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Glycolysis andKrebsCycle
JANegrn, Ph.D. Chem 4220

JANegrn n, , Ph.D. Chem 4220

Summary
Glycolysis
Glycolytic pathway Ratelimitingsteps Piruvate AcetylCoA

KrebsCycle Energyrelationships Studyquestions

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Glycolysis andHistory
1897:Hans&EduardBuchner,attemptingto manufacturecell freeextractsofyeastfor possibletherapeuticuse,addedsucroseasa preservative,anddiscoveredthatthesucrosewas fermentedintoalcohol bytheyeastjuice!!! Thesignificanceofthisfindingwasimmense. TheBuchners demonstratedforthe1sttimethat fermentation couldtakeplaceoutsidelivingcells. Itrefutedthe1860assertionofLouisPasteur,that fermentationisinextricablytiedtolivingcells. Thevitalistic dogmawasgone,andmetabolism JANegrn n, , Ph.D. becamechemistry
Chem 4220

Glycolysis andHistory
Studiesofmuscleextractsthenshowedthat manyofthereactionsoflacticacid fermentationwerethesameasalcohol fermentation. Thisdiscoveryrevealedanunderlyingunityof biochemistry. 1940:thecompleteglycolytic pathwaywas elucidated elucidated. Glycolysis isalsoknownastheEmbden Meyerhofpathway,from thepioneeringwork ofGustavEmbden &OttoMeyerhof
JANegrn n, , Ph.D. Chem 4220

OttoFritzMeyerhof

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Glycolysis
Glycolysis istheanaerobiccatabolismof glucose. l Glucoseisoxidizedtoeitherlactateorpyruvate. Underaerobicconditions,thedominant productinmosttissuesispyruvate pyruvate. . Whenoxygenisdepleted depleted,asforinstanceduring prolongedvigorousexercise,thedominant glycolytic productinmanytissuesislactate lactate.
JANegrn n, , Ph.D. Chem 4220

Glycolysis

Overallreaction:
Glucose+ 2 ADP+ 2 Pi + 2 NAD+ >2 Pyruvate+ 2 ATP+ 2 NADH+ 2 H+ + 2 H2O TheNADHgeneratedduringglycolysis isusedtofuelmitochondrialATP synthesisviaoxidativephosphorylation phosphorylation.
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ReactionsofGlycolysis
Thepathwayofglycolysis canbeseenas consistingof2separatephases. phases Thefirstisthechemicalprimingphaserequiring energyintheformofATP,andthesecondis consideredtheenergyyieldingphase. Inthefirstphase,2equivalentsofATPareused toconvertglucosetofructose1,6bisphosphate (F1 6BP) (F1,6BP). InthesecondphaseF1,6BPisdegradedto pyruvate,withtheproductionof4equivalentsof ATPand2equivalentsofNADH. NADH
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FirstsTwoReactions
Hexokinase trapsglucoseinthecellandbegins glycolysis

Isomerization ofglucose6phosphatetofructose6 phosphatebyphosphoglucose isomerase

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FormationFructose1,6bisphosphate (F1,6BP)
FormationofFructose1,6bisphosphatefrom Glucose6phosphate

Thisreactioniscatalyzedbyphosphofructokinase (PFK),anallosteric enzymethatsetsthepaceof glycolysis.

JANegrn n, , Ph.D. Chem 4220

Phosphofructokinase (PFK)isanimportant enzymeinvirtuallyall livingcells.Itperforms thecommittedstepin theglycolytic pathway. Itisamultisubunit allosteric enzyme

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Phosphofructokinase IstheKey EnzymeintheControlofGlycolysis

HighlevelsofATPallosterically inhibittheenzymeintheliver(a 340kdtetramer), ) thus h lowering l i its i affinityforfructose6phosphate. ATPelicitsthiseffectbybindingtoa specificregulatorysitethatisdistinct fromthecatalyticsite.AMPreverses theinhibitoryactionofATP ATP,andso theactivityoftheenzymeincreases whentheATP/AMPratioislowered. AfallinpH andhighconcentrationof citrate alsoinhibits phosphofructokinase activity.
JANegrn n, , Ph.D. Chem 4220

Fructose2,6bisphosphate (F2,6BP)isa potentactivatorofphosphofructokinase

Fructose2,6 bisphosphate activates phosphofructo kinase by increasingits affinityfor fructose6 phosphateand diminishingthe inhibitory effectofATP.
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Homework Howistheconcentrationof fructose2,6bisphosphate controlled?

JANegrn n, , Ph.D. Chem 4220

Regulation ofPFK

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MalignantCancersExhibitanIncrease inGlycolysis (Warburgeffect),

Mostcancercells Glucose predominantlyproduce Hypoxia + energybyglycolisis Inducible Factor followedbylacticacid (HIF1) fermentationinthe + cytosol,ratherthanby Piruvate oxidationofpyruvate in mitochondrialikemost Christofk,H.R.,etal.2008.TheM2splice normalcells. isoform ofpyruvate kinase isimportantfor Thisoccursevenif cancermetabolismandtumour growth. Nature 452,230233(13March2008). oxygenisplentiful.
JANegrn n, , Ph.D. Chem 4220

http://www.nature.com/nature/journal/v452 /n7184/full/nature06734.html

FormationofPhosphateTrioses
Two3carbon f fragments t areproduced fromone 6carbonsugar (glucose)

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Cleavage of six six-carbon sugar

(DHAP)

(GAPorPGAL)

Thealdolase reactionproceedsreadilyinthereversedirection, beingutilizedforbothglycolysis andgluconeogenesis. Thetwoproductsofthealdolase reactionequilibratereadilyina reactioncatalyzedbytriose phosphateisomerase.

JANegrn n, , Ph.D. Chem 4220

ProductionofNADH
Glyceraldehyde3P dehydrogenase (G3PDH) catalyzes l the h NAD+ dependentoxidationof G3Pto1,3 bisphosphoglycerate (1,3BPG)andNADH. TheG3PDHreactionis reversible,andthesame enzymecatalyzesthe reversereactionduring gluconeogenesis.

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InErythrocytes
Theformationof2,3 bisphosphoglycerate, 2,3BPGbytheenzyme bisphosphoglycerate mutase 2,3BPGisan importantregulatorof hemoglobin's g affinity y foroxygen.

JANegrn n, , Ph.D. Chem 4220

FirstProductionofATP
Thehighenergy phosphate h h t of f1 1,3 3 BPGisusedtoform ATPand3 phosphoglycerate (3PG)bytheenzyme phosphoglycerate kinase.
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ConversionofPhosphoacyestertoa Highenergyform
Theremainingreactionsof glycolysis areaimedat convertingtherelativelylow energyphosphoacylesterof 3PGtoahighenergyform andharvestingthephosphate asATP. The3PGisfirstconvertedto 2PGbyphosphoglycerate mutase andthe2PG conversionto phosphoenoylpyruvate (PEP) iscatalyzedbyenolase.
JANegrn n, , Ph.D. Chem 4220

SecondProductionofATP
Inthisstrongly exergonic reaction, reaction thehighenergy phosphateofPEPis conservedasATP. Thelossof phosphatebyPEP leadstothe productionof pyruvate.
JANegrn n, , Ph.D. Chem 4220

Piruvate Kinase

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Glycolysis Summary

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FateofPiruvate

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ThePyruvate Dehydrogenase (PDH)Complex

Duringtheoxidationof pyruvate toCO2 bypyruvate d h d dehydrogenase th the electronsflowfrom pyruvate tothelipoamide moietyofdihydrolipoyl transacetylase thentothe FADcofactorof dihydrolipoyl dehydrogenase andfinally toreductionofNAD+ to NADH. Theacetylgroupislinkedto coenzymeA(CoASH)ina highenergythioester bond. TheacetylCoA thenenters theTCAcycleforcomplete oxidationtoCO2 andH2O. Pyruvate+CoA+NAD+ >CO2 +acetylCoA+NADH+H+

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Pyruvate Deshydrogenase

JANegrn n, , Ph.D. Chem 4220

RegulationofthePDHComplex
PDHactivityis regulatedbyits'state ofphosphorylation, beingmostactivein the dephosphorylated state. ThePDHcomplex p is inhibitedbyacetyl CoA andNADHand activatedbynon acetylatedCoA (CoASH)andNAD+.
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KrebsCycleorTricarboxilic AcidCycle(TCA)
Sugars Commonpathwayformetabolismof allfuels FattyAcids AminoAcids CharacteristicsofTCAcycle

Occursinthemitochondriasmatrix AlsoknownastheKrebs cycle. Oxidatively stripselectronsfromfats, y andp proteins. carbohydrates

TCA

CO2

FunctionsofTCAcycle=amphibolic
Energyproduction=catabolic. Biosynthesis=anabolic.
JANegrn n, , Ph.D. Chem 4220

Amino Acids, Purines, Pirimidines

Fatty Acids

H+ +e

ElectronTransportChain

KrebsCycleorTricarboxilic AcidCycle(TCA)
AcetylCoA isacentralintermediateinlipidmetabolism Underaerobicconditions theendproductof acid glycolysis ispyruvic acid. Glucose Thenextstepisthe Aminoacids formationofacetyl Piruvate coenzymeA(acetylCoA) thisstepistechnicallynota partofthecitricacidcycle. Fatty acids AcetylCoA Ketone bodies

Triacylglycerides Phospholipids Eicosanoids

JANegrn n, , Ph.D. Chem 4220

TCA

Cholesterol

CO2 +H2O+Energy(ATP)

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KrebsCycleMainReactions
1. Condensation: CitrateSynthase
AcetylCoA +Oxaloacetate Citrate

2a.Dehydratation: Aconitase
Citrate [cisaconitate]

2b.Hydratation:Aconitase
[cisaconitate] Isocitrate

3a.Oxidation:Isocitrate Dehydrogenase
Isocitrate [Oxalosuccinate]

3b.Decarboxylation: Isocitrate Dehydrogenase

[Oxalosuccinate] Ketoglutarate

4. Oxidation,Decarboxylation: Ketoglutarate Dehydrogenase

Ketoglutarate g SuccinylCoA y

5. Hydrolysis,ATPsynthesis: Succinyl CoA Synthase

Succinyl CoA Succinate

6. Oxidation: Succinate Dehydrogenase

Succinate Fumarate

1Condensation 1Dehydratation 2Hydratation 4Oxidation(H2 removal) 3usingNAD 1usingFAD 2Decarboxylation 1ATPsynthesis

7. Hydratation:Fumarase
Fumarate Malate

8. Oxidation:Malate Dehydrogenase
Malate Oxaloacetate
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ADP+Pi ATP
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RegulationoftheTCAcycle
Regulationlikethatofglycolysis, occursatboththelevelofentryof substratesintothecycleaswellasat thekeyreactionsofthecycle. FuelenterstheTCAcycleprimarilyas acetylCoA.Thegenerationofacetyl CoA fromcarbohydratesis,therefore, amajorcontrolpointofthecycle. Thisisthereactioncatalyzedbythe Piruvate Dehydrogenase. y g
Piruvate
Ca+

Inhibition Activation

AcetylCoA

Oxaloacetate Malate F Fumarate t Succinate Succinyl CoA NADH

Citrate

TwolevelsofTCAregulation:
NAD/NADHandADP/ATP Allosteric Enzymes
9 9 9 9 PDH CitrateSinthase Isocitrate Dehydrogenase -Ketoglutarate Dehydrogenase

Isocitrate
Ca+ , ADP

Ketoglutarate
Ca+

JANegrn n, , Ph.D. Chem 4220

Isocitrate Dehydrogenase (IDE)

IDEisamajor regulatory l t point i tin i theTCAcycle.The enzymeis allosterically activated byADPandinhibited byATP.

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ClinicalConsideration
LacticAcidemia
Excessive E i exercise i Excessiveanaerobic metabolismof carbohydrates.
9Brainandmuscle severelyaffectedsince theydependonaerobic metabolism.

JANegrn n, , Ph.D. Chem 4220

LacticAcidosisinHIVpatientsreceivingNRTI

Patient:6.4mM LacticAcid Normal:12mM Mechanismsofnucleosidereversetranscriptaseinhibitor(NRTI)inducedlacticacidosis

VPatelandSSHedayati (2006)LacticacidosisinanHIVinfectedpatientreceivinghighlyactiveantiretroviraltherapy. NatClin Pract Neprol 2: 109114 doi:10.1038/ncpneph00102

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Where do all the NADHs and FADH2s Go?

JANegrn n, , Ph.D. Chem 4220

WhatisthemaximumyieldofhighenergyATPinthe aerobiccatabolismofglucoseinbacteria?
Glycolysis
glucose2pyruvate+2NADH+2ATP8ATPs 2pyruvate 2acetylCoA +2NADH6ATPs acetylCoA2CO2+3NADH+FADH2+GTP2x12ATPs

Pyruvate P t Dehydrogenase D h d TCA

TotalATPfrom1molofglucose38ATPs
30.5kJ/moleATP

=1,160kJ/moleglucose = 1,160kJ[1kcal/4.184kJ]=277kcal/moleglucose

InacalorimetertheG of combustion for 1molglucose= 2820kJ/mol;thereforethecellefficiencyis1160/2880,which isnearly40%.

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StudyQuestions
Whatstheimportanceofthe TCAcycle? Whereisthisprocesscarriedout? HowmanyATPareproduced?
Glucoseanaerobically Glucoseaerobically Piruvate toAcetylCoA ForoneAcetylCoA enteringintoTCA
Aerobically,anaerobically,total

BytheaidofdiagramsexplainthereactionsoftheTCAcycle. Writedowntheoverallstoichiometric equationfortheTCAcycle. WhatarethepointsofregulationinTCAcycle? HowisPyruvate dehydrogenase regulated? Howareaminoacid,carbohydratesandfattyacidsmetabolismrelatedtotheTCAcycle? HowdoestheTCAcyclefunctionasbiosyntheticprecursors? WhatisthecommittedstepinTCAcycle? WhyisitthatGlycolysis cantakeplaceundereitheraerobicoranaerobicconditionsbutthe citricacidcycleproceedsstrictlyunderaerobicconditions?
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