftofibrin,601–602Familialhypertrophiccardiomyopathy,carbohydratemetabolismand,123Fibrinoligase(factorXIII),600t569–570,570fcitricacidcyclein,133,134,135fFibrinolysis,604–605Fanconi’sanemia,338extramitochondrial,173FibrinopeptidesAandB,602,603f
657660/INDEXFibroblastgrowthfactorreceptor3,achon-Follicle-stimulatinghormone(FSH),437,Fructosedroplasiacausedbymutation438,439fabsorptionof,475,475fingenefor,551t,554,554fFootprinting,DNA,413indiabeticcataract,172Fibroblastgrowthfactorreceptors,chon-Forbes’disease,152tglycemicindexof,474drodysplasiascausedbymutationForensicmedicinehepaticingenefor,551t,554,554fpolymerasechainreaction(PCR)in,hyperlipidemia/hyperuricemiaand,Fibroblastgrowthfactors(FGFs),554405170–171Fibronectin,535,537–538,540,541frestrictionfragmentlengthmetabolismaffectedby,167,169fFibrousproteins,30polymorphisms(RFLPs)in,411ironabsorptionaffectedby,478collagenas,38variablenumbersoftandemlyrepeatedmetabolismof,167,169fFiglu.SeeFormiminoglutamateunits(VNTRs)in,411defectsin,171–172FinalcommonpathwayofbloodFormicacid,pK/pKavalueof,12tpyranoseandfuranoseformsof,103fcoagulation,599,601,602fFormiminoglutamate,inhistidineD-Fructose,105t,106fFingerprinting,DNA,413catabolism,250,251fFructose-1,6-bisphosphatase,156t,166FISH.SeeFluorescenceinsituhybridizationFormyl-tetrahydrofolate,493,493f,494deficiencyof,171–172Fish-eyedisease,228t43Sinitiationcomplex,inproteinsynthesis,Fructose-2,6-bisphosphatase,157,158f5′cap,mRNAmodificationand,355365,366fincovalentcatalysis,54,55fFlanking-sequenceDNA,39743Spreinitiationcomplex,inproteinFructose-1,6-bisphosphateFlavinadeninedinucleotide(FAD),86–87,synthesis,365,366fingluconeogenesis,153,154f290t,489FPA/FPB.SeeFibrinopeptidesAandBinglycolysis,137,138fincitricacidcycle,133Frameshiftmutations,363,364fFructose-2,6-bisphosphate,157–158,Flavinmononucleotide(FMN),50,86–87,ABObloodgroupand,619158f489Frameworkregions,592Fructoseintolerance,hereditary,171FlavoproteinsFreeaminoacids,absorptionof,477Fructose6-phosphateelectron-transferring,87Freeenergyfreeenergyofhydrolysisof,82tasoxidases,86–87,88fchangesin,80–81ingluconeogenesis,153,154fFlip-floprate,phospholipid,membranechemicalreactiondirectionand,inglycolysis,137,138fasymmetryand,42060–61Fructosuria,essential,163,171–172Flippases,membraneasymmetryand,420couplingand,81–82,81f,82fFSF.SeeFibrinstabilizingfactorFluidmosaicmodel,421f,422enzymesaffecting,63FSH.SeeFollicle-stimulatinghormoneFluid-phasepinocytosis,429–430,429fequilibriumstateand,60–61Fucose,inglycoproteins,516tFluidity,membrane,422redoxpotentialand,86,87tFucosidosis,532–533,533tFluorescence,ofporphyrins,273–274,transitionstatesand,61Fucosylatedoligosaccharides,selectins277fofhydrolysisofATP,82–83,82tbinding,530Fluorescenceinsituhybridization,ingeneliberationofasheat,95Fucosyltransferase/fucosyl(Fuc)transferase,mapping,406–407,407t,Freefattyacids,111,180,205,206t618635tinfattyliver,212Fumarase(fumaratehydratase),132f,133Fluoride,496tglucosemetabolismaffecting,215Fumarate,132f,133Fluoroacetate,130,132finsulinaffecting,215intyrosinecatabolism,254,255f1-Fluoro-2,4-dinitrobenzene(Sanger’sketogenesisregulationand,186–187,inureasynthesis,246f,247reagent),forpolypeptide187f,188fFumarylacetoacetate,intyrosinecatabolism,sequencing,25lipogenesisaffectedby,177–178,177f254f,2555-Fluorouracil,290,291f,297metabolismof,206–207Fumarylacetoacetatehydrolase,defectat,inFlux-generatingreaction,129starvationand,232–234,234f,234ttyrosinemia,255FMN.SeeFlavinmononucleotideFreepolyribosomes,proteinsynthesison,FunctionalgroupsFolate.SeeFolicacid498,506.SeealsoaminoacidchemicalreactionsaffectedFolatetrap,492f,494Polyribosomesby,18–20FoldingFreeradicals(reactiveoxygenspecies).Seeaminoacidpropertiesaffectedby,18polarandchargedgrouppositioningand,alsoAntioxidantsphysiologicsignificanceof,10–116hydroperoxidasesinprotectionagainst,pKof,mediumaffecting,13protein,36–37,37f88Functionalplasmaenzymes,57.Seealsoafterdenaturation,36inkwashiorkor,479EnzymesFolicacid(folate/pteroylglutamicacid),lipidperoxidationproducing,118–119,Furanoseringstructures,103f,104482t,492–494,493f120fFusionproteins,recombinant,inenzymecoenzymesderivedfrom,51inoxygentoxicity,90–91,611–613,study,58,59fdeficiencyof,250,482t,494613tFXR.SeeFarnesoidXreceptorfunctional,492,494xenobioticcellinjuryand,631,631fformsofindiet,492,493fD-Fructofuranose,103finhibitorsofmetabolismof,494Fructokinase,167,169fΔG.SeeFreeenergy0supplemental,494deficiencyof,171–172ΔG,60,61,81Folinicacid,493D-Fructopyranose,103fΔGD,61
658INDEX/661ΔGF,61Gastroenteropathy,protein-losing,582diseasecausing,recombinantDNAenzymesaffecting,63Gatedionchannels,424technologyindetectionof,G-CSF.SeeGranulocytecolony-stimulatingGaucher’sdisease,203t407–408,408f,409tfactorGDH.SeeGlutamatedehydrogenase/humangenomeinformationand,638Gprotein-coupledreceptors(GPCRs),458,L-GlutamatedehydrogenaseheterogeneousnuclearRNAprocessing459,460fGDP-Fuc,516tinregulationof,354Gproteins,459,461tGDP-Man,516t,517housekeeping,376incalcium-dependentsignalGEFs.SeeGuaninenucleotideexchangeimmunoglobulin,DNArearrangementtransduction,464f,465factorsand,325–326,393,593–594incAMP-dependentsignaltransduction,Gelelectrophoresisdouble-strandbreakrepairand,436,459,461tpolyacrylamide,forprotein/peptide337–338inJak/STATpathway,467purification,24,24f,25finducible,376inrespiratoryburst,623pulsed-field,forgeneisolation,635tknockout,412GABA.Seeγ-AminobutyrateGelfiltration,forprotein/peptideprocessed,325GAGs.SeeGlycosaminoglycanspurification,21–22,23freporter,385–386,387f,388fGal-Gal-Xyl-Sertrisaccharide,518Gemfibrozil,229targeteddisruptionof,412Gal-hydroxylysine(Hyl)linkage,518Gender,xenobiotic-metabolizingenzymesvariationsinGaltransferase,618–619,619faffectedby,630methodsforisolationof,635tGalactokinase,167,170fGene.SeeGenes;Genomenormal,recombinantDNAtechniquesinheriteddefectsin,172Genearraychips,proteinexpressionand,foridentificationof,407Galactosamine,169,171f28size/complexityand,397,399tD-Galactosamine(chondrosamine),106Geneconversion,325Geneticcode,303,358–363,359tGalactose,102,167–169,170fGenedisruption/knockout,targeted,412featuresof,358–359,360tabsorptionof,475,475fGeneexpressionL-α-aminoacidsencodedby,14,15–16tglycemicindexof,474constitutive,376,378Geneticdiseasesinglycoproteins,516tinpyrimidinenucleotidesynthesis,diagnosisofmetabolismof,167–169,170fregulationof,297–299enzymesin,57enzymedeficienciesand,172regulationof,374–395recombinantDNAtechnologyin,D-Galactose,104f,105talternativesplicingand,354,354f,407–412,408f,409t,410f,411fα-D-Galactose,104f393–394,636genetherapyfor,411Galactose1-phosphateuridyltransferase,eukaryotictranscriptionand,383–387Geneticlinkage.SeeLinkageanalysis167,170fhormonesin,458fGeneticmapping,633,634f.SeealsoGalactosemia,102,163,172negativeversuspositive,374,375t,HumanGenomeProjectGalactosidases,inglycoproteinanalysis,378,380Genetics.SeealsoHumanGenomeProject517inprokaryotesversuseukaryotes,molecular,1Galactoside,106391–395,392txenobiotic-metabolizingenzymesaffectedGalactosylceramide,116,117f,201,203fregulatoryprotein–DNAbindingby,630GalCer.SeeGalactosylceramidemotifsand,387–390,388t,Genevansystem,forfattyacidGallstones,474389f,390f,391fnomenclature,111cholesterol,219regulatoryproteinDNAbindingandGenomeGalNAc,inglycoproteins,515,516ttrans-activationdomainsand,redundancyin,320–322GalNAc-Ser(Thr)linkage390–391,392fremovalofgenefrom(targetedgeneinglycoproteins,518,519fretinoicacidin,483disruption/knockout),412inglycosaminoglycans,543temporalresponsesand,374–375,sequencing.SeealsoHumanGenomeGalNActransferase,inABOsystem,375fProject618–619,619fGenemapping,319,406–407,407t,633,approachesusedin,634,635tGamma-(γ)aminobutyrate.See634f,635tresultsof,636–637,636t,637tγ-AminobutyrateGeneproducts,diseasesassociatedwithworkingdraftof,633Gamma-(γ)carotene,482deficiencyof,407tGenomiclibrary,402,413Gamma-globulin,581fgenetherapyfor,411Genomictechnology,396.SeealsoGamma-(γ)glutamyltransferase,630Genetherapy,411RecombinantDNA/recombinantGamma-(γ)glutamyltranspeptidaseforα1-antitrypsindeficiencyemphysema,DNAtechnology(γ-glutamyltransferase),589Genomics,proteinsequencingand,28diagnosticsignificanceof,57tforureabiosynthesisdefects,248Geometricisomerism,ofunsaturatedfattyGangliosides,116Genetranscription.SeeTranscriptionacids,112–114,114faminosugarsin,106,169,171fGeneralacid/basecatalysis,52Geranyldiphosphate,incholesterolsialicacidsin,110Genessynthesis,219,221fsynthesisof,201–202,203falterationof,323–326,324f,325fGGT.Seeγ-GlutamyltransferaseGapjunctions,431amplificationof,ingeneexpressionGhosts,redcellmembraneanalysisand,614GAPs.SeeGuanineactivatingproteinsregulation,323–326,324f,Gibbsfreeenergy/Gibbsenergy.SeeFreeGastriclipase,475325fenergy
659662/INDEXGilbertsyndrome,283regulationof,155–158,156t,158fGlucose1-phosphateGK(glucokinase)gene,regulationof,355,allostericmodificationin,157freeenergyofhydrolysisof,82t355fcovalentmodificationin,157ingluconeogenesis,154f,155Gla.Seeγ-Carboxyglutamateenzymeinduction/repressionin,Glucose6-phosphateGlcCer.SeeGlucosylceramide155–157,156tfreeenergyofhydrolysisof,82tGlcNAc.SeeN-Acetylglucosaminefructose2,6-bisphosphatein,ingluconeogenesis,153,154fGlialfibrillaryacidprotein,577t157–158,158finglycogenbiosynthesis,145,146fGlibenclamide.SeeGlyburidesubstrate(futile)cyclesin,158inglycolysis,137,138fGlobin,278thermodynamicbarrierstoglycolysisGlucose-6-phosphatedehydrogenaseα-Globingene,localizationof,407tand,153–155,154fdeficiencyof,163,169–170,613,614f,β-GlobingeneGluconolactonehydrolase,163,165f619,630tlocalizationof,407tD-Glucopyranose,103finpentosephosphatepathway,156t,recombinantDNAtechnologyinαD-Glucopyranose(α-anomer),103f,104163,164f,165fdetectionofvariationsin,βD-Glucopyranose(β-anomer),103f,104Glucosetolerance,161–162,161f407–408,408f,409tGlucosamine,106f,169,171fGlucosetransporters,159,160tGlobularproteins,30inheparin,545inbloodglucoseregulation,159,160β-turnsin,32,34fGlucosan(glucan),107insulinaffecting,427β1-Globulin,581fGlucose,102,102–106redcellmembrane,611,612ttransferrinas,586absorptionof,474,475,475fGlucoside,106γ-Globulin,581fasaminosugarprecursor,169,171fGlucosuria,161Globulins,580bloodlevelsof.SeeBloodglucoseGlucosylceramide,116,201,203fGlomerularfiltration,basallaminain,540epimersof,104,104fGlucuronate/glucuronicacid,166–167,Glomerularmembrane,lamininin,540–542inextracellularandintracellularfluid,168fGlomerulonephritis,541416,416tbilirubinconjugationwith,280,280f,Glomerulus,renal,lamininin,540–542furanoseformsof,103f,104281fGlucagon,148,160–161galactoseconversionto,167–169,D-Glucuronate,105,106fingluconeogenesisregulation,157170fβ-Glucuronidases,281inlipogenesisregulation,178,178fglycemicindexof,474GlucuronidationGlucagon/insulinratio,inketogenesisinglycogenbiosynthesis,145,146fofbilirubin,280,280f,281fregulation,187inglycoproteins,516tofxenobiotics,628–629Glucagon-likepeptide,437insulinsecretionand,160,161–162Glucuronides,163Glucan(glucosan),107interconvertibilityof,231GLUT1–4.SeeGlucosetransportersGlucantransferase,inglycogenolysis,146,isomersof,102–104,103fGlutamate146f,148fasmetabolicnecessity,232carboxylationof,vitaminKascofactorGlucocorticoidreceptor-interactingproteinmetabolismof,122–123,122f,123f,for,487–488,488f(GRIP1coactivator),472,472t124–125,125f,136–140,138f,catabolismof,249,250fGlucocorticoidresponseelement(GRE),139f,140f,159,159f.Seealsoinprolinesynthesis,238,239f456,458f,459tGluconeogenesis;Glycolysissynthesisof,237,238fGlucocorticoids,437.SeealsospecifictypeATPgeneratedby,142,143ttransaminationand,243–244,243f,inaminoacidtransport,427infedstate,232244fbloodglucoseaffectedby,161freefattyacidsand,215inureabiosynthesis,243–244,243f,inlipolysis,215,216finsulinaffecting,160,161–162244fNF-κBpathwayregulatedby,468,468fbypentosephosphatepathway,123,L-Glutamatedecarboxylase,267,268freceptorsfor,471163–166,164f,165f,167fGlutamatedehydrogenase/L-glutamatesynthesisof,440,441fstarvationand,232–234,234f,234t,dehydrogenase,237,238ftransportof,454–455,455t236innitrogenmetabolism,244–245,244f,D-Glucofuranose,103fpermeabilitycoefficientof,419f245fGlucogenicaminoacids,231–232pyranoseformsof,103f,104Glutamate-α-ketoglutaratetransaminaseGlucokinase,156trenalthresholdfor,161(glutamatetransaminase),inureainbloodglucoseregulation,159–160,structureof,102,103fbiosynthesis,243–244,244f160ftransportof,159,160t,428,429f,475,Glutamate-γ-semialdehydedehydrogenase,inglycogenbiosynthesis,145,146f,156t475fblockatinhyperprolinemia,inglycolysis,137,138f,156tinsulinaffecting,427250Glucokinasegene,regulationof,355,355fD-Glucose,103f,104f,105tGlutamicacid,15tGluconeogenesis,123,125,153–162,154fα-D-Glucose,104fGlutaminase,inaminoacidnitrogenbloodglucoseregulationand,158–161,L-Glucose,103fcatabolism,245,245f159f,160fGlucose-alaninecycle,159Glutamine,15tcitricacidcyclein,133–134,134f,Glucose-6-phosphataseinaminoacidnitrogencatabolism,245,153–155,154fdeficiencyof,152t,300245fglycolysisand,136–140,138f,139f,ingluconeogenesis,156tcatabolismof,249,250f153–155,154finglycogenolysis,147synthesisof,237,238f
660INDEX/663Glutamineanalogs,purinenucleotideGlycine,15t,264glycogensynthaseandphosphorylaseinsynthesisaffectedby,293catabolismof,pyruvateformationand,regulationof,148–150,Glutaminesynthetase/synthase,237,238f,250,252f150–151,150f,151f245,245fincollagen,535Glycolipidstoragediseases,197Glutamylamidotransferase,PRPP,inhemesynthesis,264,270–273,273f,Glycolipids(glycosphingolipids),111,116,regulationof,294,295f274f,275f,276f117fγ-Glutamyltransferase,630synthesisof,238,239fABObloodgroupand,618γ-GlutamyltranspeptidaseGlycinesynthasecomplex,250aminosugarsin,169,171f(γ-glutamyltransferase),Glycinuria,250galactoseinsynthesisof,167–169,170fdiagnosticsignificanceof,57tGlycocalyx,110Glycolysis,83,122,123f,136–144,137fGlutaricacid,pK/pKavalueof,12tGlycochenodeoxycholicacid,synthesisof,aerobic,139Glutathione226fanaerobic,136,137f,139asantioxidant,611–613,613tGlycocholicacid,synthesisof,226fasmuscleATPsource,574–576,575f,inconjugationofxenobiotics,Glycoconjugate(complex)carbohydrates,575t629–630glycoproteinsas,514ATPgeneratedby,142,143tasdefensemechanism,629Glycoforms,514clinicalaspectsof,142–143functionsof,629–630Glycogen,102,107,108finerythrocytes,140,140fGlutathioneperoxidase,88,166,167f,170,incarbohydratemetabolism,123,123f,glucoseutilization/gluconeogenesisand,612,613t155136–140,138f,139f,153–155,Glutathionereductase,erythrocytecarbohydratestorageand,145,146t154f.SeealsoGluconeogenesispentosephosphatepathwayand,166,metabolismof,145–152.Seealsopathwayof,136–140,138f,139f,140f167fGlycogenesis;Glycogenolysispyruvateoxidationand,134,135f,riboflavinstatusand,490branchingin,145,147f140–142,141f,142f,143tGlutathioneS-transferases,629clinicalaspectsof,151–152,152fregulationof,140inenzymestudy,58,59fregulationofenzymesin,156tGlyburide(glibenclamide),188cyclicAMPin,147–150,148f,fructose2,6-bisphosphatein,Glycanintermediates,formationofduring149f,150f157–158,158fN-glycosylation,526glycogensynthaseandgluconeogenesisand,140,155–158,Glycemicindex,474phosphorylasein,150–151,151f156t,158fGlyceraldehyde(glycerose),DandLisomersinstarvation,234atsubcellularlevel,126,127fof,103f,104fmuscle,145,146t,573,575fthermodynamicbarrierstoreversalof,Glyceraldehyde3-phosphateGlycogenphosphorylase,145–146,146f153–155inglycolysis,137,138fpyridoxalphosphateascofactorfor,Glycolyticenzymes,inmuscle,556oxidationof,137,139f491Glycomics,533Glyceraldehyde3-phosphatedehydrogenaseregulationof,148–150,150–151,150f,Glycophorins,110,518,615–616,615f,inglycolysis,137,138f151f616f,616tinredcellmembranes,615f,616tGlycogenstoragediseases,102,145,GlycoproteinIIb-IIIa,inplateletactivation,Glycerol,114151–152,152t607,622tinlacticacidcycle,159Glycogensynthase,inglycogenmetabolism,Glycoproteinglycosyltransferases,520permeabilitycoefficientof,419f145,146f,155,156tGlycoproteins,30,109–110,109t,439f,synthesisof,155regulationof,148–150,150–151,150f,514–534,580,581–582.SeealsoGlyceroletherphospholipids,synthesisof,151fspecifictypeandPlasmaproteins199,200fGlycogensynthasea,148–150,150faminosugarsin,106,169,171fGlycerolkinase,155,197,198f,214Glycogensynthaseb,150,150fasialoglycoproteinreceptorinclearanceGlycerolmoiety,oftriacylglycerols,123Glycogenesis,124–125,145,146fof,517Glycerolphosphatepathway,198fregulationofasbloodgroupsubstances,514,618Glycerol-3-phosphatecyclicAMPin,147–150,148f,149f,carbohydratesin,109tacylglycerolbiosynthesisand,197,197f,150fclassesof,518,519f198fenzymesin,156tcomplex,521,522ffreeenergyofhydrolysisof,82tglycogensynthaseandphosphorylaseformationof,521,524triacylglycerolesterificationand,in,148–150,150–151,150f,diseasesassociatedwithabnormalitiesof,214–215,214f151f530,530t,531f,531t,532f,533tGlycerol-3-phosphateacyltransferase,198f,Glycogenin,145,146fextracellular,absorptivepinocytosisof,199Glycogenolysis,125,145–147,146f430Glycerol-3-phosphatedehydrogenase,155,bloodglucoseregulationand,158–161,infertilization,528198f,199159f,160ffunctionsof,514,515t,528–533,529tmitochondrial,87cyclicAMP-independent,148galactoseinsynthesisof,167–169,170fGlycerophosphateshuttle,99,100fcyclicAMPinregulationof,147–150,glycosylphosphatidylinositol-anchored,Glycerophospholipids,111148f,149f,150f518,519f,527–528,528tGlycerose(glyceraldehyde),DandLisomersdebranchingenzymesin,146–147,high-mannose,521,522fof,103f,104f148fformationof,521,524
661664/INDEXGlycoproteins(cont.)Glycosylphosphatidylinositol-anchoredGTP-γ-S,vesiclecoatingand,510hybrid,521,522f(GPI-anchored/GPI-linked)Guanine,288tformationof,521glycoproteins,518,519f,Guanineactivatingproteins,501,502fimmunoglobulinsas,593527–528,528tGuaninenucleotideexchangefactors,501,membraneasymmetryand,420inparoxysmalnocturnalhemoglobinuria,502fN-linked,518,519f,521–527531,531fGuanosine,287f,288tnucleotidesugars,516–517,516tGlycosyltransferases,glycoprotein,520,basepairingofinDNA,303,304,305fO-linked,518,518–520,519f,520f,526–527inuricacidformation,299,299f520t,521tGlypiation,528Guanosinediphosphatefucose(GDP-Fuc),oligosaccharidechainsof,514GM-CSF.SeeGranulocyte-macrophage516tredcellmembrane,615,615fcolony-stimulatingfactorGuanosinediphosphatemannosesugarsin,515–517,516tGM1ganglioside,116,117f(GDP-Man),516t,517techniquesforstudyof,514,515tGM3ganglioside,116Guanosinemonophosphate.SeeGMPasialoglycoproteinreceptorin,517GMP,288t,297fGuanylylcyclase,462,463glycosidasesin,517,517tcyclic,289f,290Gyrase,bacterial,306lectinsin,517–518,518tassecondmessenger,290,436,437t,Gyrateatrophyofretina,250inzonapellucida,528457,462–463Glycosaminoglycans,109,109f,542,IMPconversionto,293,296f542–547.Seealsospecifictypefeedback-regulationof,294,296fHbands,556,557f,558faminosugarsin,106PRPPglutamylamidotransferaseHbloodgroupsubstance,618,619fdeficienciesofenzymesindegradationof,regulatedby,294Hchains.SeeHeavychains545–547,546t,547fGolgiapparatusH1histones,314,315fdiseaseassociationsof,548–549coreproteinsynthesisin,543H2Ahistones,314,315distributionsof,543–545,544f,544tglycosylationand,509,524–525,525fH2Bhistones,314,315functionsof,547–549,548tinmembranesynthesis,509H3histones,314,314–315structuraldifferencesamong,543–545,inproteinsorting,498,500f,509H4histones,314,314–315544f,544t,545finVLDLformation.213fH2S.SeeHydrogensulfidesynthesisof,542–543retrogradetransportfrom,507Hsubstance,ABObloodgroupand,618Glycosidases,inglycoproteinanalysis,517Gout/goutyarthritis,299Haber-Weissreaction,612Glycosides,105–106GPIIb-IIIa,inplateletactivation,607,622tHagemanfactor(factorXII),599f,600,β-N-Glycosidicbond,286,287fGPCRs.SeeGprotein-coupledreceptors600tGlycosphingolipids(glycolipids),111,116,GPI-anchored/linkedglycoproteins,518,Hairpin,306,309f,413117f,201–202,203f519f,527–528,528tHalflifeABObloodgroupand,618inparoxysmalnocturnalhemoglobinuria,enzyme,242aminosugarsin,169,171f531,531fprotein,242galactoseinsynthesisof,167–169,170fGranulocyte-colonystimulatingfactor,610plasmaprotein,582inmembranes,417Granulocyte-macrophagecolony-Halt-transfersignal,506membraneasymmetryand,420stimulatingfactor,610Hapten,inxenobioticcellinjury,631,Glycosuria,161Granulomatousdisease,chronic,623,623f631fN-Glycosylases,inbaseexcision-repair,337,Granulosacells,hormonesproducedby,Haptoglobin,583t,584,584f337f442Hartnupdisease,258,490Glycosylatedhemoglobin(HbA1c),47Gratuitousinducers,378HATactivity.SeeHistoneacetyltransferaseGlycosylationGRE.SeeGlucocorticoidresponseelementactivityofcollagen,537GRIP1coactivator,472,472tHbA(hemoglobinA),P50of,42congenitaldisordersof,531,531tGrouptransferpotential,83,83fHbA1c(glycosylatedhemoglobin),47incovalentmodification,massincreasesofnucleosidetriphosphates,289–290,HbF(fetalhemoglobin),P50of,42and,27t289f,290f,290tHbM(hemoglobinM),46,363,614inhibitorsof,527,527tGrouptransferreactions,8HbS(hemoglobinS),46,46f,363nucleotidesugarsin,516–517,516tGrowthfactors,hematopoietic,610hCG.SeeHumanchorionicgonadotropinN-Glycosylation,521–527,523f,524f,Growthhormone,437,438HDL.SeeHigh-densitylipoproteins525f,526taminoacidtransportaffectedby,427Health,normalbiochemicalprocessesasdolichol-P-P-oligosaccharidein,localizationofgenefor,407tbasisof,2–4,3t521–524,523freceptorfor,436Heartinendoplasmicreticulum,524–525,GSH.SeeGlutathionedevelopmentaldefectsof,570525fGSLs.SeeGlycosphingolipidsmetabolismin,235tglycanintermediatesformedduring,526GST(glutathioneS-transferase)tag,inthiamindeficiencyaffecting,489inGolgiapparatus,524–525,525fenzymestudy,58,59fHeartdisease,coronary(ischemic).Seealsoinhibitionof,527,527tGTP,290Atherosclerosisregulationof,526–527,527tcyclicGMPformedfrom,462cholesteroland,227tunicamycinaffecting,527,527tinphosphorylation,85Heartfailure,556O-Glycosylation,520,521fGTPases,459inthiamindeficiency,489
662
663666/INDEXHexosemonophosphateshunt.SeePentoseHistidineF8receptorsfor,435–436,436f,471phosphatepathwayinoxygenbinding,40,41fproteinsas,436Hexoses,102,102treplacementofinhemoglobinM,46recognitionandcouplingdomainson,inglycoproteins,109tHistidinemia,250435–436metabolismof,163–166,164f,165f,Histoneacetyltransferaseactivity,ofspecificity/selectivityof,435,436f167f.SeealsoPentosephosphatecoactivators,383,472,473signaltransductionand,456–473pathwayHistonechaperones,315intracellularmessengersand,clinicalaspectsof,169–172Histonedimer,315457–468,461t,463tphysiologicimportanceof,105,105tHistoneoctamer,315,315fresponsetostimulusand,456,457fHFEmutations,inhemochromatosis,Histonetetramer,314–315,315signalgenerationand,456–457,458f,586–587Histones,314,314–315,315f,315t459f,459tHGP.SeeHumanGenomeProjectacetylationanddeacetylationof,genetranscriptionmodulationand,HhaI,399texpressionaffectedby,383468–473,470f,471f,472tHierarchicalshotgunsequencing,634HIV-I,glycoproteinsinattachmentof,533stimulusrecognitionby,456,457fHighaltitude,adaptationto,46HIVprotease,inacid-basecatalysis,52,53fstorage/secretionof,453,454tHigh-densitylipoproteins,205,206tHMG-CoA.See3-Hydroxy-3-methyl-synthesisofapolipoproteinsof,205–206,206tglutaryl-CoAchemicaldiversityof,438,439fatherosclerosisand,210–211,227HMM.SeeHeavymeromyosincholesterolin,438,438–445,439t,metabolismof,209–211,211fhMSH1/hMSH2,incoloncancer,336440fratiooftolow-densitylipoproteins,227HNCC.SeeHereditarynonpolyposiscolonpeptideprecursorsand,449–453receptorfor,210,211fcancerspecializationof,437High-densitymicroarraytechnology,412hnRNA.SeeHeterogeneousnuclearRNAtyrosinein,438,439–449,439tHigh-energyphosphates,83.SeealsoATPHolocarboxylasesynthetase,biotinastargetcellsfor,434–435,435tinenergycaptureandtransfer,82–83,coenzymeof,494transportof,454–455,454t,455t82f,82t,83fHomeostasisvitaminDas,484–486as“energycurrency”ofcell,83–85,84f,bloodinmaintenanceof,580Housekeepinggenes,37685fhormonesignaltransductioninHp.SeeHaptoglobinsymboldesignating,83regulationof,456,457fHpaI,399ttransportof,creatinephosphateshuttleHomocarnosine,264,265fHPETE.SeeHydroperoxidesin,100,101fHomocarnosinosis,264HPLC.SeeHigh-performanceliquidHigh-mannoseoligosaccharides,521,Homocysteinechromatography522fincysteineandhomoserinesynthesis,HREs.SeeHormoneresponseelementsformationof,521,524238–239,239fHRPT.SeeHypoxanthine-guanineHigh-molecular-weightkininogen,599f,600functionalfolatedeficiencyand,494phosphoribosyltransferaseHigh-performanceliquidchromatography,Homocystinurias,250hsp60/hsp70,aschaperones,36–37reversedphase,forprotein/vitaminB12deficiency/functionalfolate5HT(5-hydroxytryptamine).SeeSerotoninpeptidepurification,23–24deficiencyand,492f,494Humanchorionicgonadotropin(hCG),438Hillcoefficient,67Homodimers,34HumanGenomeProject,3–4,633–641Hillequation,66–67,67fHomogentisate,intyrosinecatabolism,approachesusedinelucidationof,634,Hinderedenvironment,forhemeiron,41,254f,255635t41fHomogentisatedioxygenase/oxidase,89futureworkand,637HindIII,399tdeficiencyof,inalkaptonuria,255goalsof,633–635HingeregionHomologyimplicationsof,637–638immunoglobulin,591,592fconservedresiduesand,54,55tmajorfindingsof,636–637,636t,637tnuclearreceptorprotein,460inproteinclassification,30proteinsequencingand,28Hippuricacid/hippurate,synthesisof,264,Homopolymertailing,399Humanimmunodeficiencyvirus(HIV-I),265fHomoserine,synthesisof,239,239fglycoproteinsinattachmentof,Histamine,621tHormone-dependentcancer,vitaminB6533formationof,265deficiencyand,491Huntersyndrome,546tHistidase,impaired,250Hormoneresponseelements,349,386,Hurlersyndrome,546tHistidine,16t,265,265f388f,456–457,459t,469,470fHurler-Scheiesyndrome,546tβ-alanyldipeptidesand,264,265fHormone-sensitivelipase,214–215,214fHyaluronicacid,109,109f,543,544f,544tcatabolismof,250,251finsulinaffecting,215diseaseassociationsand,548conservedresiduesand,55tHormones.Seealsospecifictypefunctionsof,547decarboxylationof,265inbloodglucoseregulation,159Hyaluronidase,547inoxygenbinding,40,41fclassificationof,436–437,437tHybridglycoproteins,521,522frequirementsfor,480facilitateddiffusionregulatedby,427formationof,521Histidine57,incovalentcatalysis,53–54,glycoproteinsas,514Hybridmapping,radiation,635t54flipidmetabolismregulatedby,215–217,Hybridization,397,403–404,413HistidineE7,inoxygenbinding,216finsitu,ingenemapping,406–407,407t,40,41finmetaboliccontrol,128f,129635t
664INDEX/667Hybridomas,595–596,596f17α-Hydroxylase,insteroidsynthesis,440,Hyperglycemia.SeealsoDiabetesmellitusHydrocortisone.SeeCortisol441f,442,443fglucagoncausing,161Hydrogenbonds,5,6f18-Hydroxylase,insteroidsynthesis,440,insulinreleaseinresponseto,466finDNA,303,304,305f441fHyperhomocysteinemia,folicacidsupple-Hydrogenionconcentration.SeealsopH21-Hydroxylase,insteroidsynthesis,440,mentsinpreventionof,494enzyme-catalyzedreactionrateaffected441fHyperhydroxyprolinemia,255by,64,64f27-Hydroxylase,sterol,226Hyperkalemicperiodicparalysis,569tHydrogenperoxideHydroxylasecycle,89,90fHyperlacticacidemia,212glutathioneindecompositionof,629Hydroxylases,89–90Hyperlipidemia,170–171,490ashydroperoxidasesubstrate,88–89insteroidsynthesis,438,440,441fHyperlipoproteinemias,205,228t,229productionofinrespiratoryburst,622Hydroxylationfamilial,228tHydrogensulfide,respiratorychainaffectedincollagenprocessing,537Hyperlysinemia,periodic,258by,95,96fincovalentmodification,massincreasesHypermetabolism,136,479Hydrolases,50and,27tHyperornithinemia-hyperammonemiacholesterylester,223ofxenobiotics,626,626–628,629tsyndrome,250fumarylacetoacetate,defectat,inHydroxylysine,synthesisof,240Hyperoxaluria,primary,250tyrosinemia,2555-Hydroxymethylcytosine,287,289fHyperparathyroidism,boneandcartilagegluconolactone,163,165f3-Hydroxy-3-methylglutaryl-CoAaffectedin,551tlysosomal,deficienciesof,532–533,533t(HMG-CoA)Hyperphenylalaninemias,255Hydrolysis(hydrolyticreactions),7–8.Seeinketogenesis,184–185,185fHyperprolinemias,typesIandII,249–250alsospecificreactioninmevalonatesynthesis,219,220fHypersensitivesites,chromatin,316freeenergyof,82–83,82t3-Hydroxy-3-methylglutaryl-CoAHypersplenism,inhemolyticanemia,619inglycogenolysis,146,146f,148f(HMG-CoA)lyaseHypertension,hyperhomocysteinemiaand,oftriacylglycerols,197deficiencyof,188folicacidsupplementsinHydropathyplot,419inketogenesis,185,185fpreventionof,494Hydroperoxidases,86,88–893-Hydroxy-3-methylglutaryl-CoAHyperthermia,malignant,556,564–565,Hydroperoxides,formationof,194,195f(HMG-CoA)reductase565f,569tHydrophiliccompounds,hydroxylationcholesterolsynthesiscontrolledby,220,Hypertriacylglycerolemiaproducing,627223findiabetesmellitus,205Hydrophilicportionoflipidmolecule,119,inmevalonatesynthesis,219,220ffamilial,228t120f3-Hydroxy-3-methylglutaryl-CoAHypertrophiccardiomyopathy,familial,Hydrophobiceffect,inlipidbilayerself-(HMG-CoA)synthase569–570,570fassembly,418inketogenesis,185,185fHyperuricemia,170–171,300Hydrophobicinteractionchromatography,inmevalonatesynthesis,219,220fHypervariableregions,591–592,594fforprotein/peptidepurification,p-Hydroxyphenylpyruvate,intyrosineHypoglycemia,15323catabolism,254f,255fattyacidoxidationand,180,187–188Hydrophobicinteractions,6–717-Hydroxypregnenolone,440,441ffructose-induced,171–172Hydrophobicportionoflipidmolecule,17-Hydroxyprogesterone,440,441finsulinexcesscausing,162119,120fHydroxyprolineduringpregnancyandinneonate,161Hydrostaticpressure,580synthesisof,240,240f,535–537Hypoglycin,180,188L(+)-3-Hydroxyacyl-CoAdehydrogenase,tropoelastinhydroxylationand,539Hypokalemicperiodicparalysis,569t181,182f4-Hydroxyproline,catabolismof,253f,Hypolipidemicdrugs,2293-Hydroxyanthranilatedioxygenase/255Hypolipoproteinemia,205,228t,229oxygenase,894-Hydroxyprolinedehydrogenase,defectin,Hypouricemia,300Hydroxyapatite,549inhyperhydroxyprolinemia,Hypoxanthine,289D(–)-3-Hydroxybutyrate(β-hydroxy-255Hypoxanthine-guaninephosphoribosylbutyrate),183–184,184f15-Hydroxyprostaglandindehydrogenase,transferase(HRPT)D(–)-3-Hydroxybutyratedehydrogenase,194defectofinLesch-Nyhansyndrome,300184,184f3β-Hydroxysteroiddehydrogenase,438,localizationofgenefor,407t24-Hydroxycalcidiol(24,25-dihydroxyvita-441f,442,443fHypoxia,lactateproductionand,136,137f,minD3),invitaminD17β-Hydroxysteroiddehydrogenase,442,139–140metabolism,484,485f443f25-Hydroxycholecalciferol(calcidiol),in5-Hydroxytryptamine.SeeSerotoninvitaminDmetabolism,484,485fHyperalphalipoproteinemia,familial,228tI.SeeIodine/iodide4-Hydroxydicoumarin(dicumarol),486Hyperammonemia,types1and2,247Ibands,556,557f,558fHydroxylamine,forpolypeptidecleavage,Hyperargininemia,248I-celldisease,431,432t,512t,524,530t,26tHyperbilirubinemia,281–284,284t531–532,532t,546–547,546t7α-Hydroxylase,bileacidsynthesisHypercholesterolemia,205Ibuprofen,cyclooxygenasesaffectedby,193regulatedby,226,226f,227familial,1,228t,432tICAM-1,529,529t11β-Hydroxylase,insteroidsynthesis,440,LDLreceptordeficiencyin,209,432tICAM-2,529,529t441fHyperchromicityofdenaturation,304–305ICF.SeeIntracellularfluid
665668/INDEXIcterus(jaundice),270,281–284,284tingluconeogenesisregulation,Insert/insertions,DNA,413IDDM.SeeInsulin-dependentdiabetes155–157recombinantDNAtechnologyinmellitusgratuitous,378detectionof,409Idiotypes,594inregulationofgeneexpression,376Inside-outsideasymmetry,membrane,IDL.SeeIntermediate-densitylipoproteinsInduciblegene,376419–420L-Iduronate,105,106fInfantileRefsumdisease,188,503,503tInsulators,387IEF.SeeIsoelectricfocusingInfectionnonpolarlipidsas,111IgA,591,594t,595fglycoproteinhydrolasedeficienciesand,Insulin,107–109,438,449,450fIgD,591,594t533adiposetissuemetabolismaffectedby,IgE,591,594tneutrophilsin,620,621t216–217IGF-I.SeeInsulin-likegrowthfactor-Iproteinlossand,480inbloodglucoseregulation,160–162IgG,591,592f,594trespiratoryburstin,622–623deficiencyof,161.SeealsoDiabetesdeficiencyof,595Inflammation,190mellitushypervariableregionsof,591–592,acutephaseproteinsin,583,583tfreefattyacidsaffectedby,215594fcomplementin,596genefor,localizationof,407tIgM,591,594t,595fneutrophilsin,620,621tglucagonopposingactionsof,160–161Immuneresponse,class/isotypeswitchingintegrinsand,529t,620–621,622tinglucosetransport,427and,594selectinsand,528–529,529t,530finglycolysis,137,155–157Immunoglobulingenes,593NF-κBin,468initiationofproteinsynthesisaffectedby,DNArearrangementand,325–326,393,prostaglandinsin,190367,367f593–594selectinsin,528–530,529f,529t,530finlipogenesisregulation,178–179double-strandbreakrepairand,Influenzavirusinlipolysisregulation,178–179,215,337–338hemagglutininin,calnexinbindingto,216fImmunoglobulinheavychainbinding526phosphorylasebaffectedby,148protein,508neuraminidasein,533receptorfor,436,465,466fImmunoglobulinheavychains,591,592fInformationpathway,457,459fsignaltransmissionby,465–467,466fgenesproducing,593Inhibitionstorage/secretionof,453,454tImmunoglobulinlightchains,591,592fcompetitiveversusnoncompetitive,synthesisof,449,450finamyloidosis,59067–69,67f,68f,69fInsulin-dependentdiabetesmellitusgenesproducing,593feedback,inallostericregulation,74–76,(IDDM/type1),161–162.SeeDNArearrangementand,325–326,75falsoDiabetesmellitus393,593–594irreversible,69Insulin/glucagonratio,inketogenesisImmunoglobulins,583t,591–597,593t.Inhibitor-1,148,149f,151,151fregulation,187SeealsospecifictypeunderIgInitialvelocity,64Insulin-likegrowthfactorI,receptorfor,436classswitchingand,594inhibitorsaffecting,68,68f,69fInsulinresistance,611classesof,591,593t,594tInitiationIntegralproteins,30,420,421fdiseasescausedbyover-andinDNAsynthesis,328–330,329f,330f,asreceptors,431underproductionof,594–595331fredcellmembrane,615–616,615f,616f,functionsof,593,594tinproteinsynthesis,365–367,366f616tgenesfor.SeeImmunoglobulingenesinRNAsynthesis,342,342f,343–344Integration,chromosomal,324,324fhybridomasassourcesof,595–596,596fInitiationcomplexes,inproteinsynthesis,Integrins,neutrophilinteractionsand,529t,structureof,591,592,592f,594f,595f365,366f,367620–621,622tIMP(inosinemonophosphate)Initiatorsequence,346–348,347fIntercellularjunctions,431conversionoftoAMPandGMP,293,Innermitochondrialmembrane,92,93fIntermediate-densitylipoproteins,206t296fimpermeabilityof,exchangetransportersIntermediatefilaments,577–578,577tfeedbackregulationof,294,296fand,98–100,98f,99fIntermembranespace,proteinsin,501synthesisof,293–294,295f,296f,297fproteininsertionin,501Intermittentbranched-chainketonuria,259Importins,501,502fInorganicpyrophosphatase,infattyacidInternalpresequences,501Insituhybridization/fluorescenceinsituactivation,85Internalribosomalentrysite,371,371fhybridization,ingenemapping,Inosinemonophosphate(IMP)Interphasechromosomes,chromatinfibers406–407,407t,635tconversionoftoAMPandGMP,293,in,316Inactivechromatin,316–318,383296fInterveningsequences.SeeIntronsInbornerrorsofmetabolism,1,249,545feedback-regulationof,294,296fIntestinalbacteria,inbilirubinconjugation,Inclusioncell(I-cell)disease,431,432t,synthesisof,293–294,295f,296f,297f281512t,524,530t,531–532,532tInositolhexaphosphate(phyticacid),calciumIntracellularenvironment,membranesinIndole,permeabilitycoefficientof,419fabsorptionaffectedby,477maintenanceof,415–416,416tIndomethacin,cyclooxygenasesaffectedby,Inositoltrisphosphate,464–465,464f,465fIntracellularfluid(ICF),415,416,416t193inplateletactivation,606,606f,607Intracellularmembranes,415Inducedfitmodel,52,53finrespiratoryburst,623Intracellularmessengers,457–468,461t,InducersInotropiceffects,566463t.Seealsospecifictypeandenzymesynthesisaffectedby,74Inr.SeeInitiatorsequenceSecondmessengers
666INDEX/669Intracellularsignals,457–458metabolismof,585,585fJchain,595fIntracellulartraffic,498–513.Seealsodisordersof,586,587tJackson-Weisssyndrome,551tProteinsortingnonheme,92,95f,585JAKkinases,436,467,467fdisordersduetomutationsingenestransferrinintransportof,584–586,Jak-STATpathway,436,467,467fencoding,512t,513585f,585tJamaicanvomitingsickness,188Intrinsicfactor,477,491–492Iron-bindingcapacity,total,586Jaundice(icterus),270,281–284,284tinperniciousanemia,492Irondeficiency/irondeficiencyanemia,478,Joiningregion,genefor,593Intrinsicpathwayofbloodcoagulation,497,586DNArearrangementand,393,593–594598,599f,600–601Ironporphyrins,270“JumpingDNA,”325Introns(interveningsequences),319,Ironregulatoryprotein,585Junctionaldiversity,593–594352–354,353f,358,413Ironresponseelements,586Juxtaglomerularcells,inrenin-angiotensininrecombinantDNAtechnology,397,Iron-sulfurproteincomplex,92,95fsystem,451398fIrreversiblecovalentmodifications,76–77,removaloffromprimarytranscript,77f352–354,353fIrreversibleinhibition,enzyme,69K.SeeDissociationconstantInulin,glomerularmembranepermeabilityIRS1–4,ininsulinsignaltransmission,K.SeePotassiumto,540465,466fk.SeeRateconstant“Invertsugar,”107Ischemia,136,431Kd.SeeDissociationconstantIodine/iodide,496tIsletsofLangerhans,insulinproducedby,kdeg.SeeRateofdegradationdeficiencyof,447–449160Keq.SeeEquilibriumconstantinthyroidhormonesynthesis,447,448f,Isocitratedehydrogenase,130–131,132fKm.SeeMichaelisconstant449inNADPHproduction,176,176fks.SeeRateofsynthesis5-Iodo-2′-deoxyuridine,291fIsoelectricfocusing,forprotein/peptideKw.SeeIonproductIodopsin,483purification,24,25fKappa(κ)chains,591o-Iodosobenzene,forpolypeptidecleavage,IsoelectricpH(pI),aminoacidnetchargeKartagenersyndrome,57726tand,17Karyotype,320fIodothyronylresidues,447.SeealsoIsoenzymes.SeeIsozymesKayser-Fleischerring,588Thyroxine;TriiodothyronineIsoleucine,15tKDEL-containingproteins,506–507,5-Iodouracil,290catabolismof,259,260f,261f508tIonchannels,415,423–424,425f,426t,interconversionof,240Keratansulfates,544f,544t,545568trequirementsfor,480functionsof,547incardiacmuscle,566–567,568,568tΔ5,4-Isomerase,438,441f,442,443fKeratins,577t,578diseasesassociatedwithdisordersof,568,Isomerases,50Kernicterus,282,283569tinsteroidsynthesis,438,441f,442,α-Ketoaciddecarboxylase,defect/absenceIonexchangechromatography,forprotein/443fof,inmaplesyrupurinediseasepeptidepurification,22–23Isomerism(branched-chainketonuria),259Ionproduct,8–9geometric,ofunsaturatedfattyacids,α-Ketoaciddehydrogenase,Iontransport,inmitochondria,99112–114,114fbranched-chain,259Ionizingradiation,nucleotideexcision-ofsteroids,117,118fthiamindiphosphateascoenzymefor,repairofDNAdamagecausedofsugars,102–104,103f488–489by,337Isoniazid,acetylationof,630α-KetoacidsIonophores,99,424Isopentenyldiphosphate,incholesterolaminoacidsindietreplacedby,240IP3.SeeInositoltrisphosphatesynthesis,219,221foxidativecarboxylationof,259,260f,IPTG.SeeIsopropylthiogalactosideIsopreneunits,polyprenoidssynthesized261f,262fIREG1.SeeIronregulatoryproteinfrom,118,119fKetoacidosis,180,188–189IRES.SeeInternalribosomalentrysiteIsoprenoids,synthesisof,incholesterol3-Ketoacyl-CoAthiolasedeficiency,188Iron,496tsynthesis,219,221f,222f3-Ketoacylsynthase,173,175fabsorptionof,478,584–586,585f,Isopropylthiogalactoside,378Ketogenesis,125–126,126f,183–187585tIsoprostanes(prostanoids),112,119highratesoffattyacidoxidationand,inhemochromatosis,478cyclooxygenasepathwayinsynthesisof,183–186,184fvitaminCandethanolaffecting,478,192,192–194,193f,194fHMG-CoAin,184–185,185f496Isothermicsystems,biologicsystemsas,regulationof,186–187,187f,188fdeficiencyof,49780Ketogenicaminoacids,232distributionof,585tIsotopes.Seealsospecifictypeα-Ketoglutarate,131ferrous,inoxygentransport,40–41inplasmaproteinanalysis,581inaminoacidcarbonskeletonheme,278,585Isotype(class)switching,594catabolism,249,250,250f,absorptionof,478,585,585fIsotypes,594251fhinderedenvironmentfor,41,41fIsovalericacidemia,259,259–262inglutamatesynthesis,237,238f,243,inmethemoglobinemia,46Isovaleryl-CoAdehydrogenase,inisovaleric243f,244fincorporationofintoprotoporphyrin,acidemia,259–262transportersystemsfor,99271–272,272fIsozymes,54–55inureasynthesis,244,244f
667
668INDEX/671Ligand-receptorcomplex,insignaldisordersassociatedwithabnormalitiesremnant,206t,208,209fgeneration,456–457of,431liveruptakeof,208–209Ligases,50fattyacids,111–114Liposomes,421DNA,328t,330,332,332fglycolipids,111,116,117famphipathiclipidsforming,119–121,Ligation,413interconvertibilityof,231120finRNAprocessing,352inmembranes,416–418artificialmembranesand,421Light,inactivetransport,427ratiooftoprotein,416,416fLipotropicfactor,212Lightchainsmetabolismof,122f,123–124,123f,β-Lipotropin,453,453fimmunoglobulin,591,592f125–126,126f.SeealsoLipolysisLipoxins,112,114f,190,192inamyloidosis,590infedstate,232clinicalsignificanceof,196genesproducing,593inliver,211–212,213flipoxygenasepathwayinformationof,DNArearrangementand,325–326,neutral,111192,193f,194,195f393,593–594peroxidationof,118–119,120fLipoxygenase,119,194,195fmyosin,560phospholipids,111,114–116,115freactivespeciesproducedby,119insmoothmusclecontraction,570precursor,1115-Lipoxygenase,194,195fLightmeromyosin,560–561,560fsimple,111Lipoxygenasepathway,192,193f,194,Limitdextrinosis,152tsteroids,117–118,117f,118f,119f195fLines,definitionof,413transportandstorageof,205–218Liquidchromatography,high-performanceLINEs.SeeLonginterspersedrepeatadiposetissueand,214–215,214freversed-phase,forpeptidesequencesbrownadiposetissueand,217,217fseparation,23–24Lineweaver-Burkplotclinicalaspectsof,212–214Lithium,496tinhibitorevaluationand,68,68f,69ffattyaciddeficiencyand,194–195Lithocholicacid,synthesisof,226fKmandVmaxestimatedfrom,66,66faslipoproteins,205–206,206t,207fLiverLinguallipase,475liverin,211–212,213fangiotensinogenmadein,451Linktrisaccharide,inglycosaminoglycantriacylglycerols(triglycerides),114,115fbilirubinuptakeby,280–281,280f,synthesis,543turnoverof,membranesand,511–512281f,282fLinkageanalysis,635tLipogenesis,125,173–177,174f,175fcirrhosisof,130,212inglycoproteinstudy,515tacetyl-CoAfor,176–177inα1-antitrypsindeficiency,590Linoleicacid/linoleate,113t,190,190f,fattyacidsynthasecomplexin,173–176,cytochromeP450isoformsin,627192174f,175fdisordersof,inα1-antitrypsindeficiency,inessentialfattyaciddeficiency,191malonyl-CoAproductionin,173,174f589–590,590fsynthesisof,191fNADPHfor,175f,176,176ffattyα-Linolenicacid/α-linolenate,113t,190,regulationof,178–179,178falcoholismand,212–214190f,192enzymesin,156t,173,174f,178,ofpregnancy,188inessentialfattyaciddeficiency,191178ftriacylglycerolmetabolismimbalancesynthesisof,191,191fnutritionalstatein,177–178and,212γ-Linolenicacid/γ-linolenate,113tLipolysis,125,126f,216–217,216f.Seefructoseoverloadand,170–171inessentialfattyaciddeficiency,191alsoLipids,metabolismofglycogenin,145,146tinpolyunsaturatedfattyacidsynthesis,hormone-sensitivelipasein,214–215,hemesynthesisin,272191,192f214fALAsynthaseinregulationof,Lipaseshormonesaffecting,215–216,216f272–273,276fdiagnosticsignificanceof,57tinsulinaffecting,178–179ketonebodiesproducedby,183–184,indigestion,475,476ftriacylglycerol,197184f,186intriacylglycerolmetabolism,197,Lipophiliccompounds,cytochromeP450metabolismin,124–125,125f,126f,214–215,214f,475,476fisoformsinhydroxylationof,627130,235tLipidbilayer,418–419,418f,419fLipoproteinlipase,125,126f,207–208,fattyacidoxidation,ketogenesisand,membraneproteinsand,419209f,210f183–186,184fLipidcore,oflipoprotein,205familialdeficiencyof,228tfructose,167,169fLipidrafts,422involvementinremnantuptake,208,glucose,154f,159,159–160,Lipidstoragedisorders(lipidoses),209f159f202–203,203tα1-Lipoprotein,581ffructose2,6-bisphosphateinLipids,111–121.Seealsospecifictypeβ1-Lipoprotein,581fregulationof,157–158,158famphipathic,119–121,120fLipoprotein(a)excess,familial,228tglycogen,145–147,145,146f,148asymmetryof,membraneassemblyand,Lipoproteins,30,111,125,205–206,206t,lipid,211–212,213f511,512f207f,580,583t.Seealsospecificplasmaproteinsynthesisin,125,581classificationof,111typevitaminD3synthesisin,445,446f,484,complex,111carbohydratesin,110485fincytochromeP450system,627incholesteroltransport,223–224,225fLiverphosphorylase,147derived,111classificationof,205,206tdeficiencyof,152tdigestionandabsorptionof,475–477,deficiencyof,fattyliverand,212LMM.SeeLightmeromyosin476fdisordersof,228t,229Lockandkeymodel,52
669672/INDEXLocuscontrolregions,387Lysosomalenzymes,623D-Mannose,104f,105tLonginterspersedrepeatsequencesinI-celldisease,431,432t,531–532,α-D-Mannose,104f(LINEs),321–322532fMannose-bindingprotein,deficiencyof,533Loopeddomains,chromatin,316,318,Lysosomalhydrolases,deficienciesof,Mannose6-phosphate/mannose6-Psignal,319f532–533,533t526Loops(proteinconformation),32–33LysosomesinI-celldisease,531,532,532fLooseconnectivetissue,keratansulfateIin,inoligosaccharideprocessing,524inproteinflow,507,508t545proteinentryinto,507,507f,508tMannosidosis,532–533,533tLow-densitylipoproteinreceptor-relateddisordersassociatedwithdefectsin,MAP(mitogen-activatedprotein)kinaseprotein,206512t,513ininsulinsignaltransmission,466f,467inchylomicronremnantuptake,Lysozyme,621tinJak/STATpathway,467208–209,209fLysylhydroxylaseMaplesyrupurinedisease(branched-chainLow-densitylipoproteins,205,206tdiseasescausedbydeficiencyof,538tketonuria),259apolipoproteinsof,206,206tinhydroxylysinesynthesis,240,537Marasmus,80,237,478,478–479metabolismof,209,210fLysyloxidase,537,539Marblebonedisease(osteopetrosis),552ratiooftohigh-densitylipoproteins,Lyticpathway,379,379fMarfansyndrome,fibrillinmutationsatherosclerosisand,227D-Lyxose,104f,105tcausing,539–540,540freceptorsfor,209Maroteaux-Lamysyndrome,546tinchylomicronremnantuptake,Massspectrometry,27,27f208–209,209fMac-1,529,529tcovalentmodificationsdetectedby,27,incotranslationalinsertion,505–506,α2-Macroglobulin,583t,590,62427f,27t506fantithrombinactivityof,603forglycoproteinanalysis,514,515tregulationof,223Macromolecules,cellulartransportof,tandem,27Low-energyphosphates,83428–431,429f,430ftranscript-proteinprofilingand,412β-LPH.Seeβ-LipotropinMadcowdisease(bovinespongiformMastcells,heparinin,545LRP.SeeLow-densitylipoproteinencephalopathy),37Matrixreceptor-relatedproteinMagnesium,496textracellular,535–555.SeealsospecificL-tryptophandioxygenase(tryptophaninchlorophyll,270componentpyrrolase),89inextracellularandintracellularfluid,mitochondrial,92,93f,130LTs.SeeLeukotrienes416,416tMatrix-assistedlaser-desorption(MALDI),Lungsurfactant,115,197Majorgroove,inDNA,305f,306inmassspectrometry,27deficiencyof,115,202operonmodeland,378Matrix-processingpeptidase,499Luteinizinghormone(LH),437,438,439fMalate,132f,133Matrixproteins,499LXs.SeeLipoxinsMalatedehydrogenase,132f,133diseasescausedbydefectsinimportof,LXXLLmotifs,nuclearreceptorMalateshuttle,99,100f503coregulators,473MALDI.SeeMatrix-assistedlaser-desorptionMaxamandGilbert’smethod,forDNALyases,50Maleylacetoacetate,intyrosinecatabolism,sequencing,404–405insteroidsynthesis,440–442,441f,254f,255Maximalvelocity(Vmax)443fMalicenzyme,156t,157allostericeffectson,75–76Lymphocytehoming,selectinsin,528–530,inNADPHproduction,176,176finhibitorsaffecting,68,68f,69f529f,529t,530fMalignancy/malignantcells.SeeMichaelis-MentenequationinLymphocytes.SeealsoBlymphocytes;Cancer/cancercellsdeterminationof,65–66,66fTlymphocytesMalignanthyperthermia,556,564–565,substrateconcentrationand,64,64frecombinantDNAtechnologyinstudy565f,569tMcArdle’sdisease/syndrome,152t,573of,624MalonateMechanicallygatedionchannels,568tLysine,16trespiratorychainaffectedby,95,96fMediator-relatedproteins,472t,473catabolismof,256f,258succinatedehydrogenaseinhibitionby,MedicinepIof,1767–68,67fpreventive,biochemicalresearchrequirementsfor,480Malonyl-CoA,infattyacidsynthesis,173,affecting,2Lysinehydroxylase,vitaminCascoenzyme174frelationshipoftobiochemistry,1–4,3ffor,496Malonyltransacylase,173,174f,175fMedium-chainacyl-CoAdehydrogenase,Lysis,cell,complementin,596Maltase,475deficiencyof,188Lysogenicpathway,379,379fMaltose,106–107,107f,107tMegaloblasticanemiaLysolecithin(lysophosphatidylcholine),Mammaliantargetofrapamycin(mTOR),folatedeficiencycausing,482t,492,610t116,116fininsulinsignaltransmission,vitaminB12deficiencycausing,482t,metabolismof,200–201,201f466f,467492,494,610tLysophosphatidylcholine.SeeLysolecithinMammotropin.SeeProlactinMelanocyte-stimulatinghormone(MSH),Lysophospholipase,200,201fManganese,496t453,453fLysophospholipids,116,116fMannosamine,169,171fMELAS(mitochondrialencephalomyopa-Lysosomaldegradationpathway,defectinD-Mannosamine,106thywithlacticacidosisandinlipidoses,203Mannose,inglycoproteins,516tstroke-likeepisodes),100–101
670INDEX/673Meltingpoint,ofaminoacids,186-Mercaptopurine,290,291fcontrolofquantityand,73–74Meltingtemperature/transitionMercapturicacid,629covalentmodificationand,74,76,temperature,305,422Mercuricions,pyruvatemetabolism77–78,78fMembraneattackcomplex,596affectedby,142rate-limitingreactionsand,73Membranefattyacid-transportprotein,207Meromyosinatsubcellularlevel,126,127fMembraneproteins,419,420t,514.Seeheavy,560f,561,561fattissueandorganlevels,124–126,125f,alsoGlycoproteinslight,560–561,560f126f,235tassociationofwithlipidbilayer,419MessengerRNA(mRNA),307,309–310,ofxenobiotics,626–632flowof,507,507f,508t310f,311f,341,342t,359.SeeMetachromaticleukodystrophy,203tintegral,30,420,421falsoRNAMetal-activatedenzymes,50mutationsaffecting,diseasescausedby,alternativesplicingand,354,354f,Metalions,inenzymaticreactions,50431–432,432f,432t393–394,636Metalloenzymes,50peripheral,420–421,421fcodonassignmentsin,358,359tMetalloflavoproteins,86–87redcell,614–617,615f,616f,616teditingof,356Metalloproteins,30structureof,dynamic,419expressionof,detectionofingeneMetallothioneins,588Membranetransport,423,423t,424f,isolation,635tMetaphasechromosomes,317f,318,319t426–431,426f.Seealsospecificmodificationof,355–356Metastasismechanismnucleotidesequenceof,358glycoproteinsand,514,526,530t,531Membranes,415–433mutationscausedbychangesin,membraneabnormalitiesand,432tartificial,421–422361–363,361f,362f,364fMethacrylyl-CoA,catabolismof,262fassemblyof,511–513,512f,512tpolycistronic,376Methemoglobin,46,363,613–614asymmetryof,416,419–420recombinantDNAtechnologyand,397Methemoglobinemia,46,614bilayersof,418–419,418f,419frelationshipoftochromosomalDNA,Methionine,15t,264,266fmembraneproteinassociationand,419321factive(S-adenosylmethionine),258f,biogenesisof,511–513,512f,512tstabilityof,regulationofgeneexpression259,264,266f,289,290f,290tcholesterolin,417and,394–395,394fcatabolismof,258f,259,259ffluidmosaicmodeland,422transcriptionstartingpointand,342requirementsfor,480depolarizationof,innerveimpulsevariationsinsize/complexityof,397,Methioninesynthase,492,494transmission,428399tMethotrexate,296–297,494functionof,415–416,421–422Metabolicacidosis,ammoniain,245dihydrofolate/dihydrofolatereductasefluidityaffecting,422Metabolicalkalosis,ammoniain,245affectedby,296–297,494glycosphingolipidsin,417Metabolicfuels,231–236.SeealsoDigestionMethylationGolgiapparatusinsynthesisof,509clinicalaspectsof,236incovalentmodification,massincreasesintracellular,415dietproviding,474,478and,27tlipidsin,416–418infedandstarvingstates,232–234,ofdeoxycytidineresidues,geneamphipathic,119,120f,417–418,417f233f,234f,234texpressionaffectedby,383mutationsaffecting,diseasescausedby,interconvertabilityof,231–232inglycoproteinanalysis,515t431–432,432f,432tMetabolicpathway/metaboliteflow,122,ofxenobiotics,626,630phospholipidsin,114–116,115f,122–124.Seealsospecifictypeβ-Methylcrotonyl-CoA,catabolismof,261f416–417,417fandMetabolism5-Methylcytosine,287,289fplasma.SeePlasmamembraneflux-generatingreactionsin,129α-Methyldopa,446protein:lipidratioin,416,416fnonequilibriumreactionsin,128–129Methylenetetrahydrofolate,493,493fproteinsin,419,420t.Seealsoregulationof,72,73f,126–129,128finfolatetrap,493f,494Membraneproteinscovalentmodificationin,797-Methylguanine,289fredcell,614–617,615f,615t,616f,616tunidirectionalnatureof,72,73fMethylhistidine,576hemolyticanemiasand,619,620tMetabolism,81,122–129,235t.SeealsoinWilson’sdisease,265selectivityof,415,423–426,423t,424f,specifictypeandCatalysis;Methylmalonicaciduria,155425f,426tMetabolicpathwayMethylmalonyl-CoA,accumulationofinsterolsin,417bloodcirculationand,124–126,125f,vitaminB12deficiency,492structureof,416–421,416f126fMethylmalonyl-CoAisomerase(mutase),inasymmetryand,416,419–420grouptransferreactionsin,8propionatemetabolism,155,fluidmosaicmodelof,421f,422inbornerrorsof,1,249155f,492Menadiol,486,487fintegrationof,metabolicfuelsand,Methylmalonyl-CoAmutase(isomerase),Menadioldiacetate,486,488f231–236155,155f,492Menadione,486.SeealsoVitaminKregulationof,72,73f,126–129,128fMethylmalonyl-CoAracemase,inpropi-Menaquinone,482t,486,488f.Seealsoallostericandhormonalmechanismsonatemetabolism,155,155fVitaminKin,74,74–76,75f,128f,129Methylpentose,inglycoproteins,109tMenkesdisease,588enzymesin,126–129,128fMethyl-tetrahydrofolate,infolatetrap,MEOS.SeeCytochromeP450-dependentallostericregulationand,74,493f,494microsomalethanoloxidizing74–76,75f,128f,129Mevalonate,synthesisof,incholesterolsystemcompartmentationand,72–73synthesis,219,220f,221f,222f
671674/INDEXMg.SeeMagnesiumMitochondrialencephalomyopathies,withMonounsaturatedfattyacids,112,113t.SeeMicelles,418,418flacticacidosisandstroke-likealsoFattyacids;Unsaturatedfattyamphipathiclipidsforming,119,120f,episodes(MELAS),100acids418,418fMitochondrialgenome,499dietary,cholesterollevelsaffectedby,227inlipidabsorption,475Mitochondrialglycerol-3-phosphatesynthesisof,191,191fMichaelisconstant(Km),65dehydrogenase,87Morquiosyndrome,546tallostericeffectson,75–76Mitochondrialmembraneproteins,muta-MPP.SeeMatrix-processingpeptidasebindingconstantapproximatedby,66tionsof,431MPS.SeeMucopolysaccharidosesenzymaticcatalysisrateand,65–66,66f,Mitochondrialmembranes,92,93fMRE.SeeMineralocorticoidresponse72,73fenzymesasmarkersand,92elementinhibitorsaffecting,68,69fexchangetransportersand,98–100,98f,mRNA.SeeMessengerRNAMichaelis-Mentenequationin99fMRP2.SeeMultidrugresistance-likedeterminationof,65–66,66fproteininsertionin,501protein2Michaelis-Mentenequation,65Mitochondrialmyopathies,fatalinfantile,MSH.SeeMelanocyte-stimulatinghormoneBi-Bireactionsand,70,70fandrenaldysfunction,oxidore-MstII,399tregulationofmetaboliteflowand,72,ductasedeficiencycausing,100insicklecelldisease,409,410f73fMitogen-activatedprotein(MAP)kinasemtDNA.SeeMitochondrialDNAMicrofilaments,576–577ininsulinsignaltransmission,466f,mTOR,ininsulinsignaltransmission,α2-Microglobulin,583t467466f,467Microsatelliteinstability,322inJak/STATpathway,467Mucins,519–520,520tMicrosatellitepolymorphism,322,411,Mitoticspindle,microtubulesinformationgenesfor,520413of,577O-glycosidiclinkagesin,518,519–520,Microsatelliterepeatsequences,322,413Mixed-functionoxidases,89–90,627.See519fMicrosomalelongasesystem,177,177falsoCytochromeP450systemrepeatingaminoacidsequencesin,519,Microsomalfraction,cytochromeP450ML.SeeMucolipidoses520fisoformsin,627MOAT.SeeMultispecificorganicanionMucolipidoses,546–547,546tMicrotubules,577transporterMucopolysaccharides,109,109fMigration,cell,fibronectinin,540Modeling,molecular,inproteinstructureMucopolysaccharidoses,545–547,546t,Milk(lactose)intolerance,102,474,475analysis,36547fMineralocorticoidresponseelement,459tMolecularbiology,1.SeealsoRecombinantMucoproteins.SeeGlycoproteinsMineralocorticoids,437DNA/recombinantDNAMucus,519–520receptorfor,471technologyMultidrugresistance-likeprotein2,insynthesisof,438–440,441finprimarystructuredetermination,bilirubinsecretion,280Minerals,2,496–497,496t25–26Multipassmembraneprotein,aniondigestionandabsorptionof,477–478Molecularchaperones.SeeChaperonesexchangeproteinas,615,615f,Minorgroove,inDNA,305f,306Moleculargenetics,1,396.Seealso616tMismatchrepairofDNA,336,336f,336tRecombinantDNA/recombinantMultiplemyeloma,595coloncancerand,336DNAtechnologyMultiplesclerosis,202Missensemutations,361,362–363,362fMolecularmodeling,inproteinstructureMultiplesulfatasedeficiency,203familialhypertrophiccardiomyopathyanalysis,36Multisitephosphorylation,inglycogencausedby,569–570,570fMolecularmotors,577metabolism,151MIT.SeeMonoiodotyrosineMolybdenum,496tMultispecificorganicaniontransporter,inMitchell’schemiosmotictheory.SeeMonoacylglycerolacyltransferase,198f,199bilirubinsecretion,280ChemiosmotictheoryMonoacylglycerolpathway,198f,199,Muscle,556–576,557f.SeealsoCardiacMitochondria475–477,476fmuscle;SkeletalmuscleALAsynthesisin,270,273f2-Monoacylglycerols,198f,199ATPin,556,561–562,573–574,575fcitricacidcyclein,122,122f,123f,124f,Monoclonalantibodies,hybridomasincontractionof.SeeMusclecontraction126,127f,130,133–135,134fproductionof,595–596,596finenergytransduction,556–559,557f,fattyacidoxidationin,180–181,181fMonoglycosylatedcorestructure,calnexin558f,559fiontransportin,99bindingand,526fibersin,556proteinsynthesisandimportby,Monoiodotyrosine(MIT),447,448f,449glycogenin,145,146t499–501,501tMonomericproteins,34metabolismin,125,125f,235t,576trespirationrateof,ADPincontrolof,Mononucleotides,287glycogen,14594–95,97t,98f“salvage”reactionsand,294,295f,297flactateproductionand,139respiratorychainin,92.SeealsoMonooxygenases,89–90.Seealsoasproteinreserve,576RespiratorychainCytochromeP450systemproteinsof,566t.SeealsoActin;Myosin;MitochondrialcytochromeP450,89–90,inmetabolismofxenobiotics,626Titin627.SeealsoCytochromeP450Monosaccharides,102.SeealsospecifictypeMusclecontraction,556,558f,561–565,systemandGlucose564tMitochondrialDNA,322–323,322f,absorptionof,475,475fATPhydrolysisin,561–562,561f323tphysiologicimportanceof,104–105,105tincardiacmuscle,566–568
672INDEX/675regulationofMyocardialinfarction,lactateregulationof,526–527,527tactin-based,562–563dehydrogenaseisoenzymesintunicamycinaffecting,527,527tcalciumin,562diagnosisof,57,57t,58fNa.SeeSodiumincardiacmuscle,566–568Myofibrils,556,557f,558fNa+-Ca2+exchanger,463sarcoplasmicreticulumand,Myoglobin,40–48Na+-K+ATPase,427–428,428f563–564,563f,564fα-helicalregionsof,40,41finglucosetransport,428,429finsmoothmuscle,570–571,571fβsubunitsofhemoglobinand,42NAD+(nicotinamideadeninedinucleotide),myosin-based,570oxygendissociationcurvefor,41–42,42f87,490,490fmyosinlightchainkinasein,oxygenstoredby,40,41–42,42f,573absorptionspectrumof,56,56f570–571,571fMyoglobinuria,47incitricacidcycle,133relaxationphaseof,561,564,564tMyokinase(adenylylkinase),84ascoenzyme,87,89f,290tinsmoothmuscledeficienciesof,151–152NADHcalciumin,571ingluconeogenesisregulation,157absorptionspectrumof,56,56fnitricoxidein,571–573,573fassourceofATPinmuscle,573,575fextramitochondrial,oxidationof,99,slidingfilamentcross-bridgemodelof,Myopathies,92100f557–559,558fmitochondrial,100–101fattyacidoxidationyielding,181insmoothmuscle,570–573fatalinfantile,andrenaldysfunction,inpyruvatedehydrogenaseregulation,tropomyosinandtroponinin,562oxidoreductasedeficiency141–142,142fMusclefatigue,136causing,100NADHdehydrogenase,87,93Musclephosphorylase,147Myophosphorylasedeficiency,152tNADP+(nicotinamideadeninedinucleotideabsenceof,152tMyosin,557,559,560fphosphate),87,490activationofinmusclecontraction,557–559,558f,ascoenzyme,87,89f,290tcalcium/musclecontractionand,561–562,561f,562finpentosephosphatepathway,163,148regulationofsmoothmuscle164f,165fcAMPand,147–148,149fcontractionand,570NAD(P)+-dependentdehydrogenases,inMusculardystrophy,Duchenne,556,instriatedversussmoothmuscle,572tenzymedetection,56565–566,566fstructureandfunctionof,560–561,560fNADPHMutagenesis,site-directed,inenzymestudy,Myosin-bindingproteinC,566tincytochromeP450reactions,90f,62758Myosin(thick)filaments,557,558fintramitochondrial,proton-translocatingMutations,314,323–326,323f,324f,Myosinhead,560,560ftranshydrogenaseand,99325fconformationalchangesin,inmuscleforlipogenesis,175f,176,176fbasesubstitution,361,361f,362contraction,561pentosephosphatepathwayand,163,constitutive,376Myosinheavychains,560164f,165f,169frameshift,363,364ffamilialhypertrophiccardiomyopathyNADPH-cytochromeP450reductase,627ABObloodgroupand,619causedbymutationsingenefor,NADPHoxidase,621t,622–623geneconversionand,325569–570,570fchronicgranulomatousdiseaseassociatedintegrationand,324,324fMyosinlightchainkinase,570–571,571fwithmutationsin,623,623fofmembraneproteins,diseasescausedMyosinlightchains,560NCoA-1/NCoA-2coactivators,472,472tby,431–432,432f,432tinsmoothmusclecontraction,570NCoR,472t,473missense,361,362–363,362fMyotoniacongenita,569tNDPs.SeeRibonucleosidediphosphatesfamilialhypertrophiccardiomyopathyMyristicacid,112t,510Nebulin,566tcausedby,569–570,570fMyristylation,510NEFA(nonesterifiedfattyacids).SeeFreemRNAnucleotidesequencechangescaus-incovalentmodification,massincreasesfattyacidsing,361–363,361f,362f,364fand,27tNegativenitrogenbalance,479nonsense,362Negativeregulators,ofgeneexpression,point,361374,375t,378,380recombinantDNAtechnologyinN-acetylneuraminicacid,169,171fNegativesupercoils,DNA,306detectionof,408–409,408fingangliosides,201,203fNEM-sensitivefactor(NSF),509,510frecombinationand,323–324,323f,324finglycoproteins,169,171f,515,516tNeonataladrenoleukodystrophy,503,silent,361inmucins,519f,520503tsisterchromatidexchangesand,325,325fN-linkedglycoproteins,518,519f,Neonatal(physiologic)jaundice,282–283suppressor,363521–527Neonataltyrosinemia,255transition,361,361fclassesof,521,522fNeonate,hypoglycemiain,151transpositionand,324–325synthesisof,521–527,523f,524f,525f,Nervecells.SeeNeuronstransversion,361,361f526tNerveimpulses,428Myastheniagravis,431dolichol-P-P-oligosaccharidein,NervoussystemMyelinsheets,428521–524,523fglucoseasmetabolicnecessityfor,232Myeloma,595inendoplasmicreticulumandGolgithiamindeficiencyaffecting,489Myelomacells,hybridomasgrownfrom,apparatus,524–525,526tNESs.SeeNuclearexportsignals596,596fglycanintermediatesformedduring,Netcharge,ofaminoacid,16–17,17fMyeloperoxidase,612,621t,623526Netdiffusion,423
673676/INDEXNeuAc.SeeN-AcetylneuraminicacidNightblindness,vitaminAdeficiencyNucleargenes,proteinsencodedby,499Neuraltubedefects,folicacidsupplementscausing,482t,483Nuclearlocalizationsignal(NLS),501,inpreventionof,494Nitricoxide,556,571–573,573f,574t,502f,508tNeuraminicacid,110,116607tNuclearmagneticresonance(NMR)Neuraminidasesclotting/thrombosisaffectedby,607,spectroscopydeficiencyof,532–533,533t607tforglycoproteinanalysis,514,515tinglycoproteinanalysis,517Nitricoxidesynthases,572–573,573f,574tproteinstructuredemonstratedby,influenzavirus,533Nitrite,nitricoxideformationfrom,57235–36Neurofilaments,577tNitrogen,aminoacid(α-amino)Nuclearporecomplexes,501Neurologicdiseases,proteinconformationcatabolismof,242–248Nuclearproteins,O-glycosidiclinkagesin,alterationsand,37endproductsof,242–243518Neurons,membranesofureaas,242–243,245–247,246fNuclearreceptorcoactivatorsimpulsestransmittedalong,428L-glutamatedehydrogenasein,(NCoA-1/NCoA-2),472,472tionchannelsin,424,425f244–245,244f,245fNuclearreceptorcorepressor(NCoR),472t,synapticvesiclefusionwith,511Nitrogenbalance,479473Neuropathy,sensory,invitaminB6excess,Nitroglycerin,572Nuclearreceptorsuperfamily,436,469,491NLS.SeeNuclearlocalizationsignal469–471,471f,472tNeutrallipids,111NMR.SeeNuclearmagneticresonanceNucleases,8,312Neutropenia,610(NMR)spectroscopyactivechromatinand,316Neutrophils,620–624NO.SeeNitricoxideNucleicacids.SeealsoDNA;RNAactivationof,621–622NOsynthase.SeeNitricoxidesynthasebasesof,287–289,288tbiochemicalfeaturesof,620tNoncodingregions,inrecombinantDNAdietarilynonessential,293enzymesandproteinsof,621ttechnology,397,398fdigestionof,312ininfection,620Noncodingstrand,304structureandfunctionof,303–313ininflammation,620,621tNoncompetitiveinhibition,competitiveNucleolyticprocessing,ofRNA,352integrinsand,620–621,622tinhibitiondifferentiatedfrom,Nucleophile,wateras,7–8selectinsand,528–529,529t,530f67–69,67f,68f,69fNucleophilicattack,inDNAsynthesis,proteinasesof,623–624,624tNoncovalentassemblies,inmembranes,416328,329frespiratoryburstand,622–623NoncovalentforcesNucleoplasmin,315NF-κBpathway,468,468f,469finbiomoleculestabilization,6Nucleoproteins,packingof,318,319t,320fNiacin,482t,490,490f.Seealsopeptideconformationand,20Nucleosidases(nucleosidephosphorylases),Nicotinamide;NicotinicacidNonequilibriumreactions,128–129purine,deficiencyof,300incitricacidcycle,133citricacidcycleregulationand,135Nucleosidediphosphatekinase,85deficiencyof,482t,490glycolysisregulationand,140,153–155Nucleosidetriphosphatesexcess/toxicityof,490Nonesterifiedfattyacids.SeeFreefattyacidsgrouptransferpotentialof,289–290,Nicktranslation,413Nonfunctionalplasmaenzymes,57.Seealso289f,290f,290tNickel,496tEnzymesnonhydrolyzableanalogsof,291,292fNicks/nick-sealing,inDNAreplication,indiagnosisandprognosis,57,57tinphosphorylation,85332,332fNonhemeiron,92,95f,585Nucleosides,286–287,288tNicotinamide,482t,490,490f.SeealsoNonhistoneproteins,314Nucleosomes,314,315–316,315fNiacinNon-insulindependentdiabetesmellitusNucleotideexcision-repairofDNA,336,coenzymesderivedfrom,50–51(NIDDM/type2),161337,338fdehydrogenasesand,87,89fNonoxidativephase,ofpentosephosphateNucleotidesugars,inglycoproteinbiosyn-excess/toxicityof,490pathway,163–166thesis,516–517,516t,520,521tNicotinamideadeninedinucleotideNonrepetitive(unique-sequence)DNA,Nucleotides,286–292,288t.Seealso(NAD+),87,490,490f320,320–321Purine;Pyrimidinesabsorptionspectrumof,56,56fNonsensecodons,359,361,363adenylylkinase(myokinase)inincitricacidcycle,133Nonsensemutations,361interconversionof,84ascoenzyme,87,89f,290tNonsteroidalanti-inflammatorydrugsascoenzymes,290,290tNicotinamideadeninedinucleotidecyclooxygenaseaffectedby,193DNA,deletion/insertionof,frameshiftphosphate(NADP+),87,490prostaglandinsaffectedby,190mutationsand,363,364fascoenzyme,87,89f,290tNorepinephrine,439f,447,447f.Seealsometabolismof,293–302inpentosephosphatepathway,163,CatecholaminesinmRNA,358164f,165fsynthesisof,267,267f,445–447,447fmutationscausedbychangesin,Nicotinicacid,482t,490,490f.Seealsointhermogenesis,217,217f361–363,361f,362f,364fNiacinNorthernblottransferprocedure,305–306,physiologicfunctionsof,289ashypolipidemicdrug,229403,404f,413aspolyfunctionalacids,290NIDDM.SeeNon-insulindependentingeneisolation,635tpolynucleotides,291–292diabetesmellitusNPCs.SeeNuclearporecomplexessyntheticanalogsof,inchemotherapy,Nidogen(entactin),inbasallamina,540NSF.SeeNEM-sensitivefactor290–291,291fNiemann-Pickdisease,203tNuclearexportsignals,503ultravioletlightabsorbedby,290
674INDEX/677Nucleus(cell),importinsandexportinsinOncogenes,1Overnutrition,478–479transportand,501–503,502fcyclinsand,334OxaloacetateNutrition,474–480.SeealsoDietOncoproteins,Rbproteinand,334inaminoacidcarbonskeletonbiochemicalresearchaffecting,2Oncotic(osmotic)pressure,580,584catabolism,249,250flipogenesisregulatedby,177–178Oncoviruses,cyclinsand,334inaspartatesynthesis,237–238,238fNutritionaldeficiencies,474Opencomplex,345incitricacidcycle,126,127f,130,131f,inAIDSandcancer,479Operatorlocus,377–378,377f,378133,134f,135Nutritionallyessentialaminoacids,124,Operon/operonhypothesis,375,376–378,Oxalosis,170237t,480.SeealsoAminoacids376f,377fOxidases,86,86–87,87f.SeealsospecificNutritionallyessentialfattyacids,190.SeeOpticalactivity/isomer,104typealsoFattyacidsORC.SeeOriginreplicationcomplexceruloplasminas,587abnormalmetabolismof,195–196ORE.SeeOriginreplicationelementcopperin,86deficiencyof,191–192,194–195Ori(originofreplication),326,327f,413flavoproteinsas,86–87,88fNutritionallynonessentialaminoacids,Originreplicationcomplex,326mixed-function,89–90,627.Seealso124,237,237t,480Originreplicationelement,326CytochromeP450systemsynthesisof,237–241Originofreplication(ori),326,327f,Oxidation,86–91413definitionof,86Ornithine,265,266fdehydrogenasesin,87–88,88f,89fOR.SeeRightoperatorcatabolismof,250,251ffattyacid,180–189.SeealsoKetogenesisObloodgroupsubstance,618–619,619finureasynthesis,245,246–247,246f,acetyl-CoAreleaseand,123–124,Ogene,618–619247123f,181–183,181f,182fO-glycosidiclinkageOrnithineδ-aminotransferase,mutationsβ,181–183,181f,182fofcollagen,537in,250ketogenesisregulationand,ofproteoglycans,542–543Ornithine-citrullineantiporter,defective,186–187,187f,188fO-linkedglycoproteins,518,518–520,250modified,183,183f519f,520f,520tOrnithinetranscarbamoylase/L-Ornithineclinicalaspectsof,187–189synthesisof,520,521ttranscarbamoylasehypoglycemiacausedbyimpairmentO-linkedoligosaccharides,inmucins,deficiencyof,247,300of,187–188519–520,519f,520finureasynthesis,246–247,246finmitochondria,180–181,181fObesity,80,205,231,474,478Orosomucoid(α1-acidglycoprotein),hydroperoxidasesin,88–89lipogenesisand,173583toxidasesin,86–87,87f,88fOctamers,histone,315,315fOrotatephosphoribosyltransferase,296,oxygentoxicityand,90–91,611–613,Oculocerebrorenalsyndrome,512t297,298f613t1,25(OH)2-D3.SeeCalcitriolOroticaciduria,300,301oxygenasesin,89–90,90f3β-OHSD.See3β-HydroxysteroidOrotidinemonophosphate(OMP),296,redoxpotentialand,86,87tdehydrogenase298fOxidation-reduction(redox)potential,86,Okazakifragments,327,330,331fOrotidinuria,30187tOleicacid,112,112f,113,113t,114f,190fOrphanreceptors,436,471Oxidativedecarboxylation,ofsynthesisof,191,191fOsmoticfragilitytest,617α-ketoglutarate,131,132fOligomers,importofbyperoxisomes,503Osmoticlysis,complementin,596Oxidativephase,ofpentosephosphateOligomycin,respiratorychainaffectedby,Osmotic(oncotic)pressure,580,584pathway,163,164f,165f95,96f,97fOsteoarthritis,535,551tOxidativephosphorylation,83,92–101,Oligonucleotideproteoglycansin,548122.SeealsoPhosphorylation;definitionof,413Osteoblasts,549,549f,550Respiratorychaininprimarystructuredetermination,26Osteocalcin,488,496,548tchemiosmotictheoryof,92,95–97,97fOligosaccharide:proteintransferase,523Osteoclasts,549–550,549f,550fclinicalaspectsof,100–101Oligosaccharidebranches(antennae),521Osteocytes,549,549fmusclegenerationofATPby,573,OligosaccharidechainsOsteogenesisimperfecta(brittlebones),574–576,575f,575tglycoprotein,514,515t,581–582551–552,551tpoisonsaffecting,92,95,96finN-glycosylation,524,525fOsteoid,549f,550Oxidativestress,612regulationof,526Osteomalacia,482t,484,485,551tOxidoreductases,49,86.Seealsospecifictypesugarsin,515,516tOsteonectin,548tdeficiencyof,100glycosaminoglycans,543Osteopetrosis(marblebonedisease),552Oxidosqualene:lanosterolcyclase,220,222fOligosaccharideprocessing,521,524,525fOsteoporosis,485,551t,552OxygenGolgiapparatusin,509Osteopontin,548tbinding,42,42f.SeealsoOxygenationregulationof,526,527fOuabain,106Bohreffectand,44,45fOligosaccharides,102Na+-K+ATPaseaffectedby,428histidinesF8andE7in,40,41fO-linked,inmucins,519–520,519f,Outermitochondrialmembrane,92,93fhemoglobinaffinities(P50)for,42–43,520fproteininsertionin,50143fOMP(orotidinemonophosphate),296,Ovary,hormonesproducedby,437,myoglobininstorageof,40,41–42,42f,298f442–445,444f,445f573
675678/INDEXOxygen(cont.)storage/secretionof,453,454tPeriodicacid-Schiffreagent,inglycoproteinreductiveactivationof,627synthesisof,450,451fanalysis,515ttransportof,ferrousironin,40–41Paroxysmalnocturnalhemoglobinuria,Periodichyperlysinemia,258Oxygendissociationcurve,formyoglobin432t,528,530t,531,531fPeriodicparalysisandhemoglobin,41–42,42fPartitionchromatography,forprotein/hyperkalemic,569tOxygenradicals.SeeFreeradicalspeptidepurification,21hypokalemic,569tOxygentoxicity,superoxidefreeradicalPassivediffusion/transport,423,423t,424fPeripheralproteins,420–421,421fand,90–91,611–613,613t.SeePasteureffect,157Peripherin,577talsoFreeradicalspBR322,402,402t,403fPermeabilitycoefficients,ofsubstancesinOxygenases,86,89–90PCR.SeePolymerasechainreactionlipidbilayer,418,419fOxygenationofhemoglobinPDH.SeePyruvatedehydrogenasePerniciousanemia,482t,492conformationalchangesand,42,43f,44fPDI.SeeProteindisulfideisomerasePeroxidases,88,192apoprotein,42PECAM-1,529,529tPeroxidation,lipid,freeradicalsproduced2,3-bisphosphoglyceratestabilizing,Pedigreeanalysis,409,410fby,118–119,120f45,45fPellagra,482t,490Peroxins,503highaltitudeadaptationand,46Penicillamine,forWilsondisease,589Peroxisomal-matrixtargetingsequencesmutanthemoglobinsand,46Pentasaccharide,inN-linkedglycoproteins,(PTS),503,508tOxysterols,119521,522fPeroxisomes,89,503Pentosephosphatepathway,123,163–166,absence/abnormalitiesof,503,503t164f,165f,167finZellweger’ssyndrome,188,503Pi,inmusclecontraction,561,561fcytosolaslocationforreactionsof,163biogenesisof,503P50,hemoglobinaffinityforoxygenand,enzymesof,156tinfattyacidoxidation,182–18342–43,43ferythrocytehemolysisand,169–170,Pfeiffersyndrome,551tp53protein/p53gene,339613PFK-1.SeePhosphofructokinasep160coactivators,472,472timpairmentof,169–170PGHS.SeeProstaglandinHsynthasep300coactivator/CPB/p300,461,468,NADPHproducedby,163,164f,PGIs.SeeProstacyclins469,469f,472,472t165fPGs.SeeProstaglandinsP450cytochrome.SeeCytochromeP450forlipogenesis,175f,176,176fpH,9–13.SeealsoAcid-basebalancesystemnonoxidativephaseof,163–166aminoacidnetchargeand,16,17fP450scc(cytochromeP450sidechainoxidativephaseof,163,164f,165fbufferingand,11–12,12f.Seealsocleavageenzyme),438,440f,442riboseproducedby,163,164fBuffersp/CIPcoactivator,472,472tPentoses,102,102tcalculationof,9–10Pcomponent,inamyloidosis,590inglycoproteins,109tdefinitionof,9P-selectin,529tphysiologicimportanceof,104–105,enzyme-catalyzedreactionrateaffectedPAC(P1-based)vector,401–402,402t,413105tby,64,64fPAF.SeePlatelet-activatingfactorPentosuria,essential,163,170isoelectric,aminoacidnetchargeand,17PAGE.SeePolyacrylamidegelPEPCK.SeePhosphoenolpyruvatePhagelambda,378–383,379f,380f,381f,electrophoresiscarboxykinase382fPain,prostaglandinsin,190Pepsin,477PhagesPalindrome,413inacid-basecatalysis,52forcloningingeneisolation,635tPalmitate,173,173–174Pepsinogen,477inrecombinantDNAtechnology,401Palmiticacid,112tPeptidases,inproteindegradation,242,Phagocyticcells,respiratoryburstof,Palmitoleicacid,113t,190f243f622–623Palmitoylation,incovalentmodification,Peptidebonds.SeealsoPeptidesPhagocytosis,429massincreasesand,27tformationof,7,368Pharmacogenetics,630,631–632Pancreaticinsufficiency,invitaminB12partialdouble-bondcharacterof,19–20,Pharmacogenomics,632,638deficiency,49220fPhasing,nucleosome,315–316Pancreaticislets,insulinproducedby,160Peptides,14–20,439f.SeealsoAminoPhenobarbital,warfarininteractionand,Pancreaticlipase,475,476facids;ProteinscytochromeP450inductionPanproteinaseinhibitor,α2-macroglobulinabsorptionof,477affecting,628as,590aminoacidsin,14,19,19fPhenylalanine,16tPantothenicacid,173,482t,495,495iformationof,L-α-aminoacidsin,14catabolismof,255–258,255fincitricacidcycle,133ashormoneprecursors,449–453inphenylketonuria,255,255fcoenzymesderivedfrom,51intracellularmessengersusedby,requirementsfor,480deficiencyof,482t457–468,461t,463tintyrosinesynthesis,239,240fPapain,immunoglobulindigestionby,591aspolyelectrolytes,19PhenylalaninehydroxylasePAPS.SeeAdenosine3′-phosphate-purificationof,21–24defectin,2555′-phosphosulfatePeptidylprolylisomerase,508localizationofgenefor,407tParallelβsheet,32,33fPeptidylglycinehydroxylase,vitaminCasintyrosinesynthesis,239,240fParathyroidhormone(PTH),438,450,coenzymefor,496Phenylethanolamine-N-methyltransferase451fPeptidyltransferase,368,370t(PNMT),447,447f
676INDEX/679Phenylisothiocyanate(Edmanreagent),ininneutrophilactivation,621–622PhospholipaseCβ,inplateletactivation,proteinsequencing,25,26finplateletactivation,606–607,606f606,606fPhenylketonuria,255–258Phosphatidylinositol3-kinase(PI-3kinase)PhospholipaseD,200,201fPhi(ϕ)angle,31,31fininsulinsignaltransmission,465,466fPhospholipasesPhosphagens,83,84finJak/STATpathway,467inglycoproteinanalysis,515tPhosphatasesPhosphatidylserine,115,115finphosphoglyceroldegradationandacid,diagnosticsignificanceof,57tinbloodcoagulation,601remodeling,200–201,201falkalinemembraneasymmetryand,420Phospholipids,111,205inbonemineralization,550Phosphocreatine,inmuscle,556digestionandabsorptionof,475–477,isozymesof,diagnosticsignificanceof,Phosphodiester,291476f57tPhosphodiesterases,291glycerolether,synthesisof,199,200finrecombinantDNAtechnology,incalcium-dependentsignalinlipoproteinlipaseactivity,207–208400ttransduction,463inmembranes,114–116,115f,Phosphatetransporter,99,99fincAMP-dependentsignaltransduction,416–417,417f,419,511Phosphates/phosphorus,496t461,462fmembraneasymmetryand,420,511exchangetransportersand,99,99f,100,cAMPhydrolyzedby,147inmultiplesclerosis,202101fPhosphoenolpyruvate,156tassecondmessengerprecursors,197inextracellularandintracellularfluid,freeenergyofhydrolysisof,82tsynthesisof,198f416tingluconeogenesis,133,134f,156tPhosphoproteinphosphatases,incAMP-freeenergyofhydrolysisof,82–83,82tPhosphoenolpyruvatecarboxykinasedependentsignaltransduction,high-energy,83.SeealsoATP(PEPCK),133,134f462,462finenergycaptureandtransfer,82–83,ingluconeogenesisregulation,133,134f,Phosphoproteins,incAMP-dependent82f,82t,83f153,154fsignaltransduction,461,462fas“energycurrency”ofcell,83–85,Phosphofructokinase/Phosphoricacid,pK/pKavalueof,12t84f,85fphosphofructokinase-1,156tPhosphorus.SeePhosphatessymboldesignating,83ingluconeogenesisregulation,157Phosphorylasetransportof,creatinephosphateinglycolysis,137,138f,156tinglycogenmetabolism,145–146,shuttlein,100,101fregulationand,140146flow-energy,83muscle,deficiencyof,143,152tregulationof,148–150,150–151,Phosphatidate,198f,199Phosphofructokinase-2,157,158f150f,151fintriacylglycerolsynthesis,197,197f,Phosphoglucomutase,inglycogenliver,147198,198f,199biosynthesis,145,146fdeficiencyof,152tPhosphatidatephosphohydrolase,198f,1996-Phosphogluconatedehydrogenase,156t,muscle,147Phosphatidicacid,114,115f,416–417,163,164f,165fabsenceof,152t417f3-PhosphoglycerateactivationofPhosphatidicacidpathway,476f,477inglycolysis,137,138fcalcium/musclecontractionand,Phosphatidylcholines(lecithins),114–115,inserinesynthesis,238,238f148115fPhosphoglyceratekinase,inglycolysis,137,cAMPand,147–148,149fincytochromeP450system,617138fPhosphorylasea,147,149fmembraneasymmetryand,420inerythrocytes,140,140fPhosphorylaseb,147,149fsynthesisof,197,197f,198fPhosphoglyceratemutase,inglycolysis,137,PhosphorylasekinasePhosphatidylethanolamine(cephalin),115,138fcalcium/calmodulin-sensitive,in115fPhosphoglycerides,inmembranes,glycogenolysis,148membraneasymmetryand,420416–417,417fdeficiencyof,152tsynthesisof,197,197fPhosphoglycerolsproteinphosphatase-1affecting,147Phosphatidylglycerol,115,115flysophospholipidsinmetabolismof,116,Phosphorylasekinasea,148,149fPhosphatidylinositol/phosphatidylinositide,116fPhosphorylasekinaseb,148,149f115,115fsynthesisof,197f,198f,199Phosphorylationinbloodcoagulation,601Phosphohexoseisomerase,inglycolysis,137,incovalentmodification,76,77–79,78f,GPI-linkedglycoproteinsand,527.138f78tSeealsoGlycosylphosphatidyli-Phosphoinositide-dependentkinase-1massincreasesand,27tnositol-anchored(GPI-(PDK1),ininsulinsignalmultisite,inglycogenmetabolism,151anchored/GPI-linked)transmission,465oxidative.SeeOxidativephosphorylationglycoproteinsPhospholipaseA1,200,201finrespiratoryburst,623metabolismof,464–465,464f,465fPhospholipaseA2,200,201fPhotolysisreaction,invitaminDsynthesis,assecondmessenger/secondmessengerinplateletactivation,606f,607445precursor,115,115f,437,437t,PhospholipaseC,200,201fPhotosensitivity,inporphyria,274457,463–465,463t,464f,465fincalcium-dependentsignaltransduction,Phototherapy,cancer,porphyrinsin,273synthesisof,197,197f,198f464–465,464f,465fPhylloquinone,482t,486,488f.SeealsoPhosphatidylinositol4,5-bisphosphate,115,inJak/STATpathway,467VitaminK464–465,465finrespiratoryburst,623Physicalmap,633,634f
677680/INDEXPhysiologic(neonatal)jaundice,282–283PlasmathromboplastinantecedentPolymerasesPhytanicacid,Refsum’sdiseasecausedby(PTA/factorXI),599f,600,600tDNA,326,327–328,327f,328,328taccumulationof,188deficiencyof,601inrecombinantDNAtechnology,400tPhytase,477PlasmathromboplastincomponentRNA,DNA-dependent,inRNAPhyticacid(inositolhexaphosphate),calcium(PTC/factorIX),599f,600,600tsynthesis,342–343,342f,343tabsorptionaffectedby,477coumarindrugsaffecting,604Polymorphisms,407Pi,589.Seealsoα1-Antiproteinasedeficiencyof,604acetyltransferase,630pI(isoelectricpH),aminoacidnetchargePlasmalogens,116,117f,199,200fcytochromeP450,628,630tand,17Plasmids,400–401,401f,402,402t,403f,microsatellite,322,411,413PI-3kinase413plasmaprotein,582ininsulinsignaltransmission,465,466fforcloningingeneisolation,635trestrictionfragmentlength.SeeinJak/STATpathway,467Plasmin,604–605,604fRestrictionfragmentlengthPIC.SeePreinitiationcomplexPlasminogen,604polymorphismsPIG-Agene,mutationsofinparoxysmalactivatorsof,604–605,604f,605,605f,singlenucleotide,414nocturnalhemoglobinuria,531,607tPolynucleotidekinase,inrecombinant531fPlatelet-activatingfactor,197,621tDNAtechnology,400t“Ping-Pong”mechanism,infacilitatedsynthesisof,198f,199,200fPolynucleotides,291–292diffusion,427,427fPlatelets,activation/aggregationof,598,posttranslationalmodificationof,289Ping-pongreactions,69–70,69f605–607,606fPolyol(sorbitol)pathway,172Pinocytosis,429–430aspirinaffecting,607–608PolypeptidesPIP2,inabsorptivepinocytosis,430Pleckstrin,inplateletactivation,607receptorsfor,436Pituitaryhormones,437.SeealsospecificPLP.SeePyridoxalphosphatesequencingoftypePNMT.SeePhenylethanolamine-cleavagein,25,26tbloodglucoseaffectedby,161N-methyltransferaseSanger’sdeterminationof,24–25pK/pKapOH,inpHcalculation,9Polyphosphoinositidepathway,plateletofaminoacids,15–16t,17,17f,18Pointmutations,361activationand,605–607environmentaffecting,18,18trecombinantDNAtechnologyinPolyprenoids,118,119fmediumaffecting,13detectionof,408–409,408f,409tPolyribosomes(polysomes),310,370ofweakacids,10–11,11–12,12t,13,17Poisons,oxidativephosphorylation/proteinsynthesison,498,499f,500f,PKA.SeeProteinkinaseArespiratorychainaffected506PKB.SeeProteinkinaseBby,92,95,96fplasmaproteins,581PKC.SeeProteinkinaseCPolIIsignalhypothesisofbindingof,PKU.SeePhenylketonuriaphosphorylationof,350–351503–505,504t,505fPlacenta,estriolsynthesisby,442inpreinitiationcomplexformation,Polysaccharides,102,107–110,108f,109f.Plaquehybridization,403.Seealso351–352SeealsospecifictypeHybridizationintranscription,350–351Polysomes.SeePolyribosomesPlasma,580PolarityPolytenechromosomes,318,318fPlasmacells,immunoglobulinssynthesizedofDNAreplication/synthesis,330–331Polyunsaturatedfattyacids,112,113t.Seein,591ofproteinsynthesis,364alsoFattyacids;UnsaturatedfattyPlasmaenzymes.SeealsoEnzymesofxenobiotics,metabolismand,626acidsdiagnosticsignificanceof,57,57tPoly(A)tail,ofmRNA,309,355–356dietary,cholesterollevelsaffectedby,227Plasmalipoproteins.SeeLipoproteinsininitiationofproteinsynthesis,365eicosanoidsformedfrom,190,192,Plasmamembrane,415,426–431,426f.Polyacrylamidegelelectrophoresis,for193f,194fSeealsoMembranesprotein/peptidepurification,essential,190,190fcarbohydratesin,11024,24f,25fsynthesisof,191,191f,192fmutationsin,diseasescausedby,431,Polyadenylationsites,alternative,394POMC.SeePro-opiomelanocortin432tPolyamines,synthesisof,265–266,266f(POMC)peptidefamilyPlasmaproteins,514,580–591,581f,583t.PolycistronicmRNA,376Pompe’sdisease,152tSeealsospecifictypeandPolycythemia,46Porcinestresssyndrome,565GlycoproteinsPolydystrophy,pseudo-Hurler,532,546tPorphobilinogen,270,273f,275finbone,548tPolyelectrolytes,peptidesas,19Porphyrias,274–278,277f,277tconcentrationof,580Polyfunctionalacids,nucleotidesas,290Porphyrinogens,272electrophoresisforanalysisof,580,582fPolyisoprenoids,incholesterolsynthesis,accumulationofinporphyria,274–278functionsof,583,583t220,221fPorphyrins,270–278,271f,272fhalflifeof,582Polyisoprenol,inN-glycosylation,521–522absorptionspectraof,273–274,277fininflammation,621tPolymerasechainreaction(PCR),57,hemesynthesisand,270–273,273f,polymorphismof,582405–406,406f,413,414274f,275f,276fsynthesisofingeneisolation,635treduced,272inliver,125,581inmicrosatelliterepeatsequencespectrophotometryindetectionof,onpolyribosomes,581detection,322273–274transport,454–455,454t,455t,583tinprimarystructuredetermination,26Positivenitrogenbalance,479
678INDEX/681Positiveregulators,ofgeneexpression,374,proteomicsand,28–29Promoterrecognitionspecificity,343375t,378,380Sanger’stechniqueindeterminationof,Promoters,intranscription,342,342fPosttranslationalprocessing,30,37–39,24–25alternativeuseofinregulation,354–355,38f,371Primarytranscript,342355f,393–394ofcollagen,537–538,537tPrimases,DNA,327,327f,328tbacterial,345–346,345finmembraneassembly,511–512Primosome,328,414eukaryotic,346–349,347f,348f,349f,Posttranslationaltranslocation,499Priondiseases(transmissiblespongiform384Potassium,496tencephalopathies),37Promotorsite,inoperonmodel,377f,378inextracellularandintracellularfluid,Prion-relatedprotein(PrP),37Proofreading,DNApolymerase,328416,416tPrions,37Pro-opiomelanocortin(POMC)peptidepermeabilitycoefficientof,419fProaccelerin(factorV),600t,601,602ffamily,452–453,453f.SeealsoPowerstroke,561Proaminopeptidase,477specifictypePPI.SeePeptidylprolylisomeraseProbes,402,414.SeealsoDNAprobesPro-oxidants,612.SeealsoFreeradicalsPPi.SeePyrophosphate,inorganicforgeneisolation,635tProparathyroidhormone(proPTH),450,PR.SeeProgesterone,receptorsforProbucol,229450fPravastatin,229Procarcinogens,626PropionatePRE.SeeProgestinresponseelementProcessivity,DNApolymerase,328bloodglucoseand,159Pre-β-lipoproteins,205,206t,210Prochymotrypsin,activationof,77,77fingluconeogenesis,154f,155Precursorproteins,amyloid,590Procollagen,371,496,537metabolismof,155,155fPregnancyProcollagenaminoproteinase,537Propionicacid,112testriolsynthesisin,442Procollagencarboxyproteinase,537Propionyl-CoAfattyliverof,188ProcollagenN-proteinase,diseasecausedbyfattyacidoxidationyielding,182hypoglycemiaduring,161deficiencyof,538tmethionineinformationof,259,259fironneedsduring,586Proconvertin(factorVII),599f,600t,601Propionyl-CoAcarboxylase,155,155fPregnancytoxemiaofewes(twinlambcoumarindrugsaffecting,604Proproteins,37–38,76,371disease)Prodrugs,626Propylgallate,asantioxidant/foodfattyliverand,212Proelastase,477preservative,119ketosisin,188Proenzymes,76Prostacyclins,112Pregnenolone,440frapidresponsetophysiologicdemandclinicalsignificanceof,196inadrenalsteroidogenesis,438–440,and,76clotting/thrombosisaffectedby,607,607t440f,441fProfiling,protein-transcript,412ProstaglandinE2,112,113fintesticularsteroidogenesis,442,443fProgesterone,439f,440fProstaglandinHsynthase,192Preinitiationcomplex,343,351–352bindingof,455,455tProstaglandins,112,113f,190,192assemblyof,351–352receptorsfor,471cyclooxygenasepathwayinsynthesisof,inproteinsynthesis,365,366fsynthesisof,438,442,445f192,192–194,193f,194fPrekallikrein,599f,600Progesterone(Δ4)pathway,442,443fProstanoids,112,119Premenstrualsyndrome,vitaminB6inProgestinresponseelement,459tclinicalsignificanceof,196managementof,sensoryProgestins,bindingof,455cyclooxygenasepathwayinsynthesisof,neuropathyand,491Prohormones,371192,192–194,193f,194fPrenataldiagnosis,recombinantDNAProinsulin,449,450fProstheticgroups,50technologyin,409Prokaryoticgeneexpression.SeealsoGeneincatalysis,50–51,51fPreprocollagen,537expressionProtamine,603Preprohormone,insulinsynthesizedas,449,eukaryoticgeneexpressioncomparedProteases/proteinases,8,477,624t.Seealso450fwith,391–395,392tspecifictypePreproparathyroidhormone(preproPTH),asmodelforstudy,375α2-macroglobulinbindingof,590450,451funiquefeaturesof,375–376incartilage,553Preproprotein,albuminsynthesizedas,583Prolactin,437ascatalyticallyinactiveproenzymes,Preproteins,498,581localizationofgenefor,407t76–77Presequence.SeeSignalpeptidereceptorfor,436mucinresistanceto,520Preventivemedicine,biochemicalresearchProline,16tofneutrophils,623–624,624taffecting,2accumulationof(hyperprolinemia),inproteindegradation,242,243f,477Primaquine-sensitivehemolyticanemia,613249–250StaphylococcusaureusV8,forpolypeptidePrimarystructure,21–29,31.Seealsocatabolismof,249–250,251fcleavage,25,26tProteinsequencingsynthesisof,238,239fProtein4.1,inredcellmembranes,615f,aminoacidsequencedetermining,18–19Prolinedehydrogenase,blockofproline616f,616t,617Edmanreactionindeterminationof,25,catabolismat,249–250ProteinC,inbloodcoagulation,600t,60326fProlinehydroxylase,vitaminCascoenzymeProteindisulfideisomerase,proteinfoldinggenomicsinanalysisof,28for,496and,37,508molecularbiologyindeterminationof,Proline-cis,trans-isomerase,proteinfoldingProtein-DNAinteractions,bacteriophage25–26and,37,37flambdaasparadigmfor,ofpolynucleotides,291–292Prolylhydroxylasereaction,240,240f,535378–383,379f,380f,381f,382f
679682/INDEXProteinfolding,36–37,37fimportinsandexportinsin,501–503,lossofintrauma/infection,480chaperonesand,499,507–508,508t502finmembranes,419,420t,514.Seealsoafterdenaturation,36KDELaminoacidsequenceand,Glycoproteins;MembraneProteinkinaseA(PKA),460,462f506–507,508tproteinsProteinkinaseB(PKB),ininsulinsignalmembraneassemblyand,511–513,ratiooftolipids,416,416ftransmission,465,466f512f,512tmodularprincipalsinconstructionof,30ProteinkinaseC(PKC)mitochondriain,499–501,501fmonomeric,34incalcium-dependentsignalperoxisomes/peroxisomedisordersand,phosphorylationof,76,77–79,78f,78t.transduction,464,464f503,503tSeealsoPhosphorylationinplateletactivation,606f,607proteindestinationand,507,507f,508tposttranslationalmodificationof,30,ProteinkinaseD1,ininsulinsignalretrogradetransportand,50737–39,38f,371transmission,466f,467signalhypothesisofpolyribosomebindingpurificationof,21–24Proteinkinase-phosphatasecascade,asand,503–505,504t,505freceptorsas,431,436secondmessenger,437,437tsignalsequencesand,492f,498–499,soluble,30Proteinkinases,77499fstructureof,31–36incAMP-dependentsignaltransduction,transportvesiclesand,508–511,509t,diseasesassociatedwithdisordersof,460–461,462f510f37incGMP-dependentsignaltransduction,Proteinturnover,74,242foldingand,36–37,37f463membranesaffecting,511higherordersof,30–39deficiencyof,151–152rateofenzymedegradationand,74molecularmodelingand,36DNA-dependent,indouble-strandbreakProteinases.SeeProteases/proteinasesnuclearmagneticresonancespec-repair,338Proteins.SeealsospecifictypeandPeptidestroscopyinanalysisof,35–36inglycogenmetabolism,147–148,149f,β-turnsin,32,34fprimary,21–29,31.SeealsoPrimary151,151facutephase,583,583tstructureinhormonalregulation,436,465–468negative,vitaminAas,483–484priondiseasesassociatedwithoflipolysis,215,216fL-α-aminoacidsin,14alterationof,37ininitiationofproteinsynthesis,365asymmetryof,membraneassemblyand,quaternary,33–35,35fininsulinsignaltransmission,465–467,511,512fsecondary,31,31–33,31f,32f,33f,466fbinding,454–455,454t,455t34finJak/STATpathway,467,467fcatabolismof,242–248supersecondarymotifsand,33inNF-κBpathway,468,468fclassificationof,30tertiary,33–35,35finproteinphosphorylation,77,78fconfigurationof,30x-raycrystallographyinanalysisof,35Protein-lipidrespiratorychaincomplexes,conformationof,30synthesisof,358–373.SeealsoProtein93peptidebondsaffecting,20sortingProtein-losinggastroenteropathy,582core,542,543faminoacidsin,124,124fProteinphosphatase-1,147,148,149f,151,inglycosaminoglycansynthesis,elongationin,367–370,368f151f542–543environmentalthreatsaffecting,370Proteinphosphatases,77.Seealsodegradationof,toaminoacids,242,243finfedstate,232Phosphatasesdenaturationofgeneticcode/RNAand,307–308,Proteinprofiling,412proteinrefoldingand,36309t,358–363.SeealsoGeneticProtein-RNAcomplexes,ininitiation,temperatureand,63code365–367,366fdietaryinhibitionofbyantibiotics,371–372,ProteinS,inbloodcoagulation,600t,603digestionandabsorptionof,477372fProteinsequencingmetabolismof,infedstate,232initiationof,365–367,366f,367fEdmanreactionin,25,26frequirementsfor,479–480bymitochondria,499–501,501tgenomicsand,28dimeric,34modularprinciplesin,30massspectrometryin,27,27f,27tdomainsof,33–34polysomesin,370,498,499fmolecularbiologyin,25–26encodingofbyhumangenome,636,posttranslationalprocessingand,371peptidepurificationfor,21–24637tinribosomes,126,127fpolypeptidecleavageand,25,26tinextracellularandintracellularfluid,recognitionandattachment(charging)proteomicsand,28–29416,416tin,360,360fpurificationfor,21–24,22f,23f,24f,fibrous,30recombinantDNAtechniquesfor,25fcollagenas,38407Sanger’smethodof,24–25functionof,bioinformaticsinreticulocytesin,611Proteinsorting,498–513identificationof,28–29terminationof,369f,370chaperonesand,507–508,508tfusion,inenzymestudy,58,59ftranslocationand,368cotranslationalinsertionand,505–506,globular,30virusesaffecting,370–371,371f506fGolgiapparatusinglycosylationandtransmembranedisordersduetomutationsingenessortingof,509ionchannelsas,423–424,425f,426tencoding,512t,513importof,bymitochondria,499–501,inredcells,615–616,615f,616f,Golgiapparatusin,498,500f,507,509501t616t
680INDEX/683transport,454–455,454t,455t“Puffs,”polytenechromosome,318,318foxidationof,134,135f,140–142,141f,xenobioticcellinjuryand,631Pulsed-fieldgelelectrophoresis,forgene142f,143t.SeealsoAcetyl-CoA;Proteoglycans,109,535,538,542–549,isolation,635tGlycolysis542f.SeealsoPumps,415clinicalaspectsof,142–143Glycosaminoglycansinactivetransport,427–428,428fenzymesin,156tinbone,548tPurification,protein/peptide,21–24gluconeogenesisand,153,154fcarbohydratesin,542,542f,543fPurinenucleosidephosphorylasedeficiency,Pyruvatecarboxylase,133,134f,156tincartilage,551,553300ingluconeogenesisregulation,133,134f,diseaseassociationsand,548–549Purines/purinenucleotides,286–290,286f,153,156tfunctionsof,547–549,548t289fPyruvatedehydrogenase,134,135f,140,galactoseinsynthesisof,167–169,170fdietarilynonessential,293141f,156tlinktrisaccharidein,518metabolismof,293–302deficiencyof,143Proteolysisdisordersof,300regulationof,141–142,142fincovalentmodification,76,76–77,77fgoutas,299acyl-CoAin,141–142,142f,178inprochymotrypsinactivation,77,77furicacidformationand,299,299fthiamindiphosphateascoenzymefor,Proteome/proteomics,28–29,414,synthesisof,293–294,294f,295f,296f,488636–637,637–638297fPyruvatedehydrogenasecomplex,140Prothrombin(factorII),600t,601,602fcatalystsin,293,294fPyruvatekinase,156tactivationof,601pyrimidinesynthesiscoordinatedwith,deficiencyof,143,619coumarindrugsaffecting,487,604299gluconeogenesisregulationand,157invitaminKdeficiency,487“salvage”reactionsin,294,295f,297finglycolysis,137–139,138f,156tProthrombinasecomplex,601ultravioletlightabsorbedby,290regulationand,140Protonacceptors,basesas,9Puromycin,372,372fProtondonors,acidsas,9Putrescine,inpolyaminesynthesis,266fProtonpump,respiratorychaincomplexesPyranoseringstructures,103f,104Q(coenzymeQ/ubiquinone),92,95fas,96,96f,97fPyridoxalphosphate,50,491,491fQ10(temperaturecoefficient),enzyme-Proton-translocatingtranshydrogenase,asinhemesynthesis,270catalyzedreactionsand,63sourceofintramitochondrialinureabiosynthesis,243QTinterval,congenitallylong,432tNADPH,99Pyridoxine/pyridoxal/pyridoxamineQuaternarystructure,33–35,35fProtons,transportof,byhemoglobin,44,(vitaminB6),482t,491,491fofhemoglobins,allostericpropertiesand,45fdeficiencyof,482t,49142–46Protoporphyrin,270,272fxanthurenateexcretionin,258,258fstabilizingfactorsand,35incorporationofironinto,271–272,272fexcess/toxicityof,491ProtoporphyrinIII,271,276fPyrimethamine,494ProtoporphyrinogenIII,271,276fPyrimidineanalogs,inpyrimidineRgroups,aminoacidpropertiesaffectedby,Protoporphyrinogenoxidase,271,275f,276fnucleotidebiosynthesis,29718,18tProvitaminAcarotenoids,482–483Pyrimidines/pyrimidinenucleotides,pK/pKa,18Proximalhistidine(histidineF8)286–290,286f,289fR(relaxed)state,ofhemoglobin,inoxygenbinding,40,41fdietarilynonessential,293oxygenationand,43,43f,44freplacementofinhemoglobinM,46metabolismof,293–302,301fRabproteinfamily,511Proximity,catalysisby,51diseasescausedbycataboliteRAC3coactivator,472,472tPrP(prion-relatedprotein),37overproductionand,300–301Radiation,nucleotideexcision-repairofPRPPwater-solublemetabolitesand,300,DNAdamagecausedby,337inpurinesynthesis,294,295f301fRadiationhybridmapping,635tinpyrimidinesynthesis,296,298f,299precursorsof,deficiencyof,300–301Ranprotein,501,502f,503PRPPglutamylamidotransferase,294,295fsynthesisof,296–299,298fRancidity,peroxidationcausing,118PRPPsynthetase,defectin,goutcausedby,catalystsin,296Rapamycin,mammaliantargetof(mTOR),299purinesynthesiscoordinatedwith,299ininsulinsignaltransmission,Pseudo-Hurlerpolydystrophy,532,546t,regulationof,297–299,298f466f,467547ultravioletlightabsorbedby,290RAR.SeeRetinoicacidreceptorPseudogenes,325,414Pyrophosphatase,inorganicRARE.SeeRetinoicacidresponseelementPsi(ψ)angle,31,31finfattyacidactivation,85,180Rateconstant,62PstI,399tinglycogenbiosynthesis,145,146fKeqasratioof,62–63PstIsite,insertionofDNAat,402,403fPyrophosphateRateofdegradation(kdeg),controlof,74PTA.SeePlasmathromboplastinantecedentfreeenergyofhydrolysisof,82tRate-limitingreaction,metabolismegulatedPTC.SeePlasmathromboplastininorganic,85,85fby,73componentPyrrole,40,41fRateofsynthesis(ks),controlof,74Pteroylglutamicacid.SeeFolicacidPyruvate,123Rbprotein.SeeRetinoblastomaproteinPTH.SeeParathyroidhormoneformationof,inaminoacidcarbonReactantconcentration,chemicalreactionPTSs.SeePeroxisomal-matrixtargetingskeletoncatabolism,250–255,rateaffectedby,62sequences252f,253fReactiveoxygenspecies.SeeFreeradicals
681684/INDEXRearrangements,DNARedoxstate,184collectionandoxidationofreducinginantibodydiversity,325–326,393,Reducedporphyrins,272equivalentsand,92–93,93f,94f,593–594Reducingequivalents95frecombinantDNAtechnologyinincitricacidcycle,130–133,132fdehydrogenasesin,87detectionof,409,409tinpentosephosphatepathway,166energyformetabolismprovidedby,recA,381,382frespiratorychainincollectionandoxida-93–95,98fReceptor-associatedcoactivator3(RAC3tionof,92–93,93f,94f,95foxidativephosphorylationatlevelof,94coactivator),472,472t5α-Reductase,442,444fpoisonsaffecting,92,95,96fReceptor-effectorcoupling,435–436Reduction,definitionof,86asprotonpump,96,96f,97fReceptor-mediatedendocytosis,429f,Reductiveactivation,ofmolecularoxygen,redoxpotentialofcomponentsof,43062792–93,94f,95fReceptors,431,436.SeealsospecifictypeRefsum’sdisease,188,503,503tsubstratesfor,citricacidcycleproviding,activationofinsignalgeneration,Regionalasymmetries,membrane,420131,131f456–457,458fRegulatedsecretion,498Respiratorycontrol,81,94–95,97,97t,nuclear,436,469,469–471,471f,472tRegulatoryproteins,bindingoftoDNA,98f,134–135Recognitiondomains,onhormonemotifsfor,387–390,388t,389f,Respiratorydistresssyndrome,surfactantreceptors,435390f,391fdeficiencycausing,115,202RecombinantDNA/recombinantDNARegurgitationhyperbilirubinemia,282Restrictionendonucleases/enzymes,312,technology,396–414,635tRelaxationphase397–399,399t,400f,414basepairingand,396–397ofskeletalmusclecontraction,561,564inrecombinantDNAtechnology,blottingtechniquesin,403,404fofsmoothmusclecontraction399–400,399t,400f,400t,401fchimericmoleculesin,397–406calciumin,571Restrictionenzymes.SeeRestrictioncloningin,400–402,401f,402t,403fnitricoxidein,571–573,573fendonucleasesdefinitionof,414Relaxed(R)state,ofhemoglobin,RestrictionfragmentlengthpolymorphismsDNAligasein,399–400oxygenationand,43,43f,44f(RFLPs),57,409–411,411fDNAsequencingin,404,405fReleasingfactors(RF1/RF3),inproteininforensicmedicine,411doublehelixstructureand,396,397synthesistermination,369f,370Restrictionmap,399inenzymestudy,58,59fRemnantremovaldisease,228tRetentionhyperbilirubinemia,282genemappingand,406–407,407tRenalglomerulus,laminininbasallaminaReticulocytes,inproteinsynthesis,611ingeneticdiseasediagnosis,407–412,of,540–542Retina408f,409t,410f,411fRenalthresholdforglucose,161gyrateatrophyof,250hybridizationtechniquesin,403–404Renaturation,DNA,basepairmatchingretinaldehydein,483,484flibrariesand,402and,305–306Retinal.SeealsoRetinololigonucleotidesynthesisin,404–405Renin,451,452fRetinaldehyde,482,483forganizationofDNAintogenesand,Renin-angiotensinsystem,451–452,452fRetinitispigmentosa,essentialfattyacid397,398f,399tRepeatsequences,637deficiencyand,192polymerasechainreactionin,405–406,aminoacid,519,520fRetinoblastomaprotein,333406fshortinterspersed(SINEs),321–322,Retinoicacid,482,483f.SeealsoRetinolpracticalapplicationsof,406–412414functionsof,483restrictionenzymesand,397–400,399t,Repetitive-sequenceDNA,320,321–322receptorsfor,471,483400f,400t,401fReplication/synthesis.SeeDNA,Retinoicacidreceptor(RAR),471,483terminologyusedin,413–414replication/synthesisof;Retinoicacidresponseelement,459ttranscriptionand,397,398fRNA,synthesisofRetinoidXreceptor(RXR),470,470f,471,Recombinanterythropoietin(epoetinReplicationbubbles,331–333,331f,332f,483alfa/EPO),526,610333fRetinoids,482–484,483f,484f.SeealsoRecombinantfusionproteins,inenzymeReplicationfork,327–328,327fRetinolstudy,58,59fReportergenes,385–386,387f,388fRetinol,482,482t,483f,484f.SeealsoRecombination,chromosomal,323–324,Repression,enzymeVitaminA323f,324fenzymesynthesiscontroland,74deficiencyof,482tRecruitmenthypothesis,ofpreinitiationingluconeogenesisregulation,155–157functionsof,482t,483,484fcomplexformation,352Repressorprotein/gene,lambda(cI),Retinol-bindingprotein,583tRedbloodcells,609–610,610–619.See379–383,380f,381f,382fRetrogradetransport,505,510alsoErythrocytesRepressors,348fromGolgiapparatus,507recombinantDNAtechnologyinstudyingeneexpression,374,377,378,385Retroposons/retrotransposons,321,637of,624tissue-specificexpressionand,385Retroviruses,reversetranscriptasesin,308,Redthrombus,598Reproduction,prostaglandinsin,190332–333Red(slow)twitchfibers,574–576,575tRespiration,86Reversecholesteroltransport,210,211f,Redox(oxidation-reduction)potential,86,Respiratoryburst,479,622–623219,22487tRespiratorychain,92–101.SeealsoReversetranscriptase/reversetranscription,ofrespiratorychaincomponents,92–93,Oxidativephosphorylation308,333,41494f,95fclinicalaspectsof,100–101inrecombinantDNAtechnology,400t
682INDEX/685Reversed-phasehigh-pressureclasses/speciesof,307–308,309t,341,RNApolymerases,DNA-dependent,inchromatography,forprotein/342tRNAsynthesis,342–343,342f,peptidepurification,23–24complementarityof,306,309f343tReversiblecovalentmodifications,77–79,heterogeneousnuclear(hnRNA),310RNAprimer,inDNAsynthesis,328,329f,78f,78t.SeealsoPhosphorylationgeneregulationand,354330fReye’ssyndrome,oroticaciduriain,300messenger(mRNA),307,309–310,RNAprobes,402,414RFLPs.SeeRestrictionfragmentlength310f,311f,341,342t,359RNAP.SeeRNApolymerasespolymorphismsalternativesplicingand,354,354f,RNase.SeeRibonucleasesRFs.SeeReleasingfactors393–394,636ROS(reactiveoxygenspecies).SeeFreeRheumatoidarthritis,glycosylationcodonassignmentsin,358,359tradicalsalterationsin,533editingof,356Rotorsyndrome,283Rho-dependentterminationsignals,344,expressionof,detectionofingeneRoughendoplasmicreticulum346,346fisolation,635tglycosylationin,524–525,525fRhodopsin,483,484fmodificationof,355–356inproteinsorting,498,499f,500fRiboflavin(vitaminB2),86,482t,489–490nucleotidesequenceof,358proteinsynthesisand,370incitricacidcycle,133mutationscausedbychangesin,routesofproteininsertioninto,coenzymesderivedfrom,50–51,489,490361–363,361f,362f,364f505–507,506fdeficiencyof,482t,490polycistronic,376signalhypothesisofpolyribosomedehydrogenasesdependenton,87recombinantDNAtechnologyand,bindingto,503–505,504t,505fRibonucleases,312397rRNA.SeeRibosomalRNARibonucleicacid.SeeRNArelationshipoftochromosomalDNA,RT-PCR,414Ribonucleosidediphosphates(NDPs),321fRXR.SeeRetinoidXreceptorreductionof,294,297fstabilityof,regulationofgeneRyanodine,563Ribonucleosides,286,287fexpressionand,394–395,394fRyanodinereceptor,563,564fRibonucleotidereductasecomplex,294,transcriptionstartingpointand,mutationsingenefor,diseasescausedby,297f342564–565,565f,630tRibose,102variationsinsize/complexityof,397,RYR.SeeRyanodinereceptorinnucleosides,286,287f399tpentosephosphatepathwayinmodificationof,355–356productionof,123,163,166processingof,352–355S50,67D-Ribose,104f,105t,286alternative,inregulationofgeneSphaseofcellcycle,DNAsynthesisduring,Ribosephosphate,pentosephosphatepath-expression,354,355f,393–394333–335,334f,335twayinproductionof,163,inproteinsynthesis,307–308,309tSaccharopine,inlysinecatabolism,256f,164fribosomal(rRNA),307–308,310–311,258Ribose5-phosphate,inpurinesynthesis,341,342tSalt(electrostatic)bonds(saltbridges/293–294,295faspeptidyltransferase,368,370tlinkages),7Ribose5-phosphateketoisomerase,163,processingof,355oxygenbindingrupturing,Bohreffect165fsmallnuclear(snRNA),308,309t,311,protonsand,44–45,45fRibosomaldissociation,inprotein341,342t,414“Salvage”reactionssynthesis,365,366fsmallstable,311inpurinesynthesis,294,295f,297fRibosomalRNA(rRNA),307–308,splicing,352–354,414inpyrimidinesynthesis,296310–311,341,342t.Seealsoalternative,inregulationofgeneSanfilipposyndrome,546tRNAexpression,354,354f,Sanger’smethodaspeptidyltransferase,368,370t393–394,636forDNAsequencing,404,405fprocessingof,355recombinantDNAtechnologyand,forpolypeptidesequencing,24–25Ribosomes,310,312t397,398fSanger’sreagent(1-fluoro-2,4-dinitro-bacterial,371–372structureof,306–312,308f,309f,311f,benzene),forpolypeptideproteinsynthesisin,126,127f312fsequencing,25dissociationand,370synthesisof,341–352Sarcolemma,556Ribozymes,308,311,356initiation/elongation/terminationin,Sarcomere,556–557,557fD-Ribulose,105t,106f342,342f,343–344,344fSarcoplasm,556Ribulose5-phosphate3-epimerase,163,transfer(tRNA),308,310,312f,341,ofcardiacmuscle,566165f342t,360–361,361fSarcoplasmicreticulum,calciumlevelinRichner-Hanartsyndrome,255aminoacyl,inproteinsynthesis,368skeletalmuscleand,563–564,Ricin,372,518tanticodonregionof,359563f,564fRickets,482t,484,551tprocessingandmodificationof,355,Saturatedfattyacids,111,112,112tRightoperator,379–383,380f,382f356inmembranes,417,418fRigormortis,562,564suppressor,363Saturationkinetics,64f,66RNA,303,306–312,341–357xenobioticcellinjuryand,631sigmoidsubstrate,Hillequationinascatalyst,356RNAediting,356evaluationof,66–67,67finchromatin,314RNApolymeraseIII,343tScavengerreceptorB1,210,211f
683686/INDEXScheiesyndrome,546tinglycinesynthesis,238,239fSignalhypothesis,ofpolyribosomebinding,Schindlerdisease,532–533,533tphosphorylated,264503–505,504t,505fScrapie,37synthesisof,238,238fSignalpeptidase,504,505fScurvy,482t,496tetrahydrofolateand,492–494,493fSignalpeptide,498,503–504,508tcollagenaffectedin,38–39,496,Serine195,incovalentcatalysis,53–54,54falbumin,583538–539Serinehydroxymethyltransferase,250,252f,inproteinsorting,498–499,499f,500f,SDS-PAGE.SeeSodiumdodecyl493–494503–504,505,505fsulfate-polyacrylamidegelSerineproteaseinhibitor,589.SeealsoSignalrecognitionparticle,504electrophoresisα1-AntiproteinaseSignalsequence.SeeSignalpeptideSegene,618Serineproteases.SeealsospecifictypeSignaltransducersandactivatorsofSec1proteins,511conservedresiduesand,54,55ttranscription(STATs),467,467fSec61pcomplex,504incovalentcatalysis,53–54,54fSignaltransduction,456–473Secondmessengers,76,436–437,437t,zymogensof,inbloodcoagulation,600,GPI-anchorsin,528457–468,461t,463t.Seealso600t,601hormoneresponsetostimulusand,456,specifictypeSerotonin,266–267,621t457fcalciumas,436–437,437t,457Serpin,589.Seealsoα1-Antiproteinaseintracellularmessengersin,457–468,cAMPas,147,436,437t,457,458–462,Serumprothrombinconversionaccelerator461t,463t.Seealsospecifictype460t,462f(SPCA/factorVII),599f,600t,inplateletactivation,606,606fcGMPas,290,436,437t,457,462–463601signalgenerationand,456–457,458f,diacylglycerolas,464,465fcoumarindrugsaffecting,604459f,459tinositoltrisphosphateas,464–465,464f,Sex(gender),xenobiotic-metabolizingtranscriptionmodulationand,468–473,465fenzymesaffectedby,630470f,471f,472tprecursorsofSexhormone-bindingglobulinSilencers,348phosphatidylinositolas,115,115f(testosterone-estrogen-bindingrecombinantDNAtechnologyand,397phospholipidsas,197globulin),455,455t,583tSilencingmediatorforRXRandTRSecondarystructure,31,31–33,32f,33f,SGLT1transporterprotein,475,475f(SMRT),472t,47334fSGOT.SeeAspartateaminotransferaseSilentmutations,361peptidebondsaffecting,31,31fSGPT.SeeAlanineaminotransferaseSilicon,496tsupersecondarymotifsand,33SH2domains.SeeSrchomology2(SH2)Simplediffusion,423,423t,424fSecretor(Se)gene,618domainsSimvastatin,229Secretorycomponent,ofIgA,595fSHBG.SeeSexhormone-bindingglobulinSINEs.SeeShortinterspersedrepeatSecretorygranules,proteinentryinto,507,Shortinterspersedrepeatsequencessequences507f(SINEs),321–322,414Singledisplacementreactions,69,69fSecretory(exocytotic)pathway,498Shoshinberiberi,489Singlenucleotidepolymorphism(SNP),414Secretoryvesicles,498,500fShotgunsequencing,634Single-passmembraneproteins,D-Sedoheptulose,106fSInuclease,inrecombinantDNAglycophorinsas,615–616,Selectins,528–530,529f,529t,530ftechnology,400t615f,616f,616tSelectivity/selectivepermeability,Sialicacids,110,110f,116,169,171fSingle-strandedDNA,replicationfrom,membrane,415,423–426,ingangliosides,171f,201,203f326.SeealsoDNA,423t,424f,425f,426tinglycoproteins,109t,516treplication/synthesisofSelenium,496tSialidosis,532–533,533t,546,546tSingle-strandedDNA-bindingproteinsinglutathioneperoxidase,88,166Sialoprotein,bone,548t,550(SSBs),326,327,327f,328tSialyl-LewisX,selectinsbinding,530,530fSelenocysteine,synthesisof,240,240fSisterchromatidexchanges,325,325fSelenophosphatesynthetase/synthase,240,Sialylatedoligosaccharides,selectinsSisterchromatids,318,319f240fbinding,530Site-directedmutagenesis,inenzymestudy,Self-assemblySicklecelldisease,363,61958incollagensynthesis,537pedigreeanalysisof,409,410fSite-specificDNAmethylases,398oflipidbilayer,418recombinantDNAtechnologyinSitespecificintegration,324Self-association,hydrophobicinteractionsdetectionof,408–409Sitosterol,forhypercholesterolemia,229and,6–7SidechaincleavageenzymeP450Sizeexclusionchromatography,forSensoryneuropathy,invitaminB6excess,(P450scc),438,440f,442protein/peptidepurification,491Sidechains,inporphyrins,270,271f21–22,23fSepharose-lectincolumnchromatography,Sigmoidsubstratesaturationkinetics,HillSK.SeeStreptokinaseinglycoproteinanalysis,515tequationinevaluationof,66–67,Skeletalmuscle,556,568t.SeealsoMuscle;Sequentialdisplacementreactions,69,69f67fMusclecontractionSerine,15tSignal.SeealsoSignalpeptideglycogenstoresin,573catabolismof,pyruvateformationand,generationof,456–457,458f,459f,459tmetabolismin,125,125f250,252finrecombinantDNAtechnology,414lactateproductionand,139conservedresiduesand,54,55ttransmissionof.SeealsoSignalasproteinreserve,576incysteineandhomoserinesynthesis,transductionslow(red)andfast(white)twitchfibers239,239facrossmembrane,415,431in,574–576,575t
684INDEX/687SkinSodium-potassiumpump(Na+-K+ATPase),SR-B1.SeeScavengerreceptorB1essentialfattyaciddeficiencyaffecting,427–428,428fSRC-1coactivator,472,472t194–195inglucosetransport,428,429fSrchomology2(SH2)domainsmutantkeratinsand,578Solubilitypoint,ofaminoacids,18ininsulinsignaltransmission,465,466f,vitaminD3synthesisin,445,446f,484,SolubleNSFattachmentfactor(SNAP)467485fproteins,509,510f,511inJak/STATpathway,467,467fSleep,prostaglandinsin,190Solutions,aqueous,Kwof,9SRP.SeeSignalrecognitionparticleSlidingfilamentcross-bridgemodel,ofSolvent,wateras,5,6fSRS-A.SeeSlow-reactingsubstanceofmusclecontraction,557–559,Sorbitol,indiabeticcataract,172anaphylaxis558fSorbitoldehydrogenase,167,169fssDNA.SeeSingle-strandedDNASlowacetylators,630Sorbitolintolerance,172StaphylococcusaureusV8protease,forSlow-reactingsubstanceofanaphylaxis,196Sorbitol(polyol)pathway,172polypeptidecleavage,25,26tSlow(red)twitchfibers,574–576,575tSoretband,273STAR.SeeSteroidogenicacuteregulatorySlysyndrome,546tSouthernblottransferprocedure,305–306,proteinSmallintestine403,404f,414Starch,107,108fcytochromeP450isoformsin,627Southwesternblottransferprocedure,403,glycemicindexof,474monosaccharidedigestionin,475,475f414hydrolysisof,474SmallnuclearRNA(snRNA),308,309t,SPARC(bone)protein,548tStarlingforces,580311,341,342t,414Sparteine,CYP2D6inmetabolismof,Starvation,80Smallnucleoproteincomplex(snurp),353628clinicalaspectsof,236SmallstableRNA,311SPCA.SeeSerumprothrombinconversionfattyliverand,212Smokingacceleratorketosisin,188CYP2A6metabolismofnicotineand,Specificacid/basecatalysis,51–52metabolicfuelmobilizationin,232–234,628Specificity,enzyme,49,50f234f,234tcytochromeP450inductionand,628Spectrin,615,615f,616f,616t,617triacylglycerolredirectionand,208nucleotideexcision-repairofDNAabnormalitiesof,617Statindrugs,229damagecausedby,337SpectrometrySTATs(signaltransducersandactivatorsofSmoothendoplasmicreticulum,cy-covalentmodificationsdetectedby,27,transcription),467,467ftochromeP450isoformsin,27f,27tStearicacid,112t627forglycoproteinanalysis,514,515tSteelyhairdisease(Menkesdisease),588Smoothmuscle,556,568tSpectrophotometryStemcells,differentiationoftoredbloodactin-myosininteractionsin,572tforNAD(P)+-dependentdehydrogenases,cells,erythropoietininregulationcontractionof56,56fof,610,611fcalciumin,570–571,571fforporphyrins,273–274Stereochemical(-sn-)numberingsystem,myosin-basedregulationof,570Spectroscopy,nuclearmagneticresonance114,115fmyosinlightchainphosphorylationin,(NMR)Stereoisomers.SeealsoIsomerism570forglycoproteinanalysis,514,515fofsteroids,117,118frelaxationofproteinstructuredemonstratedby,Steroidnucleus,117,117f,118fcalciumin,57135–36Steroidreceptorcoactivator1(SRC-1nitricoxidein,571–573,573fSpermidine,synthesisof,265–266,266fcoactivator),472,472tSMRT,472t,473Spermine,synthesisof,265–266,266fSteroidsulfates,201SNAP(solubleNSFattachmentfactor)Spherocytosis,hereditary,432t,617,617fSteroidogenesis.SeeSteroids,synthesisofproteins,509,510fSphingolipidoses,202–203,203tSteroidogenicacuteregulatoryproteinSNAP25,511Sphingolipids,197(STAR),442SNAREproteins,509,510f,511metabolismof,201–202,202f,203fSteroids,117–118,117f,118f,119f.SeeSNAREpins,511clinicalaspectsof,202–203,203talsospecifictypeSNP.SeeSinglenucleotidepolymorphisminmultiplesclerosis,202adrenal.SeealsoGlucocorticoids;snRNA.SeeSmallnuclearRNASphingomyelins,116,116f,201,202fMineralocorticoidsSnurp(smallnucleoprotein[snRNP]inmembranes,417synthesisof,438–442,440f,441fcomplex),353membraneasymmetryand,420calcitriolas,484Sodium,496tSphingophospholipids,111receptorsfor,436inextracellularandintracellularfluid,Sphingosine,116,116fstereoisomersof,117,118f416,416tSpinabifida,folicacidsupplementsinstorage/secretionof,453,454tpermeabilitycoefficientof,419fpreventionof,494synthesisof,123f,124,438,438–445,Sodium-calciumexchanger,463Spliceosome,353,414439t,440f,441fSodiumdodecylsulfate-polyacrylamidegelSpongiformencephalopathies,transmissibletransportof,454–455,455telectrophoresis(priondiseases),37vitaminDas,484forprotein/peptidepurification,24,24f,Squalene,synthesisof,incholesterolSterol27-hydroxylase,22625fsynthesis,219,221f,222fSterols,117redcellmembraneproteinsdeterminedSqualeneepoxidase,incholesterolsynthesis,inmembranes,417by,614–615,615f220,222fSticklersyndrome,553
685688/INDEXStickyendligation/sticky-endedDNA,299,Sucrose,106–107,107f,107tt-SNAREproteins,509,511398,400f,401f,414glycemicindexof,474T(taut)state,ofhemoglobin“Stickyfoot,”527Sugars.SeealsoCarbohydrates2,3-bisphosphoglyceratestabilizing,45,“Stickypatch,”inhemoglobinS,46,46famino(hexosamines),106,106f45fStoichiometry,60glucoseasprecursorof,169,171foxygenationand,43,43f,44fStokesradius,insizeexclusioninglycosaminoglycans,109,169,171fTtubularsystem,incardiacmuscle,566chromatography,21inglycosphingolipids,169,171fT-typecalciumchannel,567Stopcodon,369f,370interrelationshipsinmetabolismof,TAFs.SeeTBP-associatedfactorsStop-transfersignal,506171fTalin,540,541fStrain,catalysisby,52classificationof,102,102tTandem,414Streptokinase,605,605f,606tdeoxy,106,106fTandemmassspectrometry,27Streptomycin,106“invert,”107Tangierdisease,228tStriatedmuscle,556,557,557f.Seealsoisomerismof,102–104,103fTaqI,399tCardiacmuscle;Skeletalmusclenucleotide,inglycoproteinbiosynthesis,Targetcells,434–435,435tactin-myosininteractionsin,572t516–517,516treceptorsfor,435,436fStroke,withmitochondrialencephalopathy“Suicideenzyme,”cyclooxygenaseas,194Targetedgenedisruption/knockout,412andlacticacidosis(MELAS),SulfateTarui’sdisease,152t100–101active(adenosine3′-phosphate-5′-TATAbindingprotein,346,349f,350,Strongacids,9phosphosulfate),289,289f,629351Strongbases,9inglycoproteins,515TATAbox,intranscriptioncontrol,345,Structuralproteins,535inmucins,520345f,346,347f,348,348f,351tStuart-Prowerfactor(factorX),599f,600,Sulfatide,116Taurochenodeoxycholicacid,synthesisof,600tSulfation,ofxenobiotics,629226factivationof,599–600,599fSulfo(galacto)-glycerolipids,201Taut(T)state,ofhemoglobincoumarindrugsaffecting,604Sulfogalactosylceramide,2012,3-bisphosphoglyceratestabilizing,45,Substrateanalogs,competitiveinhibitionaccumulationof,20345fby,67–68,67fSulfonamides,hemolyticanemiaoxygenationand,43,43f,44fSubstratelevel,phosphorylationat,94precipitatedby,613Tay-Sachsdisease,203tSubstrateshuttlesSulfonylureadrugs,188TBG.SeeThyroxine-bindingglobulincoenzymesas,50Sulfotransferases,inglycosaminoglycanTBP.SeeTATAbindingproteininextramitochondrialNADHoxidation,synthesis,543TBP-associatedfactors,346,350,35199,100fSunlight.SeeUltravioletlightTΨCarm,oftRNA,310,312f,360,361fSubstratespecificity,ofcytochromeP450Supercoils,DNA,306,332,333fTEBG.SeeTestosterone-estrogen-bindingisoforms,627Superoxideanionfreeradical,90–91,globulinSubstrates,49611–613,613t.SeealsoFreeTelomerase,318competitiveinhibitorsresembling,radicalsTelomeres,318,319f67–68,67fproductionofinrespiratoryburst,622Temperatureconcentrationof,enzyme-catalyzedSuperoxidedismutase,90–91,119,chemicalreactionrateaffectedby,62,reactionrateaffectedby,64,611–613,613t,62262f64f,65fSupersecondarystructures,33enzyme-catalyzedreactionrateaffectedHillmodelof,66–67,67fSuppressormutations,363by,63Michaelis-Mentenmodelof,65–66,SuppressortRNA,363influidmosaicmodelofmembrane66fSurfactant,115,197structure,422conformationalchangesinenzymesdeficiencyof,115,202Temperaturecoefficient(Q10),enzyme-causedby,52,53fSV40viruses,cancercausedbycatalyzedreactionsand,63multiple,69–70Swainsonine,527,527tTemplatebinding,intranscription,342,Succinate,131–133,132fSymportsystems,426,426f342fSuccinatedehydrogenase,87,132f,Synconformers,287,287fTemplatestrandDNA,304,306,307f133Synaptobrevin,511transcriptionofinRNAsynthesis,inhibitionof,67–68,67fSyntaxin,511341–343,342fSuccinatesemialdehyde,267,268fSynthesis,rateof(ks),controlof,74Tenasecomplex,600–601Succinatethiokinase(succinyl-CoATerminaltransferase,400t,414synthetase),131,132fTerminationSuccinicacid,pK/pKavalueof,12tt1/2.SeeHalflifechainSuccinyl-CoA,inhemesynthesis,270–273,T3.SeeTriiodothyronineinglycosaminoglycansynthesis,543273f,274f,275f,276fT4.SeeThyroxineintranscriptioncycle,342,342fSuccinyl-CoA-acetoacetate-CoAtransferaseTm.SeeMeltingtemperature/transitionofproteinsynthesis,369f,370(thiophorase),133,186,186ftemperatureofRNAsynthesis,342,342f,344,344fSuccinyl-CoAsynthetase(succinateTlymphocytes,591Terminationsignals,359thiokinase),131,132ft-PA.SeeTissueplasminogenactivatorforbacterialtranscription,346,346fSucrase-isomaltasecomplex,475TΨCarm,oftRNA,310,312f,360,361fforeukaryotictranscription,349–350
686INDEX/689Tertiarystructure,33–35,35fThiamintriphosphate,489Thyroglobulin,447,449stabilizingfactorsand,35Thick(myosin)filaments,557,558fThyroid-bindingglobulin,454,583tTestes,hormonesproducedby,437,442,Thin(actin)filaments,557,558f,559fThyroidhormonereceptor-associated443f.SeealsospecifictypeThioesterase,173proteins(TRAPs),472t,473Testosterone,439f,440f6-Thioguanine,290,291fThyroidhormoneresponseelement,459tbindingof,455,455tThiokinase(acyl-CoAsynthetase)storage/secretionof,453,454tmetabolismof,442,444finfattyacidactivation,180,181fThyroidhormones,437,438synthesisof,442,443fintriacylglycerolsynthesis,199,214f,inlipolysis,215,216fTestosterone-estrogen-bindingglobulin(sex215receptorsfor,436,471hormone-bindingglobulin),455,Thiol-dependenttransglutaminase.Seesynthesisof,447–449,448f455t,583tTransglutaminasetransportof,454,454tTetracycline(tet)resistancegenes,402,Thiolesterplasmaproteinfamily,590Thyroid-stimulatinghormone(TSH),437,403fThiolase,181,182f,184438,439f,449Tetrahedaltransitionstateintermediate,ininmevalonatesynthesis,219,220fThyroperoxidase,449acid-basecatalysis,52,53fThiophorase(succinyl-CoA-Thyrotropin-releasinghormone(TRH),Tetrahydrofolate,492,493–494,493facetoacetate-CoAtransferase),438,439fTetraiodothyronine(thyroxine/T4),438,133,186,186fThyroxine(T4),438,447447Thioredoxin,294storage/secretionof,453,454tstorage/secretionof,453,454tThioredoxinreductase,294,297fsynthesisof,447–449,448fsynthesisof,447–449,448fThreonine,15ttransportof,454,454ttransportof,454,454tcatabolismof,253f,255Thyroxine-bindingglobulin,454,454tTetramersphosphorylated,264TIF2coactivator,472,472themoglobinas,42requirementsfor,480Tiglyl-CoA,catabolismof,261fhistone,314–315,315Thrombin,601,602,603fTIM.SeeTranslocase-of-the-innerTetroses,102,102tantithrombinIIIaffecting,603–604membraneTf.SeeTransferrincirculatinglevelsof,602–603Timnodonicacid,113tTFIIA,350conservedresiduesand,55tTin,496tTFIIB,350formationoffibrinand,601–602,603fTissuedifferentiation,retinoicacidin,483TFIID,346,350,351inplateletactivation,606,606fTissuefactorcomplex,601inpreinitiationcomplexformation,352ThrombolysisTissuefactor(factorIII),599f,600t,601TFIIE,350laboratorytestsinevaluationof,608Tissuefactorpathwayinhibitor,601TFIIF,350t-PAandstreptokinasein,605,605f,Tissueplasminogenactivator(alteplase/TFIIH,350606tt-PA),604–605,605,605f,TFPI.SeeTissuefactorpathwayinhibitorThrombomodulin,inbloodcoagulation,606t,607tTfR.SeeTransferrinreceptor600t,603,607,607tTissue-specificgeneexpression,385Thalassemias,αandβ,47,610tThrombosis,598–608.SeealsoCoagulationTitin,566trecombinantDNAtechnologyinantithrombinIIIinpreventionof,TMP(thymidinemonophosphate),288f,detectionof,408f,409,409t603–604288tThanatophoricdysplasia,551tcirculatingthrombinlevelsand,602–603Tocopherol,482t,486,486f.SeealsoThecacells,hormonesproducedby,442endothelialcellproductsin,607,607tVitaminETheobromine,289hyperhomocysteinemiaand,folicacidasantioxidant,91,119,486,487fTheophylline,289supplementsinpreventionof,494deficiencyof,482thormonalregulationoflipolysisand,215phasesof,598Tocotrienol,486,486f.SeealsoVitaminEThermodynamicsinproteinCorproteinSdeficiency,603Tolbutamide,188biochemical(bioenergetics),80–85.Seet-PAandstreptokinaseinmanagementTOM.SeeTranslocase-of-the-outeralsoATPof,605,605f,606tmembraneglycolysisreversaland,153–155typesofthrombiand,598Topogenicsequences,506lawsof,80–81ThromboxaneA2,113fTopoisomerases,DNA,306,328t,332,hydrophobicinteractionsand,7inplateletactivation,606f,607332fThermogenesis,217,217fThromboxanes,112,113f,190,192Totaliron-bindingcapacity,586diet-induced,217,478clinicalsignificanceof,196Toxemiaofpregnancyofewes,ketosisand,Thermogenin,217,217fcyclooxygenasepathwayinformationof,188Thiamin(vitaminB1),482t,488–489,489f192,193fToxichyperbilirubinemia,283incitricacidcycle,133Thymidine,288tToxopheroxylfreeradical,486coenzymesderivedfrom,51basepairingofinDNA,303,304,305fTpC.SeeTroponinCdeficiencyof,482t,489Thymidinemonophosphate(TMP),288tTpI.SeeTroponinIpyruvatemetabolismaffectedby,140,Thymidine-pseudouridine-cytidine(TΨC)TpT.SeeTroponinT143,489arm,oftRNA,310,312f,360,TRactivatormolecule1(TRAM-1Thiamindiphosphate,140,166,488–489,361fcoactivator),472,472t489fThymidylate,303TRAM(translocatingchain-associatedThiaminpyrophosphate,50Thymine,288tmembrane)protein,504
687690/INDEXTRAM-1coactivator,472,472tTransferrin,478,583t,584–586,585f,Transposition,324–325Transfattyacids,113–114,192585tretroposons/retrotransposonsand,321,Transaldolase,166Transferrinreceptor,586637Transaminases.SeeAminotransferasesTransfusion,ABObloodgroupand,618Transthyretin,583t,590Transamination,124,124fTransgenicanimals,385,411–412,414Transverseasymmetry,511inaminoacidcarbonskeletoncatabo-enhancers/regulatoryelementsidentifiedTransversionmutations,361,361flism,249–250,249f,250f,251fin,386TRAPs,472t,473citricacidcyclein,133–134,134fTransglutaminase,inbloodcoagulation,Trauma,proteinlossand,480inureabiosynthesis,243–244,243f600,600t,602,603fTRE.SeeThyroidhormoneresponseTranscortin(corticosteroid-bindingTranshydrogenase,proton-translocating,aselementglobulin),454–455,455tsourceofintramitochondrialTrehalase,475Transcriptprofiling,412NADPH,99Trehalose,107tTranscription,306,350–352,351t,414Transientinsertionsignal.SeeSignalTRH.SeeThyrotropin-releasinghormoneactivatorsandcoactivatorsincontrolof,peptideTriacylglycerols(triglycerides),114,115f,351,351tTransitionmutations,361,361f205bacterialpromotersin,345–346,345fTransitionstateintermediate,tetrahedal,indigestionandabsorptionof,475–477,controloffidelityandfrequencyof,acid-basecatalysis,52,53f476f344–350Transitionstates,61excessof.SeeHypertriacylglycerolemiaeukaryoticpromotersin,346–349,347f,Transitiontemperature/meltinginterconvertabilityof,231348f,349ftemperature(Tm),305,422inlipoproteincore,205,207fingeneexpressionregulation,383–387,Transketolase,163–166,165f,170metabolismof,123,123f,125–126,391,392t.SeealsoGeneerythrocyte,inthiaminnutritionalstatus126fexpressionassessment,489inadiposetissue,214–215,214fhormonalregulationof,457,458f,thiamindiphosphateinreactionsfattyliverand,212,213f468–473,470f,471f,472tinvolving,166,170,488–489hepatic,211–212,213finitiationof,342–343,342fTranslation,358,414high-densitylipoproteinsin,NF-κBinregulationof,468,469fTranslocase-of-the-innermembrane,499209–211,211fnuclearreceptorcoregulatorsin,Translocase-of-the-outermembrane,499hydrolysisin,197471–473,472tTranslocatingchain-associatedmembranereductionofserumlevelsof,drugsfor,recombinantDNAtechnologyand,397,(TRAM)protein,504229398fTranslocation,protein,499synthesisof,198f,199retinoicacidinregulationof,483Translocationcomplexes,499transportof,207,208f,209f,210freverse,414Translocon,504Tricarboxylateanions,transportersystemsinretroviruses,308,332–333Transmembraneproteins,419for,98–99inRNAsynthesis,306,307f,341–343,ionchannelsas,423–424,425f,426tlipogenesisregulationand,178342finredcells,615–616,615f,616f,616tTricarboxylicacidcycle.SeeCitricacidTranscriptioncomplex,eukaryotic,306,Transmembranesignaling,415,431cycle350–352,351tinplateletactivation,606,606fTriglycerides.SeeTriacylglycerolsTranscriptioncontrolelements,351,351tTransmissiblespongiformencephalopathiesTriiodothyronine(T3),438,447Transcriptiondomains,definitionof,(priondiseases),37storage/secretionof,453,454t387Transportproteins,454–456,454t,455t,synthesisof,447–449,448fTranscriptionfactors,351,351t583ttransportof,454,454tnuclearreceptorsuperfamily,469–471,Transportsystems/transporters.SeealsoTrimethoprim,494471f,472tspecifictypeTrinucleotiderepeatexpansions,322Transcriptionstartsites,alternative,active,423,423t,424f,426–427,Triokinase,167,169f393–394427–428,428fTriosephosphates,acylationof,123Transcriptionunit,342,345fADP/ATP,95,98fTrioses,102,102tTranscriptionalintermediaryfactor2(TIF2ATP-bindingcassette,210,211fTriphosphates,nucleoside,287,287fcoactivator),472,472tincotranslationalinsertion,506,506fTriplehelixstructure,ofcollagen,38,38f,Transcriptomeinformation,412,414disordersassociatedwithmutationsin535–539,536fTransfection,identificationofgenesencoding,512t,513Tripletcode,geneticcodeas,358,359tenhancers/regulatoryelementsexchange,98–100,98f,99ftRNA.SeeTransferRNAand,386facilitateddiffusion,423,423t,424f,Tropocollagen,38,38fTransferRNA(tRNA),308,310,312f,426–427,427,427fTropoelastin,539341,342t,360–361,361f.Seeglucose.SeeGlucosetransportersTropomyosin,557,559f,562alsoRNAininnermitochondrialmembrane,inredcellmembranes,616taminoacyl,inproteinsynthesis,36898–100,98f,99fasstriatedmuscleinhibitor,563anticodonregionof,359membrane,426–431,426fTroponin/troponincomplex,557,559f,processingandmodificationof,355,356fornucleotidesugars,517562suppressor,363Transportvesicles,498,508–511,509t,asstriatedmuscleinhibitor,563Transferases,50510fTroponinC,562
688INDEX/691TroponinI,562Ubiquinone(Q/coenzymeQ),92,95f,118UridinediphosphateN-acetylgalactosamineTroponinT,562incholesterolsynthesis,220,221f(UDP-GalNAc),516tTrypsin,477UDP-glucose.SeeUridinediphosphateUridinediphosphateN-acetylglucosamineconservedresiduesand,55tglucose(UDP-GlcNAc),516tindigestion,477UDPGal.SeeUridinediphosphategalactoseUridinediphosphategalactose(UDPGal),forpolypeptidecleavage,25,26tUDPGlc.SeeUridinediphosphateglucose167,516–517,516tTrypsinogen,477UFA(unesterifiedfattyacids).SeeFreefattyUridinediphosphategalactose(UDPGal)Tryptophan,16t,266–267,490acids4-epimerase,167,170fcatabolismof,257f,258,258fUlcers,474inheriteddefectsin,172deficiencyof,490UltravioletlightUridinediphosphateglucoseniacinsynthesizedfrom,490nucleotideabsorptionof,290(UDP/UDPGlc),145,147f,permeabilitycoefficientof,419fnucleotideexcision-repairofDNA516,516trequirementsfor,480damagecausedby,337inglycogenbiosynthesis,145,146fTryptophanoxygenase/L-tryptophanvitaminDsynthesisand,484,485fUridinediphosphateglucosedehydroge-oxygenase(tryptophanUMP(uridinemonophosphate),288f,288tnase,166,168fpyrrolase),89,257f,258Uncouplers/uncouplingproteinsUridinediphosphateglucoseTSEs.SeeTransmissiblespongiforminrespiratorychain,95,96fpyrophosphorylase,166,168fencephalopathieschemiosmotictheoryofactionof,97inglycogenbiosynthesis,145,146fTSH.SeeThyroid-stimulatinghormoneundernutritionand,479Uridinediphosphate-glucuronate/glu-α-Tubulin,577Undernutrition,474,478–479curonicacid,166–167,168f,290β-Tubulin,577Unequalcrossover,324,324fUridinediphosphatexylose(UDP-Xyl),γ-Tubulin,577Unesterifiedfattyacids.SeeFreefattyacids516tTumorcells,migrationof,hyaluronicacidUniportsystems,426,426fUridinemonophosphate(UMP),288f,and,548Unique-sequence(nonrepetitive)DNA,288tTumorsuppressorgenes,p53,339320,320–321Uridinetriphosphate(UTP),inglycogenTunicamycin,527,527tUniversaldonor/universalrecipient,618biosynthesis,145,146fβ-Turn,32,34fUnsaturatedfattyacids,111,112,113t.SeeUridyltransferasedeficiency,172Twinlambdisease.SeePregnancytoxemiaalsoFattyacidsUrobilinogensofewescisdoublebondsin,112–114,114fconjugatedbilirubinreducedto,281,Twitchfibers,slow(red)andfast(white),dietary,cholesterollevelsaffectedby,282f574–576,575t227injaundice,284,284tTwo-dimensionalelectrophoresis,proteineicosanoidsformedfrom,190,192,normalvaluesfor,284texpressionand,28193f,194fUrocanicaciduria,250TXs.SeeThromboxanesessential,190,190f,193Urokinase,605,605fTyk-2,inJak-STATpathway,467abnormalmetabolismof,195–196Uronicacidpathway,163,166–167,168fTypeAresponse,ingeneexpression,374,deficiencyof,191–192,194–195disruptionof,170375fprostaglandinproductionand,190Uronicacids,109TypeBresponse,ingeneexpression,inmembranes,417,418finheparin,545,545f374–375,375fmetabolismof,190–192UroporphyrinogenI,271,274f,275fTypeCresponse,ingeneexpression,375,oxidationof,183UroporphyrinogenIsynthase,inporphyria,375fstructuresof,190f277tTyrosine,15t,16t,267,267fsynthesisof,191,191fUroporphyrinogenIII,271,274f,275fcatabolismof,254f,255Unwinding,DNA,326,326–327Uroporphyrinogendecarboxylase,271,epinephrineandnorepinephrineformedRNAsynthesisand,344275ffrom,267,267fUracil,288tinporphyria,277tinhemoglobinM,46deoxyribonucleosidesof,inpyrimidineUroporphyrins,270,271f,272finhormonesynthesis,438,439–449,synthesis,296–297,298fspectrophotometryindetectionof,439tUrate,asantioxidant,119273–274phosphorylated,264UreaUTP,inphosphorylation,85requirementsfor,480aminoacidmetabolismand,124,124fsynthesisof,239,240fnitrogencatabolismproducing,Tyrosineaminotransferase,defectin,in242–243,245–247,246fV8protease,forpolypeptidecleavage,fortyrosinemia,255permeabilitycoefficientof,419fpolypeptidecleavage,25,26tTyrosinehydroxylase,catecholaminesynthesisof,243–244,243f,244fvi.SeeInitialvelocitybiosynthesisand,446,447fmetabolicdisordersassociatedwith,Vmax.SeeMaximalvelocityTyrosinekinase247–248Vregion/segment.SeeVariableregions/ininsulinsignaltransmission,465–467,genetherapyfor,248segments466fUricacid,289v-SNAREproteins,509,511inJak/STATpathway,467,467fpurinecatabolisminformationof,299,Valericacid,112tTyrosinemia,255299fValine,15tTyrosinosis,255Uridine,287f,288tcatabolismof,259,260f,262f
689692/INDEXValine(cont.)VitaminBcomplex.Seealsospecificvitaminincoagulation,486–488,488finterconversionof,240incitricacidcycle,133coumarinanticoagulantsaffecting,requirementsfor,480coenzymesderivedfrom,50–51,51f604Valinomycin,99VitaminB1(thiamin),482t,488–489,489fdeficiencyof,482tVanderWaalsforces,7incitricacidcycle,133VitaminKhydroquinone,487,488fVanadium,496tcoenzymesderivedfrom,51Vitamins,2,481–496,482t.Seealsospe-Variablenumbersoftandemlyrepeateddeficiencyof,482t,489cificvitaminunits(VNTRs),inforensicpyruvatemetabolismaffectedby,140,incitricacidcycle,133medicine,411143,489digestionandabsorptionof,477–478Variableregions/segments,591–592,594fVitaminB2(riboflavin),86,482t,489–490lipid-(fat)soluble,482–488genefor,593incitricacidcycle,133absorptionof,475DNArearrangementand,325–326,coenzymesderivedfrom,50–51,489,water-soluble,488–496393,593–594490VLA-1/VLA-5/VLA-6,622timmunoglobulinheavychain,591,592f,deficiencyof,482t,490VLDL.SeeVerylowdensitylipoproteins594fdehydrogenasesdependenton,87VNTRs.SeeVariablenumbersoftandemlyimmunoglobulinlightchain,325–326,VitaminB6(pyridoxine/pyridoxal/repeatedunits393,591,592f,594fpyridoxamine),482t,491,491fVoltage-gatedchannels,424,568tVascularsystem,nitricoxideaffecting,deficiencyof,482t,491vonGierke’sdisease,152t,300571–573,573f,574txanthurenateexcretionin,258,258fVonWillebrandfactor,inplateletVasodilators,556excess/toxicityof,491activation,605nitricoxideas,571–573,573f,574tVitaminB12(cobalamin),482t,491–492,VDRE.SeeVitaminDresponseelement492fVector,414absorptionof,491–492Warfarin,486,604cloning,400–402,401f,402t,403f,414intrinsicfactorin,477,491–492phenobarbitalinteractionand,expression,402deficiencyof,482t,492cytochromeP450inductionVegetariandiet,vitaminB12deficiencyand,functionalfolatedeficiencyand,492,affecting,628491494vitaminKaffectedby,487Velocityinmethylmalonicaciduria,155Water,2,5–9initial,64VitaminB12-dependentenzymes,292f,492asbiologicsolvent,5,6finhibitorsaffecting,68,68f,69fVitaminC(ascorbicacid),163,482t,biomolecularstructureand,6–7,6tmaximal(Vmax)495–496,496fdissociationof,8–9allostericeffectson,75–76asantioxidant,119inhydrogenbonds,5,6finhibitorsaffecting,68,68f,69fincollagensynthesis,38,496,535asnucleophile,7–9Michaelis-Mentenequationindeficiencyof,482t,496permeabilitycoefficientof,419fdeterminationof,65–66,66fcollagenaffectedin,38–39,496,structureof,5,6fsubstrateconcentrationand,64,64f538–539Watersolubility,ofxenobiotics,metabolismVerylowdensitylipoproteinreceptor,208ironabsorptionand,478,496and,626Verylowdensitylipoproteins,125,205,supplemental,496Watson-Crickbasepairing,7,303206t,207VitaminD,482t,484–486Waxes,111hepaticsecretionof,dietaryandhormonalincalciumabsorption,477,484,Weakacids,9statusand,211–212,213f484–485bufferingcapacityof,11–12,12fmetabolismof,125,126f,207–209,210fdeficiencyof,482t,484,485dissociationconstantsfor,10–11,12intriacylglyceroltransport,207,208f,ergosterolasprecursorfor,118,119fHenderson-Hasselbalchequation210fexcess/toxicityof,485–486describingbehaviorof,11,12fVesiclesmetabolismof,484–485,485fphysiologicsignificanceof,10–11coating,509,510freceptorfor,471pK/pKavaluesof,10–13,12t,brefeldinAaffecting,510–511VitaminD2(ergocalciferol),484Weakbases,9secretory,498,500fVitaminD3(cholecalciferol)Wernicke-Korsakoffsyndrome,482ttargeting,509,510fsynthesisofinskin,445,446f,484,485fWernicke’sencephalopathy,489transport,498,508–511,509t,510finvitaminDmetabolism,484,485fWesternblottransferprocedure,403,404f,Vimentins,577t,578VitaminD-bindingprotein,445414Vinculin,540,541fVitaminDreceptor-interactingproteinsWhitebloodcells,620–624.SeealsospecificViraloncogenes.SeeOncogenes(DRIPs),472t,473typeViruses,hostcellproteinsynthesisaffectedVitaminDresponseelement,459tgrowthfactorsregulatingproductionof,by,370–371,371fVitaminE,482t,486,486f610Vision,vitaminAin,482t,483,484fasantioxidant,91,119,486,487frecombinantDNAtechnologyinstudyVitaminA,482–484,482t,483f,484fdeficiencyof,482t,486of,624deficiencyof,482t,483–484VitaminH.SeeBiotinWhitethrombus,598excess/toxicityof,484VitaminK,482t,486–488,488f,604White(fast)twitchfibers,574–576,575tfunctionsof,482t,483calcium-bindingproteinsand,487–488,Wholegenomeshotgunapproach,634invision,482t,483488fWilliamssyndrome,539
690INDEX/693Wilsondisease,432t,587–589responsesto,630–631,630t,Zline,556,557f,558fceruloplasminlevelsin,587631tZellweger’s(cerebrohepatorenal)syndrome,genemutationsin,432t,588–589toxic,631,631f188,503,503tWobble,361Xerodermapigmentosum,337Zinc,496tXerophthalmia,vitaminAdeficiencyin,Zincfingermotif,387,388t,390,390f482t,483inDNA-bindingdomain,470X-linkeddisorders,RFLPsindiagnosisof,XP.SeeXerodermapigmentosumZonafasciculata,steroidsynthesisin,440411Xylose,inglycoproteins,516tZonaglomerulosa,mineralocorticoidX-raydiffractionandcrystallography,D-Xylose,104f,105tsynthesisin,438proteinstructureD-Xylulose,106fZonapellucida,glycoproteinsin,528demonstratedby,35L-Xylulose,105tZonareticularis,steroidsynthesisin,Xanthine,289accumulationofinessentialpentosuria,440Xanthineoxidase,87170ZP.SeeZonapellucidadeficiencyof,hypouricemiaand,300ZP1–3proteins,528Xanthurenate,excretionofinvitaminB6Zwitterions,16deficiency,258,258fZymogens,76,477Xenobiotics,metabolismof,626–632YACvector.SeeYeastartificialchromosomeinbloodcoagulation,600,600t,601conjugationin,626,628–630(YAC)vectorrapidresponsetophysiologicdemandcytochromeP450system/hydroxylationYeastartificialchromosome(YAC)vector,and,76in,626–628,629t401–402,402tZZgenotype,α1-antiproteinasedeficiencyfactorsaffecting,630forcloningingeneisolation,635tandpharmacogeneticsindrugresearchand,Yeastcells,mitochondrialproteinimportinemphysema,589631–632studiedin,499inliverdisease,590
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