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1、Chapter22Glycolysis,Outline,OverviewofGlycolysisAllglycolysisreactionsFatesofNADHandPyruvateInthepresenceofO2IntheabsenceofO2HowothersubstancesotherthanglucoseentertheglycolysisPhysiologicalfunctionofglycolysisRegulationofglycolysis,TheEmbden-Meyerhof(Warburg)PathwayEssentiallyoccurringinallcellsCel
2、lularlocation:cytosolTenreactions-sameinallcells-butratesdifferTwophases:1stphase-investingphase:glucoseF-1,6-2P2G-3-P2ndphase-harvestingphase:producestwopyruvates,ATPsandNADHThreepossiblefatesforpyruvate,OverviewofGlycolysis,Two-phasereactionsofglycolysis,EnergyInvestmentPhase,EnergyYieldingPhase,A
3、llglycolysisreactions,Dontpanic!,Allyouhavetomemorizearethetotalreaction,threerate-limitingreactions,threeinhibitors,twosubstrate-levelphosphorylationstepsandmajorregulationmechanisms.,Youarenotresponsibleforanystructures,FirstPhaseofGlycolysis,Thefirstreaction-phosphorylationofglucoseHexokinaseorgl
4、ucokinaseThisisaprimingreaction-ATPisconsumedhereinordertogetmorelaterATPmakesthephosphorylationofglucosespontaneous,1ststepinglycolysis;Glarge,negativeCatalyzedbyHexokinaseorglucokinaseFirstirreversiblereactionHexokinase(andglucokinase)acttophosphorylateglucoseandkeepitinthecellKmforglucoseis0.1mM;
5、cellhas4mMglucoseSohexokinaseisnormallyactive!Glucokinase(Kmglucose=10mM)onlyturnsonwhencellisrichinglucoseHexokinaseisregulated-allostericallyinhibitedby(product)glucose-6-P,Step1:PhosphorylationofGlucose,PhosphorylationofGlucose,SignificanceofGlucosePhosphorylation,Agoodillustrationfortheinducedfi
6、tmodel,BindingofglucosetoHexokinaseinducesalargeconformationalchangeThissamechangeinconformationisobservedforALLkinases!ItalsoaccountsforthefactthatwatercannotbeusedforhydrolysisofATPunlesswefooltheenzymebyusingxyloseinsteadofglucose.,Inducedfit,Glucose-6-PtoFructose-6-PCatalyzedbyPhosphoglucoisomer
7、aseWhydoesthisreactionoccur?nextstep(phosphorylationatC-1)wouldbetoughforhemiacetal-OH,buteasyforprimary-OHisomerizationactivatesC-3forcleavageinaldolasereactionEne-diolintermediateinthisreactionAmoonlightingprotein-doubleasakindofnervegrowthfactor,Step2:IsomerizationofPhosphoglucose,Isomerizationof
8、Phosphoglucose,Thecommittedstepinglycolysis!CatalyzedbyPhosphofructokinase(PFK)AnotherirreversiblereactionThesecondprimingreactionofglycolysisCommittedstepandlarge,-G-meansPFKishighlyregulated,Step3:FurtherPhosphorylationofPhosphoglucose,FurtherPhosphorylationofPhosphoglucose,C6cleavesto2C3s(DHAP,Gl
9、y-3-P)CatalyzedbyAldolaseAnimalaldolasesareClassIaldolasesClassIaldolasesformcovalentSchiffbaseintermediatebetweensubstrateandactivesitelysineUnderstandtheevidenceforSchiffbaseintermediate,Step4:SplittingofSix-CarbonSugar,SplittingofSix-CarbonSugar,AmechanismforClassIAldolasereaction,AmechanismforCl
10、assIIAldolasereaction,DHAPconvertedtoGly-3-PCatalyzedbyTriosePhosphateIsomerase(TIM)Anene-diolmechanism(knowit!)ActivesiteGluactsasgeneralbaseTIMisanear-perfectenzyme(why?)-Reactionrateislimitedonlybytheratethatsubstratecollideswiththeenzyme.,Step5:IsomerizationofPhosphotriose,IsomerizationofPhospho
11、triose,TIMstructureisanbarrel,orTIMbarrel.Inanbarrelthereare8parallel-strandssurroundedby8-helices.Shortloopsconnectalternating-strands&-helicesThestructureoftheTIMbarrel,andtheloopthatformsalidthatclosesovertheactivesiteafterbindingofthesubstrate.,TIM,AmechanismforTIMreaction,Glycolysis-SecondPhase
12、,Metabolicenergyproduces4ATPNetATPyieldforglycolysisistwoATPSecondphaseinvolvestwoveryhighenergyphosphateintermediates1,3BPGPhosphoenolpyruvate(PEP),Gly-3Pisoxidizedto1,3-BPGCatalyzedbyGly-3-DehydrogenaseOnlyoneoxi-reductionreactionEnergyyieldfromconvertinganaldehydetoacarboxylicacidisusedtomake1,3-
13、BPGandNADHMechanisminvolvescovalentcatalysisandNAD+-knowitArsenate(AsO43-)cansubstituteforHPO42-inthisrxnbecauseitisverysimilarinstructureandreactivity,butresulting1-arseno-3-phosphoglycerateisunstableandwillspontaneouslyhydrolyzeto3-phosphoglycerateOxidationofsubstrateisuncoupledfromATPsynthesis,so
14、glycolysisbecomesfutile-nousableenergyisextractedfromglucose:arsenateisapotentpoison!,Step6:DehydrogenationofGly-3P,DehydrogenationofGly-3P,AmechanismforGly-3-Dehydrogenasereaction,InhibitionofGly-3-DehydrogenasebyIodoacetate,ATPsynthesisfromahigh-energyphosphateCatalyzedbyPhosphoglycerateKinaseThis
15、isreferredtoassubstrate-levelphosphorylation2,3-BPG(forhemoglobin)ismadebycircumventingthePGKreaction,Step7:FirstSubstrate-levelPhosphorylation,Substrate-LevelPhosphorylation,ATPisformedwhenanenzymetransfersaphosphategroupfromasubstratetoADP.,FirstSubstrate-levelPhosphorylation,FormationandHydrolysi
16、sof2,3-BPG,Step8:Formationof2-Phosphoglycerate,PhosphorylgroupfromC-3toC-2CatalyzedbyPhosphoglycerateMutaseRationaleforthisenzyme-repositionsthephosphatetomakePEPNotethephospho-histidineintermediates!Abitof2,3-BPGisrequiredtophosphorylateHis,Formationof2-Phosphoglycerate,Amechanismforthephosphoglyce
17、ratemutasereaction,2-P-GlytoPEPCatalyzedbyEnolaseOverallGis1.8kJ/molHowcansuchareactioncreateaPEP?Energycontentof2-PGandPEParesimilarEnolasejustrearrangestoaformfromwhichmoreenergycanbereleasedinhydrolysis,Step9:FormationofPEP,FormationofPEP,Step10:Anothersubstrate-levelphosphorylation,PEPtoPyruvate
18、makesATPThethirdirreversiblereactionCatalyzedbyPyruvateKinaseThesetwoATP(fromoneglucose)canbeviewedasthepayoffofglycolysisLarge,negativeG-regulation!,AnotherSubstrate-levelPhosphorylation,TheFateofNADHandPyruvate,NADHisenergy-twopossiblefates:IfO2isavailable,NADHentersintoMitochondriabytwoshuttlepat
19、hways,whereitisre-oxidizedintheelectrontransportpathway,makingATPinoxidativephosphorylation.Inanaerobicconditions,NADHisre-oxidizedbylactatedehydrogenase(LDH),providingadditionalNAD+formoreglycolysis,Aerobicoranaerobic?,FatesofPyruvate,Go=-146kJ/mol,Potentialenergy=2840kJ/mol,Go=-235kJ/mol,Go=-196kJ
20、/mol,Go=-1160kJ/mol,Malateaspartateshuttle,Theglycerolphosphateshuttle,EnergeticsofGlycolysis,Theelegantevidenceofregulation!StandardstateGvaluesarescattered:+and-Gincellsisrevealing:Mostvaluesnearzero3of10Rxnshavelarge,negativeGLargenegativeGRxnsaresitesofregulation!,OtherSubstratesforGlycolysis,Gl
21、ycerol,Fructose,mannoseandgalactoseGycerolischangedintoDHAPFructoseandmannoseareroutedintoglycolysisbyfairlyconventionalmeans.Galactoseismoreinteresting-theLeloirpathwayconvertsgalactosetoglucose-sortof.,OtherSubstratesforGlycolysis,theLeloirpathway,SignificanceofGlycolysis,ProduceATPsProvidebiosynt
22、heticmaterialsMoonlightingfunctionGlycolysisandCancerHypoxiaandhyoxia-induciblefactor,GlycolysisProvidesbiosyntheticmaterials,Regulationofglycolysis,1.WhenATPlevelsaresufficient,glycolysisactivitydecreasesHexokinaseinhibitedbyexcessglucose6-phosphatePFK-1isinhibitedbyATPandcitratePyruvatekinaseisinh
23、ibitedbyATP2.WhenATPisneeded,glycolysisisactivatedAMP(theproductofATPconsumption)relievetheinhibitionofPFK-1byATPFructose2,6-bisphosphate(F2,6BP)relievetheinhibitionofPFK-1byATPPyruvatekinaseisactivatedbyF1,6BP,Regulationofglycolysis,Step1,Step3,Step10,RegulationofHexokinase,Hexokinasereactionismeta
24、bolicallyirreversibleG6P(product)levelsincreasewhenglycolysisisinhibitedatsitesfurtheralonginthepathwayG6PinhibitshexokinaseisozymesI,IIandIIIthatareactiveatnormalglucoseconcentrations(Km10-6to10-4M)Glucokinase(hexokinaseIV,Km10-2M)formsG6Pintheliver(forglycogensynthesis)whenglucoseisabundant(activi
25、tyismodulatedbyfructosephosphatesandaregulatoryprotein),RegulationofPFK-1,ATPisasubstrateandanallostericinhibitorofPFK-1AMPallostericallyactivatesPFK-1byrelievingtheATPinhibition(ADPisalsoanactivatorinmammaliansystems)ChangesinAMPandADPconcentrationscancontrolthefluxthroughPFK-1Elevatedlevelsofcitra
26、te(indicateamplesubstratesforcitricacidcycle)alsoinhibitPFK-1,RegulationofPFK-1byF2,6-BP,F2,6-BPisapotentactivatorofPFK-1F2,6-BPisformedfromF6Pbyphosphofructokinase-2(PFK-2),whichisabi-functionalenzyme,b-D-Fructose2,6-bisphosphate,GlucagonslowsglycolysisGlucagoninducesglucosesynthesisandconversionto
27、glucosefromglycogen,Effectofglucagononliverglycolysis,RegulationofPyruvateKinase,FourPKisozymesexistinmammaliantissuesPKisallostericallyactivatedbyF1,6BP,inhibitedbyATPGlucagonstimulatesproteinkinaseAwhichphosphorylatesPKconvertingittoalessactiveform(liverandintestine),ActivatorF1,6BP(red)boundwithP
28、K,ThePasteurEffect,Underanaerobicconditionstheconversionofglucosetopyruvateismuchhigherthanunderaerobicconditions(yeastcellsproducemoreethanolandmusclecellsaccumulatelactate)ThePasteurEffectistheslowingofglycolysisinthepresenceofoxygenMoreATPisproducedunderaerobicconditionsthanunderanaerobicconditions,thereforelessglucoseisconsumedaerobically,