北大生化课件学习VIP

CarbohydrateBiosynthesis

LecturedbyDr.Qin

Yongmei(秦咏梅)Nov.21,2007Chapter14GluconeogenesisChapter15PrincipleofMetabolicRegulation:GlucoseandGlycogenChapter20CarbohydrateBiosynthesisinPlantCanyoutellme:Whydoweneedglucoseinourbody?Whatissignificantroleofglucoseinthehumanbody?Tissuesthatsynthesizeglucose:liverandkidneyTissuesthatuseglucoseastheirprimaryenergysource:brain,muscle,erythrocytesandtestes

Inwhichkindofsituationdoweneedgluconeogenesis?

Normalphysiologysituation:-Betweenmealsandduringsleep-Exercise/workAfterheavyexerciseorwork(recyclingoflactate)Afterprotein-richdiet(glucogenicaminoacids)Starvation(glucogenicaminoacids)Gluconeogenesis2.Biosynthesisofglycogen3.Analysisofmetaboliccontrol4.Photosyntheticcarbohydratesynthesis(theCalvinCycle);5.Regulationofcarbohydratemetabolisminplants.6.BiosynthesisofstarchandsucroseContentsOverviewofHumanMetabolismGlucose-6-phosphateGlucoseGlycogenPyruvateLactateAminoacidsAcetyl-CoARibose-5-phosphateirreversible1.Gluconeogenesis

(“formationofnewsugar”)Definition:theformationofglucosefromnon-carbohydrateprecursorsSpecies:allanimals,plants,fungiandmicroorganismsOrgans(inhigheranimals):occurringlargelyinliver,asmallamountinkidneyDailyglucoserequirement(humanbeing):

160g(120gforbrain),glycogencanprovide190g.

Infastingconditions:

gluconeogenesisaccountsforupto96%oftotalglucoseproduction.SubstratesforGluconeogenesis:LactateAminoacidsGlycerolPropionateFattyacidoxidationinmammalsprovideanimportantenergysource

(andcarbonsourceinplantsandbacteria)forgluconeogenesisGlycerolAminoacidsLactateAnimalPlant1.Gluconeogenesisreferstothemetabolicpathwaythatresultsinnetglucoseproduction: 1)fromfructoseandgalactose 2)frompentosephosphates 3)fromglycogen 4)fromstarch 5)fromalanine2.Thegluconeogeneticpathwayisactive: 1)inlivercells,whenonalowcarbohydratediet 2)inmusclecellsduringintenseexercise 3)inheartcellsduringstarvation 4)inadipocytesduringfasting 5)inkidneymedullawhencirculating[Glc]islow3.Itissaidthatanimalcellscannotusefatfornetglucosebiosynthesisbecause:1)NopartwhatsoeverofatriacylglyceridemoleculecanbeconvertedintoGlc2)NopartofanyfattyacidwhatsoevercanbemadeintoGlc3)AcetylCoAiscompletelydegradedintoCO2andwaterintheTCAcycle4)AnysubstratethatreplenishestheTCAcycleiscompletelydegradedtoCO2

hexokinasePFK-1pyruvate

kinaseGlucoseGlucose-6-phosphateFructose-6-phosphateFructose-1,6-bisphosphateGlyceraldehyde3-phosphate1,3-bisphosphoglycerate3-bisphosphoglycerate2-bisphosphoglyceratephosphoenolpyruvateoxaloacetatepyruvateGlycolysisandgluconeogenesishave3differentreactionsand7commonreactions.gluconeogenesisglucoseOxaloacetateisthestartingmaterialforglucogneonesisWhatisroleofgluconeogenesis

inplant?

Activegluconeogenesisoccursingerminatingseeds,providingglucoseforthesynthesisofsucrose;Plantscanconvertacetyl-CoAderivedfromfattyacidoxidationintoglucoseviaglyoxylatecycle(occurringinglyoxysomes).Thephysicalseparationoftheseglyoxylatecycleand-oxidationenzymesfrommitochondrialcitricacidcycleenzymespreventthefurtheroxidationofacetyl-CoAtoCO2.GluconeogenesisconvertsfatsandproteinstoglucoseingerminatingseedsConversionofstoredfattyacidstosucroseingerminatingseedsGlyoxysomeMitochondrionCytosolTheintegrationofreactionsequencesinthreesubcellular

compartmentsisrequiredfortheproductionofsucrosefromstoredlipids.Gluconeogenesis

isnotareversalofglycolysisMoleculesaresynthesizedanddegradedbydifferentpathways.Eventhoughtwoopposingpathwaysmaysharemanyreversiblereactions,

atleast,onestepisuniqueandirreversibletothatoftheopposingpathway;Correspondinganabolicandcatabolicpathwaysarecontrolledatoneormoreofthereactionsuniquetoeachpathway.Energy-requiringbiosyntheticprocessesarecoupledtoenergy-yieldingbreakdownofATPinsuchawaythattheoverallprocessis

essentiallyirreversible

invivo.Inprinciple:3IrreversiblestepsinglycolyticpathwayGlucose+ATPGlucose6-phosphate+ADP G=-8.0kcalmol-1(-33kJmol-1)Fructose6-phosphate+ATP Fructose1,6-bisphosphate+ADP G=-5.3kcalmol-1(-22kJmol-1)Phosphoenolpyruvate+ADP

Pyruvate+ATP G=-4.0kcalmol-1(-17kJmol-1)HexokinasePhosphofructokinasePyruvate

kinaseConversionofpyruvatetophosphoenolpyruvate

(twoexergonicreactions)Pyruvate+ATP+GTP+H2O

phosphoenolpyruvate+ADP+GDP+Pi+2H+MitochondrialenzymeMitochondrialenzyme/cytosolicenzymeFreeenergyofcleavageofone˜PbondofATPisconservedinthecarboxylation

reactionandCO2canberemovedtopowertheformationofPEPinthedecarboxylationstep..Decarboxylationsoftendrivereactionsotherwisehighlyendergonic

Thismetabolicmotifisusedinthecitricacidcycle,thepentosephosphatepathway,andfattyacidsynthesis.Cleavageofasecond˜PbondofGTPalsocontributestodrivesynthesisofPEP.Whatshallwelearnfromthisreactionmechanism?

HCO3-+ATPHOCO2PO32-+ADP

Biotin-enzyme+HOCO2PO32-

CO2-biotin-enzyme

(activatedcarboxylgroup)+PiCO2-biotin-enzyme+pyruvate

biotin-enzyme+oxaloacetate

Biotinisacovalentlyattachedprostheticgroup,whichservesasacarrierofactivatedCO2:

-pyruvate

carboxylase-acetylCoA

carboxylase-propionyl

CoA

carboxylaseCarboxylationofpyruvate(anapleroticreaction)takesplacein3stages:PhaseIPhaseIIPyruvate

CarboxylaseThelong,flexiblechainformedbetweenbiotinandtheenzymeenablesthisprostheticgrouptorotatefromoneactivesiteoftheenzymetotheother.Avidin,aproteinineggwhitestightlybindsbiotin(Kd=10-15).Excessconsumptionofraweggscancausenutritionaldeficiencyofbiotin.ATP-activatingdomaincarrierofactivatedCO2BiotincarboxylcarrierdomainB.CytosolicNADHisrequiredforGluconeogenesis-ketoglutarate-ketoglutarateC.Conversionoffructose1,6-bisphosphatetofructose6-phosphateG’0=-16,3kJ/molD.Conversionofglucose6-phosphateto freeglucoseG’0=-13,8kJ/molTheenzymeisnotpresentinmuscleorinthebrain,andgluconeogenesisdoesnotoccurinthesetissues.Coordinatedregulationofgluconeogenesisandglycolysis

Glycolysis

andgluconeogenesispathwaysarebothspontaneous.Ifbothpathwaysweresimultaneouslyactivewithinacellitwouldconstitutea“futilecycle”thatwouldwasteenergy.FutilecyclesincarbohydratemetabolismconsumeATPGluconeogenesis:2Pyruvate+4ATP+2GTP+2NADH+4H2O

glucose+4ADP+2GDP+6Pi+2NAD++2H+

Glycolysis:Glucose+2ADP+2Pi+2NAD+

2Pyruvate+2ATP+2NADH+2H++2H2OAfutilecycleconsistingofbothpathwayswouldwaste4~Pbondspercycle.Futilecyclehasregulationsignificance:Itisawaytoturnglycolysisoffand

gluconeogenesisonwhenithasanadequateATPsupply.

Oritisawaytoturnglycolysisonand

gluconeogenesisoffwhenATPisinshortsupply.Pasteureffect:LOUISPASTEUR(1822-1895)

Pasteurobservedthatdecreaseintherateofcarbohydratebreakdownthatoccursinyeastwhenswitchedfromanaerobictoaeroticconditions.ThebasicphenomenonisacompetitionbetweenglycolysisandoxidativephosphorylationfortheavailableADPandinorganicphosphate.

Highbloodglucose,fedstate

Liver:fuelconservation

Glycogenissynthesized;

Glycolyticpathway&pyruvate

dehydrogenase

areactivated

FAbiosynthesisandfatstorageLowbloodglucose,faststate

Glycogenbreakdown

Gluconeogenesis

Hexokinase(32,9)/glucose6-phosphatase(-5,1)PFK-1(24,5)/FBP-1(-8,6)

Puruvate

kinase(26,4)/pyruvatecarboxylase-PEPCK(-22,6)Example:Ginthedirectionofgluconeogenesis

inliverunderphysiologicalconditions:(1).Hexokinase

isozymesofmuscleandliverareaffecteddifferentlybyG6P(inmuscle,lowKm)(inliver,sigmoidcurve)[glucose]inblood=4-5mMWhenbloodglucoserisesabove5mM,hexokinaseIVincreases,buthexokinaseIisgettingsaturatedandcannotrespondtoanincreaseinglucoseconcentration.HexokinaseIVissubjecttoinhibitionbyreversiblebindingofaregulatoryproteinspecifictoliverinhibited(allosteric

effectorofhexokinaseIV)Duringafast,[glucose]below5mM,F6Ptriggersinhibitionof

hexokinaseIVbyassociationwiththeregulatorprotein.Inthisway,liverdoesnotcompetewithotherorgansfortheglucose.(competewithF6pforbindingtohexokinaseIV)(2).PFK-1isundercomplexallostericregulationActivesiteallostericsiteADPF1,6BPADPAllostericregulationofmusclePFK-1byATPATPinhibitsPFK-1bybindingtoanallostericsiteandloweringtheaffinityoftheenzymeforF6P.PFK-1:allostericenzymeBindingofallostericinhibitororactivatordoesnoteffectVmax,butdoesalterKm;AllostericenzymedoesnotfollowM-Mkinetics.(3).Pyruvate

kinaseisallostericallyinhibitedbyATPIsozymesdifferintheirtissuedistributionandtheirresponsetomodulators.Mformisnotaffectedbythisphosphorylationmechanism.cAMP-dependentproteinkinase

Whenlow[glucose]causesglucagonrelease,PKAphosphorylatedtheLform(inactivation).Themechanismpreventstheliverfromconsumingglucoseandsparesitforotherorgans.thefateofpyruvateacetyl-CoA:activates

pyruvate

carboxylase(gluconeogenesis),inactivatesthepyruvate

dehydrogenasecomplex(glycolysis).(4).Gluconeogenesisisregulatedbyacetyl-CoAphosphofructokinase

(PFK-1)Fructose1,6-bisphosphatase(FBPase-1)glucose6-phosphatasehexokinase(5).F2,6BPisapotentregulatorofglycolysisandgluconeogenesisF2,6BP

hasoppositeeffects

onthe

enzymaticactivities

of

PFK-1

and

FBPase.Fructose-2,6-bisphosphateissynthesizedanddegradedbyPFK2/FBPase2PFK2/FBPase2isaBifunctionalProteinThePFK2/FBPase2probablyarosebythefusionofgenesencodingthekinaseandphosphatase

domainsPFK-1CarbohydratemetabolismasaspecificexampleofsignaltransductionsecondmessengercAMP-adrenergicreceptorPFK2/FBPase2PFK2/FBPase2GluconeogenesisGlycolysis

ProteinkinaseAPhosphoproteinPhosphatase

(activatedby

xylulose5P)fructose6-phosphatefructose2,6-bisphosphatefructose2,6bisphosphate(stimulatePFK-1)fructose6-phosphate(noPFK-1stimulation)cAMP-dependentphosphorylationofPFK2/FBPase2

activatesFBPase2andinhibitsPFK2.[Fructose-2,6-bisphosphate]decreasesinlivercellsinresponsetoaglucagon-activatedcAMPsignalcascade.Downstreameffectsinclude:Glycolysisslowsbecausefructose-2,6-bisphosphateisnotavailabletoactivatePFK-1.Gluconeogenesisincreasesbecauseofthedecreasedconcentrationoffructose-2,6-bisphosphate,whichwouldstimulatethefructose-1,6-bisphosphatase.Reciprocalregulationbyfructose-2,6-bisphosphate(6).Xylulose5-phosphateisakeyregulatorofcarbohydrateandfatmetabolism.InmammalianliverX5P,involvedinpentosephosphatepathway(productionofNADPH),mediatestheincrease

inglycolysisthatfollowsaningestionofahigh-carbohydratemeal.X5PPhosphoproteinphosphatase

PFK2/FBPase2F2,6BPactivateglycolysisAcetyl-CoAFattyacidsynthesisItwasalsofoundthatX5Pincreasedthesynthesisofalltheenzymesrequiredforfattyacidsynthesis.PFK-1FBPase-1Thedistinctiveenzymesareregulated.Certaineffectorsactivateanenzymeofonepathway,butinhibitanenzymeoftheotherpathwaytoavoidsubstratefutilecycle.glucagonHighNADH/NAD+ratioLowNADH/NAD+ratioCoricycle:theliverfurnishesglucosetocontractingskeletalmuscle,whichderivesATPfromtheglycolyticconversionofglucoseintolactate.Contractingskeletalmusclesupplieslactatetotheliver,whichuseittosynthesizeglucose.Glucose-alaninecycleAlanine

bringsbothcarbonandnitrogenfrommuscletoliver.

ThemechanismsallowmusclecellstoproduceATPwithhighratesattheexpenseofregeneratingglucosefromlactateandalanineintheliver.WhatisacommonfeatureforCori

cylcle

andGlc-AlaCycle?2.BiosynthesisofglycogenAnimals:Glycogenisthestorageformofglucose.Glycogenissynthesizedandstoredmainlyintheliverandthemuscles.Plants:

Plantsmakestarchandcellulosethroughthephotosynthesisprocesses.

Starchvs.GlycogenAnimalsandhumaneatplantmaterialsandproducts.Digestionisaprocessofhydrolysiswherethestarchisbrokenultimatelyintothevariousmonosaccharides.Amajorproductisofcourseglucosewhichcanbeusedimmediatelyformetabolismtomakeenergy.Theexcessglucoseisconvertedintheliverandmusclesintoglycogenforstorage.Anyglucoseinexcessoftheneedsforenergyandstorageasglycogenisconvertedtofat.GlycogenisaD-glucosepolymer:

(14)linkage

(16)linkageBranchesevery

8-14residuesGlycogenSynthesisGlycogenSynthesisGlycogensynthesisisnotadirectreverseofthephosphorolysisreaction.Formationofasugarnucleotide:whichisthesubstrateforpolymerizationofmonosaccharidesintopolysaacharides

Asugarnucleotideisformedthrougha

condensationreaction

betweenaNTPandasugarphosphate.Hexose1-PSeveralimportantpropertiesofsugarnucleotides:Theformationofsugarnucleotideismetabolically

irreversible,contributingtotheirreversibilityofthesyntheticpathwaysinwhichtheyareintermediates.Thenucleotidemoietyhasmanygroupsthatcanundergo

noncovalentinteractionswithenzymes;theadditionalfreeenergyofbindingcancontributesignificantlytocatalyticactivity.Thenucleotidylgroup(UMP,AMP)isanexcellentleavinggroup,facilitatingnucleophilicattackbyactivatingthesugarcarbontowhichitisattached.Cellcansetsugarnucleotideasideforonepurpose(glycogensynthesis),todistinguishfromsugarphosphateusedforglycolysis.UDP-glucoseisformedfromGlc-1-PandUTPUDP-glucosepyrophosphorylase(substrate)Glycogensynthesisglycogensynthaseprotein-tyrosine-Glucosyltransferase(glycogenin)complexofglycogeninandglycogensynthaseInitiationofaglycogenparticlebyglycogenin

catalyzingitsownglycosylationglycogensynthaseglycogensynthase,glycogen-branchingenzymesglycogeninglycogeninGlycogeninhas

glucosyltransferaseactivity

whichcatalyzestheassemblyofthefirst8residuesStructureofGlycogenParticleabout55,000glucoseresiduesinamoleculeofabout21nmdiameterandMr107Glycogensynthaseandglycogenphosphorylase

arereciprocallyregulatedallosterically

andhormonally.whenoneprocessisstimulatedandtheotherisinhibited,ifnot,resultinginafutilecycle.Glycogenphosphorylase:enzymeusedinglycogenolysis

(glycogenbreak-down)-stimulatedbylowbooldglucose;Glycogensynthase:enzymeusedinglycogenesis

(glycogensynthesis)-stimulatedbyhighbloodglucose;Coordinatedregulationofglycogensynthesis

andbreakdownAllostericregulationofmuscleglycogenphosphorylaseTheglycogenphosphorylasesofliverandmuscleareisozymes,encodedbydifferentgenesanddifferingintheirregulationproperties.Ca2+bindstoandactivatesphosphorylasebkinasethroughsubunitthatiscalmodulin.AMPbindsandactivatesphosphorylase,speedingthereleaseofG1Pfromglycogen.Glycogenphosphorylaseofliverasaglucosesensor,regulatedhormonallyandallosterically.indirectlywhen[glucose]ishighsecretionofinsulin

PP1activateglycogenbreakdowndecreaseglucoseasanallostericinhibitorCarlF.Cori(1896-1984)&GertyT.Cori(1896-1957)“Polysaccharidephosphorylase”NobelPrizeinMedicineandPhysiologyin1947CarlF.Cori(1896-1984)&GertyT.Cori(1896-1957)“Polysaccharidephosphorylase”NobelPrizeinMedicineorPhysiologyin1947Originalpublications:TheNobelPrizeinPhysiologyandMedicine1992EarlW.SutherlandUSA

1915-1974(trainedinCorislab)"forhisdiscoveriesconcerningthemechanismsoftheactionofhormones"NobelPrizeinMedicineorPhysiologyin1971AfterreadingabookaboutLouisPasteurinhighschoolhedecidedtogointomedicalresearch.Heisolatedapreviouslyunknowncompound,calledcyclicadeninemonophosphate(cAMP)andprovedthatithadanintermediaryroleinmanyhormonalfunctions.Leloirdiscoveredin1949thatonesugarwastransformedtoanothersugarvia

sugarnucleotide,later,hefoundthatglycogenwassynthesizedfromUDP-glucosein1959.(trainedinCorislab)TheNobelPrizeinPhysiologyandMedicine1992EldwinG.KrebsEdmondH.Fischer

USA

USA

1920-(trainedinCorislab)1918-TheyshowedthatepinephrineandcAMPstimulateglycogenbreakdownbyactivatingglycogenphosphorylaseviaaproteinkinaseNobel

PrizeinMedicineorPhysiology1992Glycogensynthaseisalsoregulatedbyphosphorylation

anddephosphorylationGSK3(glycogensynthase

kinase)

addsphosphorylgrouptothreeSerresidues,stronglyinactivatingit.PP1(inliver):

phosphoprotein

phosphataseGSK3actionrequirespriorphosphorylation(priming)bycaseinkinase(CKII).

PrimingofGSK3

phosphorylationofglycogensynthaseN-terminusC-terminus(inactivateform)phosphorylatedbyPKAorPKBActivationofGSK3requiresremovaloftheprimingphosphorylgroupbyPP1.

GSK3

mediatestheactionof

insulinTheinactivationofGSK3allowsPP1todephosphorylateglycogensynthase,convertingittoitsactiveform.

PP1iscentraltoglycogenmetabolismAsingleenzymecanremovephosphorylgroupsfromallthreeenzymesphosphorylatedinresponsetoglucagon(liver)andepinephrine(liver&muscle):phosphorylase

kinase,glycogenphosphorylaseandglycogensynthase(allformabigcomplex).

GM:glycogen-targetingproteinPP1doesnotexistfreeinthecytosol.

Insulin

GM-P

PP1

activateactivateEpinephrine

PKA

DissociationofPP1fromthecomplex

Inhibitor-PbindsandinactivatesPP1GM-2P

Inhibitor-PEnzyme

Stimulatedby

InhibitedbyGlycogenphosphorylase

glucagon,epinephrine,Insulin,ATP

cAMP,Ca2+,AMP

glucose

phosphorylationGlycogensynthase

Insulin,glucagon,

glucose-6-phosphateepinephrine

cAMP,Ca2+,AMP

phosphorylationThebalancebetweenglycogensynthesisand

breakdownin

liver

iscontrolledbythe

hormonesglucagon,epinephrineandinsulin.Overallshiftsincarbohydratemetabolismthatoccurinthewell-fedstateinhepatocyteOverallshiftsincarbohydratemetabolismthatoccurduringfastinginhepatocyteDifferenceintheregulationofcarbohydratemetabolisminliverandmuscleGlycogenolysis:glycogenglucose6-phosphate;Glycogenesis:glucoseglycogenThephysiologyandcarbohydratemetabolismofskeletalmuscle

differsfromthatofliverinthreeways:(1).Muscleusesitsstoredglycogenonlyforitsownneeds.(2).Glycolysisoccurswhenmusclegoesfromresttovigorouscontraction.-Pyruvate

kinase(M)isnotphosphorylatedbyPKA,so

glycolysisisnotturnoffwhen[cAMP]ishigh.(3).Musclelacksgluconeogenesis

pathway.-myocyteslackreceptorsforglucagon

3.AnalysisofmetaboliccontrolForeverycomplexproblemthereisasimplesolution.Anditisalwayswrong.-H.L.Mencken,AMenckenChrestomathy,1949Is“singlerate-determiningstep”hypothesisright?(1).Thecontrolcoefficient(C)quantifiestheeffectofachangeinenzymeactivityonmetabolitefluxthroughapathwayRangeofC:0-1.0Cexpressestherelativecontributionofeachenzymetosettingtherateatwhichmetabolitesflowthroughthepathway.C:

-notaconstant-afunctionofthewholesystemofenzymes.-dependsontheconcentrationsofsubstratesandeffectors(2).ThecontributionofeachenzymetofluxthroughapathwayisexperimentallymeasurableDependenceofglycolyticfluxinaratliverhomogenateonaddedenzymes.BothhexokinaseIVandPFK-1increasetherateofglycolysis,andhexokinasecontributesmorethanPFK-1does.C=0.79C=0.21C=0(C:fluxcontrolcoefficient)(3).Theelasticitycoefficient()isrelatedtoanenzyme’sresponsivenesstochangesinmetaboliteorregulatorconcentrations.Hyperboliccurve:-quantitativelytheresponsivenessofasingleenzymetochangesintheconcentrationofametaboliteorregulator;

-afunctionofenzyme’sintrinsickineticproperties(4).Theresponsecoefficie