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GlycolysisGlycolysis andKrebsCycleandKrebsCycle
JANegrn, Ph.D.
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
Summary Glycolysis
Glycolytic pathway
Ratelimitingsteps
Piruvate
AcetylCoA
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
.
GlycolysisGlycolysis isalsoknownastheisalsoknownastheEmbdenEmbden
MeyerhofMeyerhofpathway,frompathway,from thepioneeringworkthepioneeringwork
ofGustavofGustavEmbdenEmbden &OttoMeyerhof&OttoMeyerhof
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Glycolysis
Glycolysis istheanaerobiccatabolismanaerobiccatabolismof
g ucose.
Glucoseisoxidizedtoeitherlactateorpyruvate.
Underaerobicconditions,thedominant
productinmosttissuesispyruvatepyruvate..
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
,
prolongedvigorousexercise,thedominantglycolytic productinmanytissuesislactatelactate.
Glycolysis
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
, .
InthesecondphaseF1,6BPisdegradedtopyruvate,withtheproductionof4equivalentsofproductionof4equivalentsofATPand2equivalentsofNADHATPand2equivalentsofNADH.
Firsts
Two
ReactionsFirsts
Two
Reactions Hexokinase trapsglucoseinthecellandbegins
glycolysis
Isomerization ofglucose6phosphatetofructose6
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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FormationFormationFFructose1,6ructose1,6bisphosphatebisphosphate
(F1,6BP)(F1,6BP) FormationofFructose1,6bisphosphatefrom
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
Thisreactioniscatalyzedbyphosphofructokinase
(PFK),anallosteric enzymethatsetsthepaceofglycolysis.
Phosphofructokinase
enzymeinvirtuallyall
livingcells.Itperforms
thecommittedstepin
theglycolytic pathway.
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
. 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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
phosphateanddiminishingtheinhibitoryeffectofATP.
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HomeworkHomework
Howistheconcentrationof
fructose2,6bisphosphate
controlled?
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
Regulation of
PFK
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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MalignantCancersExhibitanIncrease
in
Glycolysis (Warburg
effect),
Mostcancercellsredominantl roduce
Glucose
energybyglycolisisfollowedbylacticacidfermentationinthecytosol,ratherthanbyoxidationofpyruvate inmitochondrialikemost
Piruvate
+
+
ypox a
Inducible
Factor
(HIF1)
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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Cleavage of sixCleavage of six--carbon sugarcarbon sugar
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
foroxygen.foroxygen.
First
Production
of
ATPFirst
Production
of
ATP Thehighenergy
p osp ateo ,
BPGisusedtoform
ATPand3
phosphoglycerate
(3PG)bytheenzyme
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
phosphoglyceratekinase.
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ConversionofConversionofPhosphoacyPhosphoacyestertoaestertoa
HighHighenergyformenergyform Theremainingreactionsof
glycolysis areaimedatconvertingtherelativelylowenergyphosphoacylesterof3PGtoahighenergyformandharvestingthephosphateasATP.
The3PGisfirstconvertedto
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
mutase andthe2PG
conversiontophosphoenoylpyruvate (PEP)iscatalyzedbyenolase.
Second
Production
of
ATPSecond
Production
of
ATP Inthisstrongly
,
thehighenergy
phosphateofPEPis
conservedasATP.
ThelossofPiruvatePiruvate KinaseKinase
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
leadstotheproductionof
pyruvate.
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GlycolysisGlycolysis SummarySummary
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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FateofFateofPiruvatePiruvate
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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+
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
NADH.
Theacetylgroupislinkedto
coenzymeA(CoASH)ina
highenergythioester bond.
TheacetylCoA thenenters
theTCAcycleforcomplete
oxidationtoCO2 andH2O.
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PyruvatePyruvate DeshydrogenaseDeshydrogenase
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
Regulation
of
the
PDH
ComplexRegulation
of
the
PDH
Complex
PDHactivityisregulatedbyits'stateofphosphorylation,beingmostactiveinthedephosphorylatedstate.
ThePDHcom lexis
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
byATP.
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ClinicalConsiderationClinicalConsideration
LacticAcidemia
Excessiveexercise
Excessiveanaerobicmetabolismofcarbohydrates.
9Brainandmuscleseverelyaffectedsince
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
theydependonaerobic
metabolism.
LacticAcidosisinHIVpatientsreceivingNRTILacticAcidosisinHIVpatientsreceivingNRTI
Patient:6.4mM LacticAcid
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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?
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
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
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
== 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?
JAJANegrNegrnn, Ph.D., Ph.D.
ChemChem42204220
HowisPyruvate dehydrogenase regulated?
Howareaminoacid,carbohydratesandfattyacidsmetabolismrelatedtotheTCAcycle? HowdoestheTCAcyclefunctionasbiosyntheticprecursors?
WhatisthecommittedstepinTCAcycle?
WhyisitthatGlycolysis cantakeplaceundereitheraerobicoranaerobicconditionsbutthe
citricacidcycleproceedsstrictlyunderaerobicconditions?
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