Glycolysis and Krebs Cycle

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GlycolysisGlycolysis andKrebsCycleandKrebsCycle

JANegrn, Ph.D.

JAJANegrNegrnn, Ph.D., Ph.D.

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Summary Glycolysis

Glycolytic pathway

Ratelimitingsteps

Piruvate

AcetylCoA


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Energyrelationships

Studyquestions


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Glycolysis andHistory

1897:Hans&EduardBuchner,attemptingto

manufacture cell free extracts of east for

possibletherapeuticuse,addedsucroseasa

preservative,anddiscoveredthatthesucrosewas

fermentedintoalcohol bytheyeastjuice!!!

Thesignificanceofthisfindingwasimmense.

TheBuchners demonstratedforthe1sttimethat


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could

take

place

outside

living

cells. Itrefutedthe1860assertionofLouisPasteur,that

fermentationisinextricablytiedtolivingcells.

TheThevitalisticvitalistic dogmawasgone,andmetabolismdogmawasgone,andmetabolism

becamechemistrybecamechemistry

Glycolysis and

History Studiesofmuscleextractsthenshowedthat

man of the reactions of lactic acid

fermentationwerethesameasalcohol

fermentation.

Thisdiscoveryrevealedanunderlyingunityof

biochemistry.

1940:thecompleteglycolytic pathwaywas OttoFritzMeyerhof


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.

GlycolysisGlycolysis isalsoknownastheisalsoknownastheEmbdenEmbden

MeyerhofMeyerhofpathway,frompathway,from thepioneeringworkthepioneeringwork

ofGustavofGustavEmbdenEmbden &OttoMeyerhof&OttoMeyerhof


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Glycolysis

Glycolysis istheanaerobiccatabolismanaerobiccatabolismof

g ucose.

Glucoseisoxidizedtoeitherlactateorpyruvate.

Underaerobicconditions,thedominant

productinmosttissuesispyruvatepyruvate..


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,

prolongedvigorousexercise,thedominantglycolytic productinmanytissuesislactatelactate.

Glycolysis


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Glucose+Glucose+ 22 ADP+ADP+ 22 PPii ++ 22 NADNAD++>2>2 Pyruvate+Pyruvate+ 22 ATP+ATP+ 22 NADH+NADH+ 22 HH++ ++ 22 HH22OO

Overallreaction:

TheNADHgeneratedduringglycolysis isusedtofuelmitochondrialATP

synthesisviaoxidativeoxidativephosphorylationphosphorylation.


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ReactionsofReactionsofGlycolysisGlycolysis

Thepathwayofglycolysis canbeseenasconsistin of2se arate hases2se arate hases.

ThefirstisthechemicalprimingphasechemicalprimingphaserequiringenergyintheformofATP,andthesecondisconsideredtheenergyyieldingphase.

Inthefirstphase,2equivalentsofATPareusedtoconvertglucosetofructose1,6bisphosphate


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, .

InthesecondphaseF1,6BPisdegradedtopyruvate,withtheproductionof4equivalentsofproductionof4equivalentsofATPand2equivalentsofNADHATPand2equivalentsofNADH.

Firsts

Two

ReactionsFirsts

Two

Reactions Hexokinase trapsglucoseinthecellandbegins

glycolysis

Isomerization ofglucose6phosphatetofructose6


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FormationFormationFFructose1,6ructose1,6bisphosphatebisphosphate

(F1,6BP)(F1,6BP) FormationofFructose1,6bisphosphatefrom


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Thisreactioniscatalyzedbyphosphofructokinase

(PFK),anallosteric enzymethatsetsthepaceofglycolysis.

Phosphofructokinase

enzymeinvirtuallyall

livingcells.Itperforms

thecommittedstepin

theglycolytic pathway.


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allosteric enzyme


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PhosphofructokinasePhosphofructokinase IstheKeyIstheKey

Enzyme

in

the

Control

of

Enzyme

in

the

Control

of

GlycolysisGlycolysis HighlevelsofATPHighlevelsofATPallosterically

inhibittheenzymeintheliver(a340kdtetramer),thusloweringitsaffinityforfructose6phosphate.

ATPelicitsthiseffectbybindingtoaspecificregulatorysitethatisdistinctfromthecatalyticsite.AMPreversesAMPreversestheinhibitoryactionofATPtheinhibitoryactionofATP,andsotheactivityoftheenzymeincreases


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. A

fall

in

pHfall

in

pH and

high

concentration

of

citratecitrate alsoinhibitsphosphofructokinase activity.

FFructose2,6ructose2,6bisphosphatebisphosphate (F(F2,62,6BP)isaBP)isapotentactivatorofpotentactivatorofphosphofructokinasephosphofructokinase Fructose2,6Fructose2,6

bis hos hatebis hos hateactivatesphosphofructokinase byincreasingitsaffinityforfructose6


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phosphateanddiminishingtheinhibitoryeffectofATP.


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HomeworkHomework

Howistheconcentrationof

fructose2,6bisphosphate

controlled?


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Regulation of

PFK


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MalignantCancersExhibitanIncrease

in

Glycolysis (Warburg

effect),

Mostcancercellsredominantl roduce

Glucose

energybyglycolisisfollowedbylacticacidfermentationinthecytosol,ratherthanbyoxidationofpyruvate inmitochondrialikemost

Piruvate

+

+

ypox a

Inducible

Factor

(HIF1)


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normalcells.

Thisoccursevenifoxygenisplentiful.

Christofk,H.R.,etal. 2008. TheM2splice

isoform of

pyruvate kinase is

important

for

cancermetabolismandtumour growth.

Nature 452,230233(13March2008).

http://www.nature.com/nature/journal/v452

/n7184/full/nature06734.html

Formation

of

Phosphate

Trioses Two3carbon

ragments

areproduced

fromone

6carbonsugar


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Cleavage of sixCleavage of six--carbon sugarcarbon sugar


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Thealdolase reactionproceedsreadilyinthereversedirection,beingutilizedforbothglycolysis andgluconeogenesis.

Thetwoproductsofthealdolase reactionequilibratereadilyinareactioncatalyzedbytriose phosphateisomerase.

(DHAP) (GAPorPGAL)

Production

of

NADHProduction

of

NADH Glyceraldehyde3P

dehydrogenase (G3PDH)catalyzestheNAD+dependentoxidationofG3Pto1,3bisphosphoglycerate (1,3BPG)andNADH.

TheG3PDHreactionisreversible,andthesame


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reversereactionduringgluconeogenesis.


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InErythrocytesInErythrocytes

Theformationof2,3

bis hos ho l cerate

2,3BPGbytheenzyme

bisphosphoglyceratebisphosphoglycerate

mutasemutase

2,3BPGisan

importantregulatorof

hemo lobin'saffinit hemo lobin'saffinit


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foroxygen.foroxygen.

First

Production

of

ATPFirst

Production

of

ATP Thehighenergy

p osp ateo ,

BPGisusedtoform

ATPand3

phosphoglycerate

(3PG)bytheenzyme


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phosphoglyceratekinase.


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ConversionofConversionofPhosphoacyPhosphoacyestertoaestertoa

HighHighenergyformenergyform Theremainingreactionsof

glycolysis areaimedatconvertingtherelativelylowenergyphosphoacylesterof3PGtoahighenergyformandharvestingthephosphateasATP.

The3PGisfirstconvertedto


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mutase andthe2PG

conversiontophosphoenoylpyruvate (PEP)iscatalyzedbyenolase.

Second

Production

of

ATPSecond

Production

of

ATP Inthisstrongly

,

thehighenergy

phosphateofPEPis

conservedasATP.

ThelossofPiruvatePiruvate KinaseKinase


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leadstotheproductionof

pyruvate.


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GlycolysisGlycolysis SummarySummary


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FateofFateofPiruvatePiruvate


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TheThePyruvatePyruvate DehydrogenaseDehydrogenase (PDH)Complex(PDH)Complex

Duringtheoxidationof

pyruvate toCO2 bypyruvate

Pyruvate+CoA+NAD+ >CO2 +acetylCoA+NADH+H+

e y rogenase t e

electronsflowfrom

pyruvate tothelipoamide

moietyofdihydrolipoyl

transacetylase thentothe

FADcofactorof

dihydrolipoyl

dehydrogenase andfinally+


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NADH.

Theacetylgroupislinkedto

coenzymeA(CoASH)ina

highenergythioester bond.

TheacetylCoA thenenters

theTCAcycleforcomplete

oxidationtoCO2 andH2O.


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PyruvatePyruvate DeshydrogenaseDeshydrogenase


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Regulation

of

the

PDH

ComplexRegulation

of

the

PDH

Complex

PDHactivityisregulatedbyits'stateofphosphorylation,beingmostactiveinthedephosphorylatedstate.

ThePDHcom lexis


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inhibited

by

acetyl

CoA andNADHandactivatedbynonacetylatedCoA(CoASH)andNAD+.


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KrebsCycleorKrebsCycleorTricarboxilicTricarboxilic AcidCycle(TCA)AcidCycle(TCA)

Commonpathwayformetabolismof SugarsSugars

CharacteristicsofTCAcycle

Occursinthemitochondriasmatrix

AlsoknownastheKrebs cycle.

Oxidatively stripselectronsfromfats,carbohydratesandproteins.

TCA

a y c sa y c s m no c sm no c s

CO2


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FunctionsofTCAcycle=amphibolicamphibolic

Energyproduction=catabolic.

Biosynthesis=anabolic.H+ + e

ElectronTransportChain

Amino

Acids,

Purines,

Pirimidines

Fatty

Acids

Krebs

Cycle

or

Krebs

Cycle

or

TricarboxilicTricarboxilic Acid

Cycle

(TCA)Acid

Cycle

(TCA) Underaerobicconditions

theendproductof

AcetylCoA isacentralintermediateinlipidmetabolism

.ThenextstepistheformationofacetylcoenzymeA(acetylCoA) thisstepistechnicallynotapartofthecitricacidcycle.

AcetylCoAKetone

bodiesFatty

acids

Piruvate

Aminoacids

ucose


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Triacylglycerides

Phospholipids

Eicosanoids

CO2 + H2O +Energy(ATP)CO2 + H2O +Energy(ATP)

CholesterolTCA


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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 SuccinylCoA

1Condensation1Condensation

11DehydratationDehydratation

22HydratationHydratation

4Oxidation(H4Oxidation(H22 removal)removal)

3usingNAD3usingNAD


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5. Hydrolysis,ATPsynthesis: Succinyl CoA Synthase

Succinyl CoA Succinate

6. Oxidation: Succinate Dehydrogenase Succinate Fumarate

7. Hydratation:Fumarase Fumarate Malate

8. Oxidation:Malate Dehydrogenase Malate Oxaloacetate

22DecarboxylationDecarboxylation

1ATPsynthesis1ATPsynthesis


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ADP+Pi

ATP


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RegulationoftheTCAcycleRegulationoftheTCAcycle

Regulationlikethatofglycolysis,

occursatboththelevelofentryof

Piruvate

InhibitionActivation

Ca+

thekeyreactionsofthecycle.

FuelenterstheTCAcycleprimarilyas

acetylCoA.Thegenerationofacetyl

CoA fromcarbohydratesis,therefore,

amajorcontrolpointofthecycle.

Thisisthereactioncatalyzedbythe

Piruvate Dehydrogenase. NADH

CitrateOxaloacetate

Malate


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TwolevelsofTCAregulation:

NAD/NADHandADP/ATP

Allosteric Enzymes9 PDH

9 CitrateSinthase

9 Isocitrate Dehydrogenase

9 -Ketoglutarate Dehydrogenase

umara e

KetoglutarateSuccinyl CoA

SuccinateCa+ ,

ADP

Ca+

IsocitrateIsocitrate DehydrogenaseDehydrogenase (IDE)(IDE) IDEisamajor

regu atorypo nt n

theTCAcycle.The

enzymeis

allosterically activated

byADPandinhibited


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byATP.


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ClinicalConsiderationClinicalConsideration

LacticAcidemia

Excessiveexercise

Excessiveanaerobicmetabolismofcarbohydrates.

9Brainandmuscleseverelyaffectedsince


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theydependonaerobic

metabolism.

LacticAcidosisinHIVpatientsreceivingNRTILacticAcidosisinHIVpatientsreceivingNRTI

Patient:6.4mM LacticAcid


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VPatelandSSHedayati (2006)LacticacidosisinanHIVinfectedpatientreceivinghighlyactiveantiretroviraltherapy.

NatClin Pract Neprol2: 109114 doi:10.1038/ncpneph00102

Mechanismsofnucleosidereversetranscriptaseinhibitor(NRTI)inducedlacticacidosis

Normal:12mM


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Where do all the NADHs andWhere do all the NADHs and

FADHFADH22s Go?s Go?


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WhatisthemaximumyieldofhighenergyATPintheWhatisthemaximumyieldofhighenergyATPinthe

aerobic

catabolism

of

glucose

in

bacteria?aerobic

catabolism

of

glucose

in

bacteria?

Glycolysis glucose2pyruvate+2NADH+2ATP 8ATPs

yruva e e y rogenase 2pyruvate 2acetylCoA +2NADH 6ATPs

TCA acetylCoA2CO2+3NADH+FADH2+GTP 2x12ATPs

TotalATPfrom1molofglucose 38ATPs 30.5kJ/moleATP

=1,160kJ/moleglucose1,160kJ/moleglucose


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== 1,160kJ[1kcal/4.184kJ]=277kcal/moleglucose1,160kJ[1kcal/4.184kJ]=277kcal/moleglucose

Inacalorimeter theInacalorimeter theG of combustion forG of combustion for1molglucose=1molglucose=2820kJ/mol;2820kJ/mol;thereforethecellefficiencyis1160/2880,whichthereforethecellefficiencyis1160/2880,whichisnearly40%.isnearly40%.


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StudyQuestionsStudyQuestions

Whatstheimportanceofthe TCAcycle?

Whereisthisprocesscarriedout?

Glucoseanaerobically

Glucoseaerobically

Piruvate toAcetylCoA

ForoneAcetylCoA enteringintoTCA Aerobically,anaerobically,total

BytheaidofdiagramsexplainthereactionsoftheTCAcycle.

Writedowntheoverallstoichiometric equationfortheTCAcycle.

WhatarethepointsofregulationinTCAcycle?


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HowisPyruvate dehydrogenase regulated?

Howareaminoacid,carbohydratesandfattyacidsmetabolismrelatedtotheTCAcycle? HowdoestheTCAcyclefunctionasbiosyntheticprecursors?

WhatisthecommittedstepinTCAcycle?

WhyisitthatGlycolysis cantakeplaceundereitheraerobicoranaerobicconditionsbutthe

citricacidcycleproceedsstrictlyunderaerobicconditions?

Glycolysis and Krebs Cycle

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