生物化学英文版课件：Chapter 15 Principles of Metabolic regulation

Chapter15PrinciplesofMetabolicregulationasfoundinglucosemetabolismBiochemistryLectureforNov.22,2012Theanimalbodywasbelievedtomaintainaconstantinternalenvironment(Milieuintérieur

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Theconstancyoftheinternalenvironmentistheconditionforafreeandindependentlife.Thelivingbodyisrelativelyindependentofitssurroundingenvironment.Thisindependencederivesfromthefactthatthetissuesareinfactwithdrawnfromdirectexternalinfluencesandareprotectedbyaveritableinternalenvironmentwhichisconstitutedbythefluidscirculatinginthebody.Allthevitalmechanisms,variedastheyare,haveonlyoneobject,thatofpreservingconstanttheconditionsoflifeintheinternalenvironment.ClaudeBernard(1813-1878)

Bernarddiscoveredglycogenasthe"sugar-formingsubstance“inliver,whichgeneratesglucosewhenadogwasonlyfedwithmeat.Theconstancyofthebodywasconsideredasadynamicbalance“Physiologicalactivityisconstantlydisturbingtheinternalenvironment.Whatisactuallymaintainedisadynamicbalancebetweenthedisturbingandrestorativeactivities’’(JohnScottHaldane,1922)Haldane,J.S.(1917).Organismandenvironmentasillustratedby thephysiologyofbreathing.YaleUniversityPress.Haldane,J.S.(1922).Respiration.YaleUniversityPress.Haldane,J.S.(1929).ClaudeBernard’sconceptionoftheinternalenvironment.Science,64,453–454.Haldane(1860–1936)Earlyobservationsonhormoneactiontoregulateanimalphysiology“Strongemotionalstatesstimulatethesecretionofadrenalinefromthemedullaoftheadrenalglandandthehormoneactsonperipheraltissuesinsuchawayastopreparetheanimalforvigorousaction,eitherfightingorfleeinginlife-threateningemergencies.”(WalterCannon,1914).Cannon,W.B.(1914).Theemergencyfunctionoftheadrenalmedullainpainandthemajoremotions.AmericanJournalofPhysiology,33,356–372.Canon(1871-1945)Animalbodyproposedtobehomeostatic(dynamicsteadystate)Bodyconstancyismaintainedbycertainmechanisms:anytendencytowardchangeautomaticallymeetswithfactorsthatresistchange(WalterCanon,1926).Homeostaticcategories:Materialsuppliesforcellularneeds.1.Glucose,protein,fat.2.Water.3.Sodiumchlorideandotherinorganicconstituentsexceptcalcium.4.Calcium.5.Oxygen.6.Internalsecretionshavinggeneralandcontinuouseffects.Environmentalfactorsaffectingcellularactivity.1.Osmoticpressure.2.Temperature.3.Hydrogen-ionconcentration.Canon(1871-1945)CanoninBeijing(1935)CannonWB.OrganizationForPhysiologicalHomeostasis.

PhysiolRev.1929;9:399-431.

W.B.Cannon.‘‘Physiologicalregulationofnormalstates:sometentativepostulatesconcerningbiologicalhomeostatics.’’IN:A.Pettit(ed.).ACharlesRichet:sesamis,sescollègues,sesélèves,p.91.Paris:ÉditionsMédicales,1926.ThefeedbackconceptofelectronicswasappliedinunderstandingregulationinlivingorganismsFirstinhormoneproduction(endocrinology,1940s)andtheninthebiosynthesisofaminoacidsandnucleotides(1950s).Foundtooccurviaallostericregulationofanenzymeatthemolecularlevelforbiosynthesis.Wiener,N.(1948)Cybernetics,ortheControlandCommunicationintheAnimalandtheMachine.NewYork:Wiley.Hoskins,R.G.(1949).Thethyroid-pituitaryapparatusasaservo(feed-back)mechanism.JournalofClinicalEndocrinology9:1429-1451.Yates,R.A.,andPardee,A.B.(1956)ControlofpyrimidinebiosynthesisinEscherichiacolibyafeedbackmechanism.J.Biol.Chem.221:757-770.Umbarger,H.E.(1956)Evidenceforanegative-feedbackmechanisminthebiosynthesisofisoleucine.

Science123:848.NorbertWiener(1894-1964)

MolecularHomeostasisformetabolitesachievedviadynamicregulationofcatabolicandanabolicreactionnetworksAllthemetabolicpathwaysareinextricallyintertwined,formingamultidimensionalnetworkofreactions.Negativefeedbackregulationloopsmaintaintheconstancy.Understandinghowthewholenetworkisdynamicallyregulatedisstillachallengingissue.BothamountandcatalyticactivityofenzymeberegulatedforcontrollingmetabolismTimescaleofregulation:millisecondstosecondstohours.ManyprinciplesofmetabolismregulationwerelearnedbystudyingsugarmetabolismConstancyofglucoselevelinblood(homeostasis).Organcooperationinglucoseusesandbiosynthesis.Hormone(insulin,glucagon,epinephrine)regulationofglucosemetabolism.AllostericregulationofglycogenphosphorylaseactivitybyAMP.Reversiblephosphorylationasawaytoregulateglycogenphosphorylaseactivity.YeastadjustmentofglucosemetabolismobservedbyPasteur(1857)

ThePasteurEffect:InthepresenceofO2,yeastcellsgrewmuchfaster,butfarlessglucoseconsumed!NosignificantchangesintheconcentrationsofATPormostoftheintermediates.Hintedthatcellmetabolismisadjustedinresponsetoconditionsbothinsideandoutsidethecell.ThefluxthroughtheglycolyticpathwayisbelievedtoberegulatedatthreestepsAstepwithalargenegativechangeinfreeenergyisassumedtoberegulated(thushighlyirreversiblewith

abuild-upofreactants).Threeenzymesfoundtoberegulated:hexokinase(step1),phosphofructokinase-1(step3),andpyruvatekinase(step10).Thechangeinfreeenergyforeachstepofglycolysisestimatedfromtheconcentrationofmetabolitesinanerythrocyte.ΔG=ΔG°'+RTlnQ

Glycogenphosphorylasefoundtobeallostericallyregulated(Cori&Cori,1930s)Glycogenphosphorylasecatalyzesthephosphorolysisofglycogen,usinginorganicphosphate(notATP!),producingGlc1-P.AMP(NOTcAMP)activatesandATPandGlc6-Pinactivatethemuscleenzyme.Rabitmuscleglycogenphosphorylase-AMPcomplex(ahomodimer)

Blue:glycogenbindingsite,Red:catalyticsiteYellow:AMPallostericsiteOrange:phosporylatedSer14+Pior+Glc6-PNobelprize1947Coriester

Glycogenphosphorylasefoundtobereversiblyphosphorylated(1950s)Theactiveformisphosphorylatedandtheinactiveformdephosphorylated,andsuchphosphorylationisregulatedbyhormones.Krebs,E.G.,andFischer,E.H.(1956)Thephosphorylasebtoaconvertingenzymeofrabbitskeletalmuscle.Biochim.Biophys.Acta20,150-157EdwinKrebs(1918-2009)

EdmondFischer(1920-,borninShanghai)Nobelprize1992cAMPfoundtomediateliverglycogenphosphorylaseactivationbyhormonesWhenepinephrineorglucagonwasaddedtointactdoglivercells,thephosphorylaseactivityfoundincreased(byphosphorylation).cAMPwasfoundtomediatethisactivationprocess.SutherlandEW,RallTW.(1958)Fractionationandcharacterizationofacyclicadenineribonucleotideformedbytissueparticles.JBiolChem.232:1077-91.Nobelprize1971Liverglycogenphosphorylaseregulatedbyallostericeffectors&reversiblephosphorylation(cascadeseffectingalargeamplificationoftheinitialsignal)PKA:cAMPdependentproteinkinaseIntrinsiccontrol

Extrinsiccontrol(Mobilizeglucoseinliver)PKAandPPI-1alsoregulateglycogensynthase,butreciprocally!LiverglycogenphosphorylaseactsasaglucosesensorGlucosebindstophosphorylasea,facilitatingtheactionofthephosphataseandconversiontophosphorylaseb(liverglycogenolysisstopedwhenbloodglucoselevelishigh).Glycogenphosphorylaseathusactsastheglucosesensoroftheliver,slowingthebreakdownofglycogenwheneverthelevelofbloodglucoseishigh.Glucose,butnotAMPisanallostericregulator!Onlyinliver:Glucose-6-phosphate+H2O

glucose+PiGlycogensynthasewasdiscoveredandfoundtobealsoregulatedbyreversiblephosphorylation(thephosphorylatedformbeinginactive)!

(Glycogenphosphorylasedoesnotcatalyzeglycogensynthesis!!!)GlycogensynthaseusesUDP-glucose

(notGlc-1-P!)Uridinediphosphate(UDP)foundtobethecarrierofactivesugars(1950s)OriginallyidentifiedasthethermostablecoenzymeofGal-1-PandGlc-1-Pisomerization(inyeast).ThenUDP-Glcfoundparticipatingintrehalose,sucroseandglycogen(inliverandmuscle)synthesis.Cardini,C.E.,Paladini,A.C.,Caputto,R.,andLeloir,L.F.(1950)Uridinediphosphateglucose:thecoenzymeofthegalactose-glucosephosphateisomerization.Nature165,191–193Leloir,L.F.,andCardini,C.E.(1957)Biosynthesisofglycogenfromuridinediphosphateglucose.J.Am.Chem.Soc.79,6340UDPNobelprize1970Glycogenin,foundasaself-glucosylatingproteinthatprimesglycogensynthesis(1984),

providesaTyrresidueforattachingthefirstglucoseresidueinaglycogenmolecule.UDP-glucose.Mn2+

Tyr194Theglycogensynthaseisphosphorylatedatmultiplesitesbytheactionofmultiplekinases(atleast11)CaseinkinaseIIaddsthefirstphosphorylgroup(“priming”)beforeglycogensynthasekinase3cananother3.Thehumangenomecontainsabout500proteinkinasegenes(modifyingabout30%ofthehumanproteins)!Thephosphorylatedformisinactiveanddephosphorylatedformactive.Glc-6-PandGlcactivatesglycogensynthaseb.Glycogensynthasealsoregulatedbyreversiblephosphorylation&allostericeffectorsAGlc-6-PsensorBindingofinsulintoreceptors(1)oncellsthenactivatesacascadeofproteinkinases(2)thatcausethecellstotakeupglucose(3)andconvertitintostoragemoleculesasglycogenandfattyacids(4,56).

(PKAforglucagon)PhosphorylatedCarbohydratemetabolisminliverasregulatedbyinsulinandglucagon.Insulinpromotessynthesisofglycogen;Epinephrine(inmuscle)andglucagon(inliver)promotesdegradationofglycogen.InsulinactivatesbutepinephrineinactivatesphosphoproteinPhosphatase1(PP1),whichinturndephosphorylates

glycogenphosphorylasekinase,glycogenphosphorylase,andglycogensynthaseGM-glycogentargetingprotein(subunitofPP1)Well-fedstate(hyperglycemia)Fastingstate(hypoglycemia)Epinephrinestimulatesglycogenolysisandglycolysisinmuscle,suppressesglycolysisinliver.Preparingtheanimalsfor“fight-or-flight”MultipleenzymesareregulatedintheglycolyticpathwayThehexokinaseisozymesinmuscleandliverworkfordifferentpurposesMuscleisozymeshaslowKm(0.1mM)andallostericallyinhibitedbyGlc6-P.LiverisozymehashighKm(10mM)andnotinhibitedbyGlc-6-Pbutbyaregulatoryprotein.Muscleisozyme(forATPproduction)Liverisozyme(forbloodglucosehomeostasis)Bloodglucoselevel(5mM)HexokinaseIVinliverpreparesthehighlevelofbloodglucoseforglycogenandfattyacidsynthesis.Activityofphosphofructokinase-1regulatedbynegative&positiveallostericeffectorsPFK-1isinhibitedbyATP(?)andcitrate(?),butactivatedbyfructose2,6-bisphosphate,AMPandADP.Commitsglucosetoglycolysis(nolongeravailableforPPP&glycogenesisActivesiteAllostericsiteItisdoubtfulwhetherATPandcitrateactaullyplaysuchregulatoryrolesinlivingcells!Fructose2,6-bisphosphatefoundasapotent

allostericstimulatorofliverphosphofructokinase-1(1980)

Studyofmechanismofactionofglucagononlivergluconeogenesisledtothediscovery.Concentrationofalow-molecular-weightstimulatorofphosphofructokinasegreatlyincreasedinhepatocytesinpresenceofglucoseanddecreasedinpresenceofglucagon.VanSchaftingen,E.,Hue,L.andHers,H.-G.(1980)Controlofthefructose-6-phosphate/fructose1,6-bisphosphatecycleinisolatedhepatocytesbyglucoseandglucagon.Roleofalow-molecular-weightstimulatorofphosphofructokinase.Biochem.J.192,887–895VanSchaftingen,E.,Hue,L.andHers,H.-G.(1980)Fructose2,6-bisphosphate,theprobablestructureoftheglucose-andglucagon-sensitivestimulatorofphosphofructokinase.Biochem.J.192,897–901Henri-GeryHers(1923-2008-Belgium)

Fructose2,6-bisphosphatefoundtoinhibitfructose1,6-bisphosphataseandthusgluconeogenesisHenri-GeryHers(1923-2008-Belgium)

LevelofF-2,6-BPsetbytherelativeactivitiesofphosphofructokinase-2(PFK-2)andfructose2,6-bisphosphatase(FBPase-2)ofasinglebifunctionalprotein.

ItexhibitsFBPase-2activitywhenphosphorylatedandPFK-2activitywhendephosphorylated(atasingleSerresidue).Rapidhormonalregulationofglycolysisandgluconeogenesisismediatedbyfructose2,6-bisphosphateInsulinpromotesdephosphorylation,thustheincreaseofF2,6-BP.Glucagonpromotesthephosphorylation,thusdecreaseofF2,6-BP;Observationinratliverextract:

HexokinaseandPFK-1bothcontribute

tosettingthefluxthroughtheglycolyticpathway(hexokinase

morethanPFK-1!),andthatphosphohexoseisomerase

doesnot

Theconventionalsimplesolution