10Glycolysis(I)2024/2/3GlycolyticPathwayRegulationoftheGlycolyticPathwayGluconeogenesisPPP2024/2/3glycolysis(fromtheGreekglykys,meaning“sweet〞〕lysis,meaning“splitting〞Glycolysis------isacatabolicpathwaywhereinglucoseisconvertedtotwomoleculesofpyruvatewiththegenerationoftwomoleculesofATP.2024/2/3Goals:

-Tounderstandtheoverallreaction

ofglycolysis-Tolearnthedetailed10stepsofglycolysisreaction

-Toknowtheenzymecatalyzingeachstepofglycolysisreaction,

particularlyinsteps#1,3,6,7,102024/2/3Metabolism2024/2/3CatabolismEnergy-yieldingreactionsFornon-photosyntheticorganisms,twosourcesoffuelDietFats,carbonhydrates,proteinsStoredcompoundsFats,starch,glycogen2024/2/3Glycolysis:EmbdenMeyerhofpathway1854-1864,LouisPaster,fermentation—〞vitalforce〞1897,HansBuchnerandEduardBuchnerYeastcell-freeextracts,sucroseFirsttimethatfermentationcouldoccuroutsidecellThehistory:2024/2/31905ArthurHardenandWilliamYoungYeastcell-freeextracts;Glucose;Inorganicphosphate;Heat-labile,nondialyzablecomponent(zymase)Heat-stable,dialyzable可透析的fraction(cozymase)2024/2/32024/2/31940,GustavEmbden,OttoMeyerhof,CarlNewberg,JacobParnas,OttoWarburg,Gerty

Cori,andCarlCori

elucidatethecompleteglycolyticpathway2024/2/32024/2/3CarlandGertyCorisharedtheNobelPrizeinPhysiologyorMedicinein1947withBernardoHoussayofArgentina,whowascitedforhisstudiesofhormonalregulationofcarbohydratemetabolism.2024/2/3TheCorilaboratoriesinSt.Louis-----aninternationalcenterofbiochemicalresearch1940s-1950ssixscientistsgotNobellaureates:①ArthurKornberg(forDNAsynthesis,1959),②SeveroOchoa(forRNAsynthesis,1959),③LuisLeloir(fortheroleofsugarnucleotidesinpolysaccharidesynthesis,1970)2024/2/3④EarlSutherland(forthediscoveryofcAMPintheregulationofcarbohydratemetabolism,1971)⑤ChristiandeDuve(forsubcellularfractionation,1974)⑥EdwinKrebs(forthediscoveryofphosphorylasekinase,1991).2024/2/3ImportanceofGlycolysisAnalmostuniversalcentralenergyyieldingpathProvidesprecursorsformanybiosyntheticpathsThestartingpoint－－－glucoseTheprocessends－－－twopyruvatemoleculesAdditionalproductsofglycolysisincludetwoATPsandtwoNADHsIllustratesenzymemechanismsIllustratesregulatorymechanisms2024/2/3sprints,requiresasourceofenergythatcanberapidlyccessed.Theanaerobicmetabolismofglucosetheprocessofglycolysisprovidessuchasourceofenergyforshort,intenseboutsofexercise.2024/2/3GlycolysisThesequenceofreactionsfromglucosetopyruvateacidiscommontocarbohydratemetabolismunderbothaerobicandanaerobicconditions.Glycolysistakesplaceinthecytosol.2024/2/3Someorganisms(yeast)underanaerobicconditionsconvertpyruvatetoethanol------alcoholicfermentationSomemicroorgnism(lacticacidbacteria)------convertpyruvatetolacticacid

------lacticacidfermentationInhumanunderaerobicconditionstheend-productispyruvicacid.PyruvateiscompletelyoxidizedtoCO2intheTCAcycleandlargeamountsofATParesubsequentlyproduced.Underanaerobicconditionstheendproductislacticacid;--------lacticacidfermentation.2024/2/3SomePointsAboutGlucoseGlucoseisverysolublesourceofquickandreadyenergy.Itisarelativelystableandeasilytransported.Inmammals,thebrainusesonlyglucoseundernon-starvationconditions.Glucoseistheonlysourceofenergyinredbloodcells.2024/2/32024/2/32024/2/3Glycolysishas10Steps2024/2/3Hexose

stage:2ATPareconsumedperglucoseTriose

stage:

4ATPareproducedperglucose Net:2ATPproducedperglucoseEachchemicalreactionpreparesasubstrateforthenextstepintheprocessATPisbothconsumedandproducedinglycolysis

2024/2/3TwophaseFirstphase----預(yù)先支出階段---ATPenergyisinvestedSecondphase----收入階段---ATPenergyisgenerated2024/2/32024/2/32024/2/3ImportanceofPhosphorylatedIntermediates1.Plasmamembranegenerallylackstransportersforphosphorylatedglycolyticintermediates.2.Phosphorylgroupsareessentialcomponentsintheenzymaticconservationofmetabolicenergy.Energyreleasedinthebreakageofphosphoanhydridebonds(ATP)ispartiallyconservedintheformationofphosphateesters(glucose6-phosphate).High-energyphosphatecompounds(1,3-bisphosphoglycerate,phosphoenolpyruvate)donatephosphorylgroupstoADPtoformATP.3.Bindingenergyresultingfromthebindingofphosphategroupstotheactivesitesofenzymeslowerstheactivationenergyandincreasesthespecificityoftheenzymaticreactions.2024/2/31stStageof

GlycolysisGlycolysis1stStage2024/2/3Step1.Hexokinasereaction

?Go=–16.7kJ/mol2024/2/3Transferstheg-phosphorylofATPtoglucoseC-6oxygentogenerateglucose6-phosphate(G6P)Mechanism:attackofC-6hydroxyloxygenofglucoseontheg-phosphorousofMgATP2-displacingMgADP-.Fourkinasesinglycolysis:steps1,3,7,and10,allofwhich

requireMg2+andhaveasimilarmechanism.2024/2/3PropertiesofhexokinasesBroad

substrate

specificity-hexokinasescanphosphorylateglucose,mannose甘露糖andfructose果糖

Yeasthexokinaseundergoesaninduced-fit

conformational

changewhenglucosebinds40000倍2024/2/3HexokinaseReactionRecallthe“inducedfit〞ATPandADPalwaysbindtoenzymesasacomplexwiththemetalionMg2+.2024/2/32024/2/3Isozymes-multiple

formsofhexokinase

HexokinasesI,II,IIIareactiveatnormalglucoseconcentrations(Kmvalues~10-6to10-4M)HexokinaseIV(Glucokinase,Km~10-2M)inthehepatocyte

isactiveathigherglucoselevels,allowsthelivertorespondtolargeincreasesinbloodglucoseglycogen2024/2/3Step2.ConversionofG6PtoF6P

?Go=1.7kJ/mol2024/2/3Convertsglucose6-phosphate(G6P)(analdose)tofructose6-phosphate(F6P)(aketose)Enzymepreferentiallybindsthea-anomerofG6P(convertstoopenchainformintheactivesite)EnzymeishighlystereospecificforG6PandF6PIsomerasereactionisnear-equilibriumincells,i.e.,DGisclosetozero.

2024/2/3Step3.Phosphofructokinase-1(PFK-1)Reaction

?Go=–14.2kJ/mol2024/2/3CatalyzestransferofaphosphorylgroupfromATPtotheC-1hydroxylgroupofF6Ptoformfructose1,6-bisphosphate(F1,6BP)PFK-1ismetabolicallyirreversibleandacriticalregulatory

pointforglycolysisinmostcells(PFK-1isthefirstcommittedstepofglycolysis)Asecondphosphofructokinase(PFK-2)synthesizesfructose2,6-bisphosphate(F2,6BP)2024/2/3PhosphofructokinaseComplexenzymeMW360,000Rate-limitingstepinglycolysisMajorcontrolpoint:allostericregulationHighATPinhibitsHighAMP,ADPstimulatesOther“fuels〞alteractivityFru-2,6-bisPhormonalsignal2024/2/3Radioisotopictracerstudiesshow:

OneGAPmolecule:C1,2,3fromGlucoseC4,5,6

SecondGAP:C1,2,3fromGlucoseC3,2,1Step4.AldolaseReaction

2024/2/3AldolasecleavesthehexoseF-1,6-BPintotwotriosephosphates:glyceraldehyde3-phosphate(GAP)anddihydroxyacetonephosphate(DHAP)MechanismiscommonforcleavingC-Cbondsinbiologicalsystems(andC-Cbondformationinthereversedirection)2024/2/3aldolasereaction(thedirectionoffructose1,6-bisphosphatecleavage)stronglypositivealdolasereactionisreadilyreversible.2024/2/32024/2/3TwoclassesofaldolasesClassIaldolasesinanimalsandplants,formtheSchiffbaseintermediateClassIIaldolasesinfungiandbacteria,azincionattheactivesiteiscoordinatedwiththecarbonyloxygenatC-22024/2/3TheclassIaldolasereaction.2024/2/32024/2/3Step5.ReactionofTriosephosphateisomeraseConversionofDHAPintoglyceraldehyde3-phosphate(GAP)allowstobemetabloizedviaglycolyticenzymes.Reactionisveryfast2024/2/32ndStageof

GlycolysisGlycolysis

2ndStage變位酶烯醇化酶脫氫酶2024/2/3?Go=+6.3kJ/molGAPconvertedto1,3BPGStep6.ReactionofGlyceraldehyde3-PhosphateDehydrogenase(GAPDH)2024/2/3ConversionofGAPto1,3-bisphosphoglycerate(1,3BPG).MoleculeofNAD+isreducedtoNADHConservationofoxidativeenergy:EnergyfromoxidationofGAPaldehydeisconservedinacid-anhydride(酸酐)linkageof1,3BPG.OxidationofthealdehydegroupofGAPproceedswithlargenegativefree-energychange.Nextstepofglycolysisusesthehigh-energyphosphateof1,3BPGtoformATPfromADP.2024/2/3Step7.Phosphoglyceratekinasereaction

?Go=–18.5kJ/molTransferofphosphorylgroupfromtheenergy-rich1,3BPGtoADPyieldsATPand3-phosphoglycerate(3PG)2024/2/3PhosphoglycerateKinase親核攻擊2024/2/3Substrate-levelphosphorylation-Steps6and7coupleoxidationofanaldehydetoacarboxylic羧基acidwiththephosphorylationofADPtoATP2024/2/3Substratechannel2024/2/3Step8.Phosphoglyceratemutase變位酶reaction

?Go=+4.4kJ/mol2024/2/3Thephosphoglyceratemutasereaction.2024/2/3CatalyzestransferofaphosphorylgroupfromonepartofasubstratemoleculetoanotherReactionoccurswithoutinputofATPenergyMechanismrequires2phosphoryl-grouptransferstepsAcovalentenzyme-phosphateintermediate(P-histidine)isinvolved2024/2/3Step9.Enolase烯醇酶(2-phosphoglyceratedehydratase脫水酶)reaction?Go=+7.5kJ/mol2024/2/33-Phosphoglycerate(3PG)isdehydratedtophosphoenolpyruvate(PEP)EliminationofwaterfromC-2andC-3yieldstheenol-phosphatePEPPEPhasaveryhighphosphorylgrouptransferpotentialbecauseitexistsinitsunstableenol

烯醇form2024/2/3Step10.PyruvatekinasereactionCatalyzesasubstrate-levelphosphorylation

MetabolicallyirreversiblereactionRegulationbothbyallosteric

modulatorsandbycovalent

modificationPyruvate

kinasegenecanberegulatedbyvarioushormonesandnutrientsPEP+ADP

Pyruvate+ATP?Go=–31.4kJ/mol2024/2/3NetreactionofglycolysisTwomoleculesofATPareproducedTwomoleculesofNAD+arereducedtoNADH

Glucose+2ADP+2NAD++2Pi

2Pyruvate+2ATP+2NADH+2H++2H2ONote:allincytosol2024/2/3TheConversionofGlucosetoPyruvate(Review)TheEnergyreleasedfromtheanaerobicconversionofglucosetopyruvateis--------47kcalmol-1.Underaerobicconditionsmuchmorechemicalbondenergycanbeextractedfrompyruvate.2024/2/3Underanaerobicconditionspyruvateisconvertedtolactate.InexercisingmuscleTheredoxbalanceismaintainedTheNAD+thatisconsumedintheglyceraldehyde3-phosphatereactionisproducedinthelactateDHreaction.Theactivitiesofglyceraldehyde3-phosphateDHandLactateDHarelinkedmetabolically.2024/2/3TheNAD+thatisconsumedintheglyceraldehyde3-phosphatereactionisproducedinthelactateDHreaction.Thus,redoxbalanceismaintained.TheNADHthatisproducedintheglyceraldehyde3-phosphatereactionisconsumedinthelactateDHreaction.Thus,redoxbalanceismaintained.Remember!Glucose+2Pi+2ADP→2lactate+2ATP+2H2O2024/2/32024/2/3Inanaerobicyeast,pyruvate→ethanolPyruvateisdecarboxylated.Acetaldehyde乙醛isreduced.2024/2/3Industrial-scalefermentation2024/2/3Variationsonathemeinalcoholicfermentation.Herealso,thereisnonetoxidationreduction.2024/2/3Whathappenswhenweingest

whattheyeastexcretes.“Itprovokesthedesire,buttakesawaytheperformance.〞

WilliamShakespeare

WilliamShakespeare2024/2/32024/2/3Whathappenswhenweingestwhattheyeastexcretes

inthetermofBiochemistryEthanolismetabolizedtoacetaldehydeandthentoacetateviaalcoholDHandacetaldehydeDH.BothenzymesconsumeNAD+andproduceNADH.Thus,withtheconsumptionofalcoholtheNADH/NAD+ratioisincreased(highNADHinhibitsTCAcycle).Also,asignificantincreaseinfattyacidsynthesisandglycerol3-phosphate,resultingintheaccumulationtriacylglycerolsproducingafattyliver.2024/2/3Lacticacidosis酸毒癥肝(實(shí)質(zhì))細(xì)胞乙醛雙硫醒[治療慢性酒精中毒藥]

2024/2/3Awordonhepaticcirrhosis肝硬化BothalcoholDHandacetaldehydeDHareeasilysaturatedsoafixedquantityofalcoholisslowlymetabolized;about7g/hour.Theaccumulationofacetaldehyde乙醛occurswith“heavydrinking〞.Thehighlyreactiveacetaldehydeistoxiccausingmuchofthetissuedamageinchronicalcoholism急性醇中毒.Itbindscovalentlytoaminogroups,nucleotides,andphospholipidstoformadducts加合物.2024/2/32024/2/3Routesforutilizingsubstratesotherthanglucoseinglycolysis

乳糖半乳糖麥芽糖2024/2/31.PathwayofAtomsinFermentationA“pulse-chase〞experimentusing14C-labeledcarbonsourcesiscarriedoutonayeastextractmaintainedunderstrictlyanaerobicconditionstoproduceethanol.Theexperimentconsistsofincubatingasmallamountof14C-labeledsubstrate(thepulse)withtheyeastextractjustlongenoughforeachintermediateinthefermentationpathwaytobecomelabeled.Thelabelisthen“chased〞throughthepathwaybytheadditionofexcessunlabeledglucose.Thechaseeffectivelypreventsanyfurtherentryoflabeledglucoseintothepathway.problems2024/2/3(a)If[1-14C]glucose(glucoselabeledatC-1with14C)isusedasasubstrate,whatisthelocationof14Cintheproductethanol?Explain.(b)Wherewould14Chavetobelocatedinthestartingglucosetoensurethatallthe14Cactivityisliberatedas14CO2duringfermentationtoethanol?Explain.2024/2/32.GlycolysisShortcutSupposeyoudiscoveredamutantyeastwhoseglycolyticpathwaywasshorterbecauseofthepresenceofanewenzymecatalyzingthereaction:Wouldshorteningtheglycolyticpathwayinthiswaybenefitthecell?Explain.2024/2/34.RequirementforPhosphateinEthanolFermentationIn1906HardenandYoung,inaseriesofclassicstudiesonthefermentationofglucosetoethanolandCO2byextractsofbrewer’syeast,madethefollowingobservations.Inorganicphosphatewasessenti