Chapter19

TheTricarboxylicAcidCycle[tra?,kɑ?b?k's?l?k]Chapter19

TheTricarboxylicA食品生物化學(xué)Chapter-19-The-Tricarboxylic-Acid-Cycle課件Aerobiccellsuseametabolicwheel--thetricarboxylicacidcycle

三羧酸循環(huán)

(alsocalledthecitricacidcycle

檸檬酸循環(huán),orKrebscycle)—togenerateenergybyacetyl-CoAoxidation.Aerobiccellsuseametabolic19.1HansKrebsandtheDiscoveryoftheTCACycle19.2TheTCACycle—ABriefSummary19.3TheBridgingStep:OxidativeDecarboxylationofPyruvate19.4EntryintotheCycle:TheCitrateSynthaseReaction19.5TheIsomerizationofCitratebyAconitase19.6IsocitrateDehydrogenase—TheFirstOxidationintheCycle19.7-KetoglutarateDehydrogenase—ASecondDecarboxylation19.8Succinyl-CoASynthetase—ASubstrate-LevelPhosphorylation19.9SuccinateDehydrogenase—AnOxidationInvolvingFAD19.10FumaraseCatalyzesTrans-HydrationofFumarate19.11MalateDehydrogenase—CompletingtheCycle19.12ASummaryoftheCycle19.13TheTCACycleProvidesIntermediatesforBiosyntheticPathways19.14TheAnaplerotic,or“FillingUp,”Reactions19.15RegulationoftheTCACycle19.16TheGlyoxylateCycleofPlantsandBacteriaOUTLINE19.1HansKrebsandtheDiscoTricarboxylicAcidCycleTricarboxylicAcidCyclePyruvateproducedinglycolysisisoxidizedinthetricarboxylicacid(TCA)cycle.ElectronsliberatedinthisoxidationflowthroughtheelectrontransportchainanddrivethesynthesisofATPinoxidativephosphorylation.Ineukaryoticcells,thisoverallprocessoccursinmitochondria.Pyruvateproducedinglycolysi19.1HansKrebsandtheDiscoveryoftheTCACycleIn1932Krebswasstudyingtheratesofoxidationofsmallorganicacidsbykidneyandlivertissue.Onlyafewsubstanceswereactiveintheseexperiments—notablycitrate,succinate,fumarate,malate,andacetate.Lateritwasfoundthatoxaloacetate

couldbemadefrompyruvateinsuchtissues,andthatitcouldbefurtheroxidizedliketheotherdicarboxylicacids.琥珀酸鹽延胡索酸鹽醋酸鹽蘋(píng)果酸鹽檸檬酸鹽草酰乙酸鹽,丁酮二酸鹽ThesesubstanceswerethepiecesintheTCApuzzlethatKrebsandotherseventuallysolved.19.1HansKrebsandtheDisco食品生物化學(xué)Chapter-19-The-Tricarboxylic-Acid-Cycle課件In1935inHungary,acrucialdiscoverywasmadebyAlbertSzent-Gy?rgyi,

whowasstudyingtheoxidationofsimilarorganicsubstratesbypigeonbreast

muscle,anactiveflightmusclewithveryhighratesofoxidationandmetabolism.

Carefullymeasuringtheamountofoxygenconsumed,heobservedthat

additionofanyofthreefour-carbondicarboxylicacids—succinate,

fumarate,ormalate—causedtheconsumptionofmuchmoreoxygenthanwasrequired

fortheoxidationoftheaddedsubstanceitself.Heconcludedthatthesesubstances

werelimitinginthecelland,whenprovided,stimulatedoxidationofendogenousglucoseandother

carbohydratesinthetissues.

Healsofoundthatmalonate丙二酸

,acompetitiveinhibitorofsuccinatedehydrogenase,inhibitedtheseoxidativeprocesses;thisfindingsuggestedthatsuccinateoxidationisacrucialstep.AlbertSzent-Gy?rgyi

hypothesizedthatthesedicarboxylicacidswerelinked

byanenzymaticpathwaythatwasimportantforaerobicmetabolism.In1935inHungary,acrucialAnotherimportantpieceofthepuzzlecamefromtheworkofCarlMartiusandFranzKnoop,whoshowedthatcitricacidcouldbeconvertedtoisocitrateandthento

-ketoglutarate.Thisfindingwassignificantbecauseitwasalreadyknownthat-ketoglutaratecouldbeenzymaticallyoxidizedtosuccinate.Atthisjuncture,thepathwayfromcitratetooxaloacetateseemedtobeasshownhere.Whereasthepathwaymadesense,thecatalyticeffectofsuccinateandtheotherdicarboxylicacidsfromSzent-Gy?rgyi’sstudiesremainedapuzzle.AnotherimportantpieceoftheIn1937Krebsfoundthatcitratecouldbeformedinmusclesuspensionsifoxaloacetateandeitherpyruvateoracetatewereadded.Hesawthathenowhadacycle,notasimplepathway,andthatadditionofanyoftheintermediatescouldgeneratealloftheothers.The

existenceofacycle,togetherwiththeentryofpyruvateintothecycleinthesynthesisofcitrate,providedaclearexplanationfortheacceleratingpropertiesofsuccinate,fumarate,andmalate.Ifalltheseintermediatesledtooxaloacetate,whichcombinedwithpyruvatefromglycolysis,theycouldstimulatetheoxidationofmanysubstancesbesidesthemselves.(Kreb’sconceptualleaptoacyclewasnothisfirst.TogetherwithmedicalstudentKurtHenseleit,hehadalreadyelucidatedthedetailsoftheureacyclein1932.)In1937KrebsfoundthatcitraThecompletetricarboxylicacid(Krebs)cycle,asitisnowunderstood,isshown.Thecompletetricarboxylicaci19.2TheTCACycle—ABriefSummaryTheentryofnewcarbonunitsintothecycleisthroughacetyl-CoA.Thisentrymetabolitecanbeformedeitherfrompyruvate(fromglycolysis)orfromoxidationoffattyacids.Transferofthetwo-carbonacetylgroupfromacetyl-CoAtothefour-carbonoxaloacetatetoyieldsix-carboncitrateiscatalyzedbycitratesynthase.Adehydration–rehydrationrearrangementofcitrateyieldsisocitrate.Twosuccessivedecarboxylationsproduce-ketoglutarateandthensuccinyl-CoA,aCoAconjugateofafour-carbonunit.Severalstepslater,oxaloacetateisregeneratedandcancombinewithanothertwo-carbonunitofacetyl-CoA.Thus,carbonentersthecycleasacetyl-CoAandexitsasCO2.Intheprocess,metabolicenergyiscapturedintheformofATP,NADH,andenzyme-boundFADH2.19.2TheTCACycle—ABriefSu19.3TheBridgingStep:OxidativeDecarboxylationofPyruvatePyruvateproducedbyglycolysisisasignificantsourceofacetyl-CoAfortheTCAcycle.Because,ineukaryoticcells,glycolysisoccursinthecytoplasm,whereastheTCAcyclereactionsandallsubsequentstepsofaerobicmetabolismtakeplaceinthemitochondria,pyruvatemustfirstenterthemitochondriatoentertheTCAcycle.19.3TheBridgingStep:OxidaThereactioniscatalyzedbypyruvatedehydrogenase,amultienzymecomplex.Thepyruvatedehydrogenasecomplex(PDC)isanoncovalentassemblyofthreedifferentenzymesoperatinginconcerttocatalyzesuccessivestepsintheconversionofpyruvatetoacetyl-CoA.Theactivesitesofallthreeenzymesarenotfarremovedfromoneanother,andtheproductofthefirstenzymeispasseddirectlytothesecondenzymeandsoon,withoutdiffusionofsubstratesandproductsthroughthesolution.Thereactioniscatalyzedbyp19.4EntryintotheCycle:TheCitrateSynthaseReaction草酰乙酸檸檬酸ThefirstreactionwithintheTCAcycle,theonebywhichcarbonatomsareintroduced,isthecitratesynthasereaction.Theoverall?G°is-31.4kJ/mol,andunderstandardconditionsthereactionisessentiallyirreversible.Althoughthemitochondrialconcentrationofoxaloacetateisverylow(muchlessthan1μM),thestrong,negative?G°drivesthereactionforward.19.4EntryintotheCycle:ThTheStructureofCitrateSynthaseCitratesynthaseinmammalsisadimerof49-kDsubunits.Oneachsubunit,oxaloacetateandacetyl-CoAbindtotheactivesite,whichliesinacleftbetweentwodomainsandissurroundedmainlyby-helicalsegments.Bindingofoxaloacetateinducesaconformationalchangethatfacilitatesthebindingofacetyl-CoAandclosestheactivesite,

sothatthereactivecarbanion負(fù)碳離子

ofacetyl-CoAisprotectedfromprotonationbywater.Inthemonomerofcitratesynthase,citrateisshowningreen,andCoAisred.InducedFitmodelTheStructureofCitrateSynthRegulationofCitrateSynthaseCitratesynthaseisthefirststepinthismetabolicpathway,andasstatedthereactionhasalargenegative?G.Asmightbeexpected,itisahighlyregulatedenzyme.NADH,aproductoftheTCAcycle,isanallostericinhibitor

ofcitratesynthase,asissuccinyl-CoA,theproductofthefollowingstepinthecycle(andanacetyl-CoAanalog).RegulationofCitrateSynthase19.5TheIsomerizationofCitratebyAconitase順烏頭酸酶Citrateitselfposesaproblem:itisapoorcandidateforfurtheroxidationbecauseitcontainsatertiaryalcohol,whichcouldbeoxidizedonlybybreakingacarbon–carbonbond.Anobvioussolutiontothisproblemistoisomerizethetertiaryalcoholtoasecondaryalcohol.Citrateisisomerizedtoisocitratebyaconitase.19.5TheIsomerizationofCit19.6IsocitrateDehydrogenase—TheFirstOxidationintheCycleInthenextstepoftheTCAcycle,isocitrateisoxidativelydecarboxylated脫羧byIsocitrateDehydrogenase異檸檬酸脫氫酶toyield-ketoglutarate酮戊二酸鹽,withconcomitantreductionofNADtoNADH.Thereactionhasanet?G°of-8.4kJ/mol,anditissufficientlyexergonictopulltheaconitasereactionforward.19.6IsocitrateDehydrogenaseIsocitrateDehydrogenaseIsocitratedehydrogenaseprovidesthefirstconnectionbetweentheTCAcycleandtheelectrontransportpathwayandoxidativephosphorylation,viaitsproductionofNADH.Asaconnectingpointbetweentwometabolicpathways,isocitratedehydrogenaseisaregulatedreaction.NADHandATPareallostericinhibitors,whereasADPactsasanallostericactivator,loweringtheKmforisocitratebyafactorof10.TheenzymeisvirtuallyinactiveintheabsenceofADP.IsocitrateDehydrogenase19.7-KetoglutarateDehydrogenase—ASecondDecarboxylationAsecondoxidativedecarboxylationoccursinthe-ketoglutaratedehydrogenasereaction.-酮戊二酸鹽乙酰琥珀酸-ketoglutaratedehydrogenaseisamultienzymecomplex—consistingof-ketoglutaratedehydrogenase,dihydrolipoyltranssuccinylase,anddihydrolipoyldehydrogenase—thatemploysfivedifferentcoenzymes.Thefreeenergychangesforthesereactionsare-29to-33.5kJ/mol.ThisreactionproducesNADHandsuccinyl-CoA.Succinyl-CoAandNADHproductsareenergy-richspeciesthatareimportantsourcesofmetabolicenergyinsubsequentcellularprocesses.19.7-KetoglutarateDehydroge19.8Succinyl-CoASynthetase—ASubstrate-LevelPhosphorylationSuccinyl-CoA乙酰琥珀酸

isitselfahigh-energyintermediateandisutilizedinthenextstepoftheTCAcycletodrivethephosphorylationofGDPtoGTP(inmammals)orADPtoATP(inplantsandbacteria).Succinyl-CoAsynthetaseprovidesanotherexampleofasubstrate-levelphosphorylation

(likePEPinChapter19),inwhichasubstrate,ratherthananelectrontransportchainorprotongradient,providestheenergyforphosphorylation.ItistheonlysuchreactionintheTCAcycle.19.8Succinyl-CoASynthetase—TheGTPproducedbymammalsinthisreactioncanexchangeitsterminalphosphorylgroupwithADPviathenucleosidediphosphatekinasereaction:TheGTPproducedbymammalsinTheFirstFiveStepsoftheTCACycleProduceNADH,CO2,GTP(ATP),andSuccinateAtwo-carbonacetylgrouphasbeenintroducedasacetyl-CoAandlinkedtooxaloacetate,草酰乙酸andtwoCO2moleculeshavebeenliberated.ThecyclehasproducedtwomoleculesofNADHandoneofGTPorATP,andhasleftamoleculeofsuccinate琥珀酸.TheTCAcyclecannowbecompletedbyconvertingsuccinatetooxaloacetate.TheFirstFiveStepsoftheTC19.9SuccinateDehydrogenase—AnOxidationInvolvingFADTheoxidationofsuccinatetofumarateiscarriedoutbysuccinatedehydrogenase.IncontrastwithalloftheotherenzymesoftheTCAcycle,whicharesolubleproteinsfoundinthemitochondrialmatrix,succinatedehydrogenaseisanintegralmembraneprotein

tightlyassociatedwiththeinnermitochondrialmembrane

andisactuallypartoftheelectrontransportchain.19.9SuccinateDehydrogenase—19.10Fumarase延胡索酸酶CatalyzesTrans-HydrationofFumarate蘋(píng)果酸鹽延胡索酸鹽Fumarateishydrated結(jié)合水inastereospecificreactionbyfumarasetogiveL-malate.Thereactioninvolvestrans-additionoftheelementsofwateracrossthedoublebond.19.10Fumarase延胡索酸酶Catalyze19.11MalateDehydrogenase脫氫酶—CompletingtheCycle草酰乙酸鹽蘋(píng)果酸鹽InthelaststepoftheTCAcycle,L-malateisoxidizedtooxaloacetatebymalatedehydrogenase.Thisreactionisveryendergonic,withaG°of+30kJ/mol.Consequently,theconcentrationofoxaloacetateinthemitochondrialmatrixisusuallyquitelow.Thereaction,however,ispulledforwardbythefavorablecitratesynthasereaction.OxidationofmalateiscoupledtoreductionofyetanothermoleculeofNAD+,thethirdoneofthecycle.19.11MalateDehydrogenase脫氫19.12ASummaryoftheCycleThenetreactionaccomplishedbytheTCAcycle,asfollows,showstwomoleculesofCO2,oneATP,andfourreducedcoenzymesproducedperacetategroup

oxidized.Thecycleisexergonic,withanet?G°foronepassaroundthecycleofapproximately-40kJ/mol.Glucosemetabolizedviaglycolysisproducestwomoleculesofpyruvateandthustwomoleculesofacetyl-CoA,whichcanentertheTCAcycle.CombiningglycolysisandtheTCAcyclegivesthenetreactionshown:19.12ASummaryoftheCycleT食品生物化學(xué)Chapter-19-The-Tricarboxylic-Acid-Cycle課件食品生物化學(xué)Chapter-19-The-Tricarboxylic-Acid-Cycle課件AllsixcarbonsofglucoseareliberatedasCO2,andatotaloffourmoleculesofATPareformedthusfarinsubstrate-levelphosphorylations.Atotalof3ATPperNADHand2ATPperFADH2maybeproducedthroughtheprocessesofelectrontransportandoxidativephosphorylation.Thusthe12reducedcoenzymesproduceduptothispointcaneventuallyproduceamaximumof34moleculesofATPintheelectrontransportandoxidativephosphorylationpathways.Together,38moleculesofATP.Butitisnotthefinalanswer.Allsixcarbonsofglucoseare19.13TheTCACycleProvidesIntermediatesforBiosyntheticPathwaysUntilnowwehaveviewedtheTCAcycleasacatabolicprocessbecauseitoxidizesacetateunitstoCO2andconvertstheliberatedenergytoATPandreducedcoenzymes.TheTCAcycleis,afterall,theendpointforbreakdownoffoodmaterials,atleastintermsofcarbonturnover.However,four-,five-,andsix-carbonspeciesproducedintheTCAcyclealsofuelavarietyofbiosyntheticprocesses.

-Ketoglutarate,succinyl-CoA,fumarate,andoxaloacetateareallprecursorsofimportantcellularspecies.Inordertoparticipateineukaryoticbiosyntheticprocesses,however,theymustfirstbetransportedoutofthemitochondria.琥珀酸鹽延胡索酸鹽醋酸鹽蘋(píng)果酸鹽檸檬酸鹽草酰乙酸鹽,丁酮二酸鹽19.13TheTCACycleProvidesTheTCAcycleprovidesintermediatesfornumerousbiosyntheticprocessesinthecell卟啉

TheTCAcycleprovidesintermeExportofcitratefrommitochondriaandcytosolicbreakdownproducesoxaloacetateandacetyl-CoA.Oxaloacetateisrecycledtomalateorpyruvate,whichre-entersthemitochondria.Thiscycleprovidesacetyl-CoAforfattyacidsynthesisinthecytosol.Exportofcitratefrommitocho19.14TheAnaplerotic補(bǔ)給,or“FillingUp,”ReactionsInasortofreciprocalarrangement,thecellalsofeedsmanyintermediatesbackintotheTCAcyclefromotherreactions.Thecatabolismofaminoacidsprovidespyruvate,acetyl-CoA,oxaloacetate,fumarate,-ketoglutarate,andsuccinate,allofwhichmaybeoxidizedbytheTCAcycle.19.14TheAnaplerotic補(bǔ)給,or“TheAnaplerotic補(bǔ)給,or“FillingUp,”ReactionsTheAnaplerotic補(bǔ)給,or“FillingPEPcarboxylaseandpyruvatecarboxylasesynthesizeoxaloacetate.Pyruvatecarboxylaseisthemostimportantanapleroticreactions.Itexistsinthemitochondriaofanimalcellsbutnotinplants,anditprovidesadirectlinkbetweenglycolysisandtheTCAcycle.Pyruvatecarboxylasehasanabsoluteallostericrequirementforacetyl-CoA.Thus,whenacetyl-CoAlevelsexceedtheoxaloacetatesupply,allostericactivationofpyruvatecarboxylasebyacetyl-CoAraisesoxaloacetatelevels,sothattheexcessacetyl-CoAcanentertheTCAcycle.PEPcarboxylaseandpyruvatec19.15RegulationoftheTCACycleSituatedasitisbetweenglycolysisandtheelectrontransportchain,theTCAcyclemustbecarefullycontrolledbythecell.Ifthecyclewerepermittedtorununchecked,largeamountsofmetabolicenergycouldbewastedinoverproductionofreducedcoenzymesandATP;conversely,ifitrantooslowly,ATPwouldnotbeproducedrapidlyenoughtosatisfytheneedsofthecell.Also,asjustseen,theTCAcycleisanimportantsourceofprecursorsforbiosyntheticprocessesandmustbeabletoprovidethemasneeded.19.15RegulationoftheTCAC食品生物化學(xué)Chapter-19-The-Tricarboxylic-Acid-Cycle課件WhatarethesitesofregulationintheTCAcycle?Baseduponourexperiencewithglycolysis,wemightanticipatethatsomeofthereactionsoftheTCAcyclewouldoperatenearequilibriumundercellularconditions(with?G≈0),whereasothers—thesitesofregulation—wouldbecharacterizedbylarge,negative?Gvalues.Threereactionsofthecycle—citratesynthase,isocitratedehydrogenase,and-ketoglutaratedehydrogenase—operatewithlarge,negative?Gvaluesundermitochondrialconditionsandarethustheprimarysitesofregulationinthecycle.Theprincipalregulatory“signals”areacetyl-CoA,ATP,NAD,andNADH.WhatarethesitesofregulatiTheTCAcycleisturnedonwheneithertheADP/ATPorNAD/NADHratioishigh,anindicationthatthecellhasrunlowonATPorNADHthusreflectingtheenergystatusofthecell.NADHinhibitsPyruvatedehydrogenase,citratesynthase,isocitratedehydrogenase,and-ketoglutaratedehydrogenaseTheTCAcycleisturnedonwheAcetyl-CoA

actsasasignaltotheTCAcyclethatglycolysisorfattyacidbreakdownisproducingtwo-carbonunits.Acetyl-CoAactivatespyruvatecarboxylase,theanapleroticreactionthatprovidesoxaloacetate,theacceptorforincreasedfluxofacetyl-CoAintotheTCAcycle.Ontheotherhand,

succinyl-CoA

isanintracycleregulator,inhibitingcitratesynthaseand-ketoglutaratedehydrogenase.Acetyl-CoAactsasasignaltoSupplementaldataSupplementaldata食品生物化學(xué)Chapter-19-The-Tricarboxylic-Acid-Cycle課件19.16TheGlyoxylateCycle乙醛酸循環(huán)(支路)ofPlantsandBacteria--organismsuseacetate醋酸鹽astheirsolecarbonsourceTCAcyclegivesoff2CO2foreverytwo-carbonacetategroupthatentersthuscannoteffectthenetsynthesisofTCAcycleintermediates.

PlantsandbacteriaemployamodificationoftheTCAcyclecalledtheglyoxylatecycle

toproducefour-carbondicarboxylicacids(andeventuallyevensugars)fromtwo-carbonacetateunits.TheglyoxylatecyclebypassesthetwooxidativedecarboxylationsoftheTCAcycle,andinsteadroutesisocitratethroughtheisocitratelyaseandmalatesynthasereactions19.16TheGlyoxylateCycle乙醛TheGlyoxylateCycleOperatesinSpecializedOrganellesTheenzymesoftheglyoxylatecycleinplantsarecontainedinglyoxysomes,乙醛酸循環(huán)體

organellesdevotedtothiscycle.Yeastandalgaecarryouttheglyoxylatecycleinthecytoplasm.TheenzymescommontoboththeTCAandglyoxylatepathwaysexistasisozymes,withspatiallyandfunctionallydistinctenzymesoperatingindependentlyinthetwocycles.TheGlyoxylateCycleHelpsPlantsGrowintheDarkTheexistenceoftheglyoxylatecycleexplainshowcertainseedsgrowunderground(orinthedark),wherephotosynthesisisimpossible.Manyseeds(peanuts,soybeans,andcastorbeans,forexample)arerichinlipids;andmostorganismsdegradethefattyacidsoflipidstoacetyl-CoA.Glyoxysomesforminseedsasgerminationbegins,andtheglyoxylatecycleusestheacetyl-CoAproducedinfattyacidoxidationtoprovidelargeamountsofoxaloacetateandotherintermediatesforcarbohydratesynthesis.OncethegrowingplantbeginsphotosynthesisandcanfixCO2toproducecarbohydrates,theglyoxysomesdisappear.TheGlyoxylateCycleOperatesChapter19

TheTricarboxylicAcidCycle[tra?,kɑ?b?k's?l?k]Chapter19

TheTricarboxylicA食品生物化學(xué)Chapter-19-The-Tricarboxylic-Acid-Cycle課件Aerobiccellsuseametabolicwheel--thetricarboxylicacidcycle

三羧酸循環(huán)

(alsocalledthecitricacidcycle

檸檬酸循環(huán),orKrebscycle)—togenerateenergybyacetyl-CoAoxidation.Aerobiccellsuseametabolic19.1HansKrebsandtheDiscoveryoftheTCACycle19.2TheTCACycle—ABriefSummary19.3TheBridgingStep:OxidativeDecarboxylationofPyruvate19.4EntryintotheCycle:TheCitrateSynthaseReaction19.5TheIsomerizationofCitratebyAconitase19.6IsocitrateDehydrogenase—TheFirstOxidationintheCycle19.7-KetoglutarateDehydrogenase—ASecondDecarboxylation19.8Succinyl-CoASynthetase—ASubstrate-LevelPhosphorylation19.9SuccinateDehydrogenase—AnOxidationInvolvingFAD19.10FumaraseCatalyzesTrans-HydrationofFumarate19.11MalateDehydrogenase—CompletingtheCycle19.12ASummaryoftheCycle19.13TheTCACycleProvidesIntermediatesforBiosyntheticPathways19.14TheAnaplerotic,or“FillingUp,”Reactions19.15RegulationoftheTCACycle19.16TheGlyoxylateCycleofPlantsandBacteriaOUTLINE19.1HansKrebsandtheDiscoTricarboxylicAcidCycleTricarboxylicAcidCyclePyruvateproducedinglycolysisisoxidizedinthetricarboxylicacid(TCA)cycle.ElectronsliberatedinthisoxidationflowthroughtheelectrontransportchainanddrivethesynthesisofATPinoxidativephosphorylation.Ineukaryoticcells,thisoverallprocessoccursinmitochondria.Pyruvateproducedinglycolysi19.1HansKrebsandtheDiscoveryoftheTCACycleIn1932Krebswasstudyingtheratesofoxidationofsmallorganicacidsbykidneyandlivertissue.Onlyafewsubstanceswereactiveintheseexperiments—notablycitrate,succinate,fumarate,malate,andacetate.Lateritwasfoundthatoxaloacetate

couldbemadefrompyruvateinsuchtissues,andthatitcouldbefurtheroxidizedliketheotherdicarboxylicacids.琥珀酸鹽延胡索酸鹽醋酸鹽蘋(píng)果酸鹽檸檬酸鹽草酰乙酸鹽,丁酮二酸鹽ThesesubstanceswerethepiecesintheTCApuzzlethatKrebsandotherseventuallysolved.19.1HansKrebsandtheDisco食品生物化學(xué)Chapter-19-The-Tricarboxylic-Acid-Cycle課件In1935inHungary,acrucialdiscoverywasmadebyAlbertSzent-Gy?rgyi,

whowasstudyingtheoxidationofsimilarorganicsubstratesbypigeonbreast

muscle,anactiveflightmusclewithveryhighratesofoxidationandmetabolism.

Carefullymeasuringtheamountofoxygenconsumed,heobservedthat

additionofanyofthreefour-carbondicarboxylicacids—succinate,

fumarate,ormalate—causedtheconsumptionofmuchmoreoxygenthanwasrequired

fortheoxidationoftheaddedsubstanceitself.Heconcludedthatthesesubstances

werelimitinginthecelland,whenprovided,stimulatedoxidationofendogenousglucoseandother

carbohydratesinthetissues.

Healsofoundthatmalonate丙二酸

,acompetitiveinhibitorofsuccinatedehydrogenase,inhibitedtheseoxidativeprocesses;thisfindingsuggestedthatsuccinateoxidationisacrucialstep.AlbertSzent-Gy?rgyi

hypothesizedthatthesedicarboxylicacidswerelinked

byanenzymaticpathwaythatwasimportantforaerobicmetabolism.In1935inHungary,acrucialAnotherimportantpieceofthepuzzlecamefromtheworkofCarlMartiusandFranzKnoop,whoshowedthatcitricacidcouldbeconvertedtoisocitrateandthento

-ketoglutarate.Thisfindingwassignificantbecauseitwasalreadyknownthat-ketoglutaratecouldbeenzymaticallyoxidizedtosuccinate.Atthisjuncture,thepathwayfromcitratetooxaloacetateseemedtobeasshownhere.Whereasthepathwaymadesense,thecatalyticeffectofsuccinateandtheotherdicarboxylicacidsfromSzent-Gy?rgyi’sstudiesremainedapuzzle.AnotherimportantpieceoftheIn1937Krebsfoundthatcitratecouldbeformedinmusclesuspensionsifoxaloacetateandeitherpyruvateoracetatewereadded.Hesawthathenowhadacycle,notasimplepathway,andthatadditionofanyoftheintermediatescouldgeneratealloftheothers.The

existenceofacycle,togetherwiththeentryofpyruvateintothecycleinthesynthesisofcitrate,providedaclearexplanationfortheacceleratingpropertiesofsuccinate,fumarate,andmalate.Ifalltheseintermediatesledtooxaloacetate,whichcombinedwithpyruvatefromglycolysis,theycouldstimulatetheoxidationofmanysubstancesbesidesthemselves.(Kreb’sconceptualleaptoacyclewasnothisfirst.TogetherwithmedicalstudentKurtHenseleit,hehadalreadyelucidatedthedetailsoftheureacyclein1932.)In1937KrebsfoundthatcitraThecompletetricarboxylicacid(Krebs)cycle,asitisnowunderstood,isshown.Thecompletetricarboxylicaci19.2TheTCACycle—ABriefSummaryTheentryofnewcarbonunitsintothecycleisthroughacetyl-CoA.Thisentrymetabolitecanbeformedeitherfrompyruvate(fromglycolysis)orfromoxidationoffattyacids.Transferofthetwo-carbonacetylgroupfromacetyl-CoAtothefour-carbonoxaloacetatetoyieldsix-carboncitrateiscatalyzedbycitratesynthase.Adehydration–rehydrationrearrangementofcitrateyieldsisocitrate.Twosuccessivedecarboxylationsproduce-ketoglutarateandthensuccinyl-CoA,aCoAconjugateofafour-carbonunit.Severalstepslater,oxaloacetateisregeneratedandcancombinewithanothertwo-carbonunitofacetyl-CoA.Thus,carbonentersthecycleasacetyl-CoAandexitsasCO2.Intheprocess,metabolicenergyiscapturedintheformofATP,NADH,andenzyme-boundFADH2.19.2TheTCACycle—ABriefSu19.3TheBridgingStep:OxidativeDecarboxylationofPyruvatePyruvateproducedbyglycolysisisasignificantsourceofacetyl-CoAfortheTCAcycle.Because,ineukaryoticcells,glycolysisoccursinthecytoplasm,whereastheTCAcyclereactionsandallsubsequentstepsofaerobicmetabolismtakeplaceinthemitochondria,pyruvatemustfirstenterthemitochondriatoentertheTCAcycle.19.3TheBridgingStep:OxidaThereactioniscatalyzedbypyruvatedehydrogenase,amultienzymecomplex.Thepyruvatedehydrogenasecomplex(PDC)isanoncovalentassemblyofthreedifferentenzymesoperatinginconcerttocatalyzesuccessivestepsintheconversionofpyruvatetoacetyl-CoA.Theactivesitesofallthreeenzymesarenotfarremovedfromoneanother,andtheproductofthefirstenzymeispasseddirectlytothesecondenzymeandsoon,withoutdiffusionofsubstratesandproductsthroughthesolution.Thereactioniscatalyzedbyp19.4EntryintotheCycle:TheCitrateSynthaseReaction草酰乙酸檸檬酸ThefirstreactionwithintheTCAcycle,theonebywhichcarbonatomsareintroduced,ist