chem_482_annotations_2.ppt

Chapter10-IntroductiontoMetabolism,Ahummingbirdhasarapidrateofmetabolism,butitsbasicmetabolicreactionsarethesameasthoseinmanydiverseorganisms,10-1,Metabolicprocessinallcellsandorganismsareverysimilar,independentofspecialneeds,Somecellsdospecializethough,andthisisdeterminedgenetically.,A.Metabolism:theSumofCellularReactions,Metabolism-theentirenetworkofchemicalreactionscarriedoutbylivingcellsMetabolites-smallmoleculeintermediatesinthedegradationandsynthesisofpolymersCatabolicreactions-degrademoleculestocreatesmallermoleculesandenergyAnabolicreactions-synthesizemoleculesforcellmaintenance,growthandreproduction,10-2,Metabolismcanbedividedintotwophases.,Catabolismisthebreakdownoffoodstuffstoextractenergy.Wasteproductsmustbeexcreted.,AnabolismusessimpleprecursorstosynthesizeAA,fats,nucleotidesandsugarsformacromoleculesynthesis.,Anabolismandcatabolism(Fig10.1),10-3,Illustratedhere,catabolismproducesenergy.,Andanabolismusesthatenergy.,Catabolisminvolvesbreakingdownmacromoleculesandproduceswaste.,Notethatanabolismusesfoodandbuildingblocks,butnotwaste.,Commonthemesoforganisms,1.Organismsorcellsmaintainspecificinternalconcentrationsofinorganicions,metabolitesandenzymes2.Organismsextractenergyfromexternalsourcestodriveenergy-consumingreactions3.Organismsgrowandreproduceaccordingtoinstructionsencodedinthegeneticmaterial(continuednextslide),10-4,Cellsaredynamic,takinginessentialnutrients,synthesizingrequiredmetabolites,andexcretingwaste.,Metabolismrequiresenzymes,allofwhicharecodedforinDNAandsynthesizedbytranscriptionandtranslation.,CommonThemes(continued),4.Organismsrespondtoenvironmentalinfluences5.Cellsarenotstatic,andcellcomponentsarecontinuallysynthesizedanddegraded(i.e.undergoturnover),10-5,Turnoverisadynamicterm,andimpliescontrolledkineticprocessesinvolvingthesynthesisanddegradationofallbiomolecules.,Formsofmetabolicpathways(Fig10.2,(a)Linear(b)Cyclic,10-6,Pathwayshaveastartingmetaboliteandendingmetabolite,andarenamedtoreflectsuch.,Abetterexampleofalinearpathwayisglycolysis,whichisglucosepyruvate+“energy”in10steps.,ThebestexampleofacyclicpathwayistheTCAcycleacetyl-CoAoxaloacetate.,Fig.10.2(continued),(c)Spiralpathway(fattyacidbiosynthesis),10-7,Fattyacidsynthesisanddegradationarespiralpathways.,B.MetabolismProceedsbyDiscreteSteps(Fig10.3),Single-stepvsmulti-steppathwaysAmultistepenzymepathwayreleasesenergyinsmalleramountsthatcanbeusedbythecell,10-8,Pathwaysrequirediscretesteps,witheachstepasimplechemicaltransformation.,Stepsreleasingenergy(-DG)allowtheenergytobestored(e.g.,asATP,NADH,Acetyl-CoA,etc.),Ifallenergywerereleasedatonce,thenitcouldntbecapturedandstored.,Thecellwouldsimplyburnfromheat.,Incells,glucose6CO2requiresover25discretesteps,withproductionofover30ATP.,Eachdiscretestepiscatalyzedbyasingleenzyme(normally),C.MetabolicPathwaysAreRegulated,MetabolismishighlyregulatedtopermitorganismstorespondtochangingconditionsMostpathwaysareirreversibleFlux-flowofmaterialthroughametabolicpathwaywhichdependsupon:(1)Supplyofsubstrates(2)Removalofproducts(3)Pathwayenzymeactivities,10-9,Pathwaysareregulatedbycooperativity,allosterism,covalentmodification,etc.,Irefertofluxas“massflow”.Whenoperative,apathwaypushesmassfromstartingpointtoendpoint.,Massflowiscontrolledbyavailabilityofsubstrates,needforproduct,andcontrolenzymeregulation.,Feedbackinhibition,Productofapathwaycontrolstherateofitsownsynthesisbyinhibitinganearlystep(usuallythefirst“committed”step(uniquetothepathway),10-10,Typically,thecontrolenzymeisthefirstcommittedstepinthepathway.,Atypicalallostericcontrolisnegativeallosterismofthecontrolenzymebythefinalproductofthepathway.,Compartmentationofmetabolicprocesses(Fig.10.6),10-11,Ineukaryotes,compartmentalizationisatypeofcontrol.,Forexample,acetylCoAisproducedinthemitochondria.,Butmustbetransportedforuseislipidsynthesis.,Intheoppositedirection,pyruvateisproducedinthecytosol,Butmustbetransportedtomitochondriaforfurtherdegradation.,D.ThermodynamicsandMetabolism,Free-energychange(DG)isameasureofthechemicalenergyavailablefromareactionDG=Gproducts-GreactantsDH=changeinenthalpyDS=changeinentropy,A.Free-EnergyChange,10-12,Rememberlawofconservationofenergy:energyisnotcreated,butconvertedfromoneformtoanother.,FromChem121,enthalpychangeisheatflowatconstantpressureDE+PDV=DH=qp.,Thecommonformsofenergyinchemicalsystemsisheat(q)andwork(w):DE=q+w,wherew=PVwork.,Freeenergy(DG)istheenergyavailabletodousefulwork(nonTDScomponentofDH),FromChem122,entropyisincreaseinrandomness(disorder).,Relationshipbetweenenergyandentropy,10-13,Therelationshipbetweenfreeenrgy,enthalpy,andentropyisgivenhere.,DSisnotusefulwork,althoughitisusefulenergy(considerthehydrophobiceffectinfolding),DHistotalenergychangeinachemicalreaction.,DGistheusefulenergy.Thisistheenergyusedtopropelmetabolism.,AnegativeDGmeansthereactionreleasesenergy(asheat),andisexothermic.,ApositiveDGmeansthereactionmustabsorbenergy(asheat),andisendothermic.,TheStandardState(DGo)Conditions,Reactionfree-energydependsuponconditionsStandardstate(DGo)-definedreferenceconditionsStandardTemperature=298K(25oC)StandardPressure=1atmosphereStandardSoluteConcentration=1.0MBiologicalstandardstate=DGoStandardH+concentration=10-7(pH=7.0)ratherthan1.0M(pH=1.0),10-14,Freeenergychangeisextrinsic,i.e.,itisdependentonamount(asanyoneona“diet”knows),Sovaluesarereportedrelativetoastandardstate(1.0M,pH7,25degCand1atmpressure).,EquilibriumConstantsandStandardFree-EnergyChange,DGreaction=DGoreaction+RTln(CD/AB),Atequilibrium:Keq=CD/ABandDGreaction=0,sothat:,DGoreaction=-RTlnKeq,10-15,Manymetabolicreactionsfunctionatequilibrium,whereforwardrate=reverserateandDG=0.,Asareminder,fromtheLawofMassAction:Keq=CD/AB,themassactionratio.,Forreactionsnotinthestandardstate,DGissumofthestandardenergychange(DGo)andthemassactionratio.,Atequilibrium(butoutsidestandardstate),themassactionratio=KeqandDG=0,sothestandardfreeenergyisrelatedtoKeq.,NotethatKeqandDGoarerelatedbyanexponential,soasmallchangeinDGoisalargechangeinKeq.,Anotherform:10-DG/2.303RT=Keq,soa2foldchangeinDGogivesa100foldchangeinKeq.,ActualFree-EnergyChangeDeterminesSpontaneityofCellularReactions,Whenareactionisnotatequilibrium,theactualfreeenergychange(DG)dependsupontheratioofproductstosubstratesQ=themassactionratio,DG=DGo+RTlnQWhereQ=CD/AB,10-16,Thefreeenergychangeforreactionsnotatstandardstateandnotatequilibriumissumofstandardstateenergyandmassactionratioe(Q).,E.Catabolismproduceshighenergycompoundsforuseinanabolism,Threetypesofcompoundsareproducedthatmediatethereleaseofenergy(1)AcetylCoAandotherthioesters(2)Nucleosidetriphosphates(e.g.ATP)(3)Reducedcoenzymes(NADH,FADH2,QH2),10-17,Themajorclassesofhighenergycompounds(verynegativeDGo)arethioesters,phospho-andmixedanhydrides,andreducedcoenzymes.,E1.TheFreeEnergyofATP,EnergyfromoxidationofmetabolicfuelsislargelyrecoveredintheformofATP,10-18,ATPisthecellsfuelsource,andmanyotherhighenergycompoundsarederivedfromATPenergy.,RememberATPhastwophosphoanhydrides,thealpha/betaandbeta/gammaphosphoanhdyrides.,Table10.1,10-19,ThehydrolysisofeachgivesDGoofabout7.5kcal/mol(about4kJ=1kcal).,Incontrast,phosphoestersarelowerinenergy,about3.5kcal/mol.,ComplexesbetweenATPandMg2+(Fig10.8),10-20,ATPnormallyfunctionsasamagnesiumcomplex.Thealpha,betacomplexisthemorecommon.,Energyofphosphoanhydrides,ElectrondeficientcentraloxygenElectrostaticrepulsionamongnegativelychargedoxygensofphosphoanhydridesofATP(3)ProductsaremorestablethanreactantsTherearemoredelocalizedelectronsonADP,PiorAMP,PPithanonATP(4)Solvationofproducts(ADPandPi)or(AMPandPPi)isbetterthansolvationofreactantATP,10-21,Theanhydrideoxygen(PO-O-PO)isbondedtotwoelectrondeficientphophorusatoms.,Thenegativechargesrepeleachphosphorylgroup,buttheyalsorepelwateroxygen,soincreasestabilitytowater.,Thisisagroundstatestabilizationoftheproductwhileincreasingenergycontentofstartingmaterial.,Theproductcharge(-3+-2)isbettersolvatedthanreactantcharge(-4).,Phosphoryl-GroupTransfer,Phosphoryl-group-transferpotential-theabilityofacompoundtotransferitsphosphorylgroupEnergy-richorhigh-energycompoundshavegrouptransferpotentialsequaltoorgreaterthanthatofATPLow-energycompoundshavegrouptransferpotentialslessthanthatofATP,10-22,AlthoughATPisthebenchmarkandmajorenergystore,othermetabolitescanhavesameorhigherenergy.,Table10.3,10-23,PEP(entry1),isanintermediateinglycolysis,hasaphosphoryl-enolmixedanhydride.,Phoshoglyceratehasamixedanhydridebetweenanacidandaphosphate(calledacyl-phosphate),Phosphocreatineisusedasanenergysourceinmuscle,butisderivedfromATP.,Acetyl-CoAisathioester.NoteitsenergyisequivalenttoATP.,Phosphateestersarelowenergy(andquitestable),E2.ThioestersHaveHighFreeEnergiesofHydrolysis,Thioestersareenergy-richcompounds(10.22)AcetylCoAhasaDGo=-31kJmol-1(10.23),10-24,TheenergyofhydrolysisofCoAestersisabout7kcal/mol.Whysomuchenergy?,Reactivityordersisacylchloride(RCO-Cl),acylanhydride(RCO-O-COR),thioesters(RCO-SR),followedbyesters(RCO-OR),Thioestersarereactivebecauseofthesizeofthesulfur.Itusesouter3porbitalstooverlapthecarbonyl2pelectrons,sopooroverlap.,E3ReducedCoenzymesConserveEnergyfromBiologicalOxidations,10-25,Redoxchemistryisverycommoninbiochemistry,sobefamiliarwiththeterms.,Anoxidant,gainselectrons,soitisreduced.,Areductantloseselectrons,soitisoxidized.,Foodstuffsareelectronrich,andlosetheirelectrons.Foodstuffscanbetermedreducingagents.,Theultimateoxidizingagentismolecularoxygen(O2),anditisreducedtowater.,Electronmovementfromfoodstuffs(Ared)tooxygen(Box),releaseslotsofenergy.,Theentireprocessisorganizedinpathwayssotheenergycanbecaptured!,ElectronTransferfromNADHProvidesFreeEnergy,10-26,ElectronsfromfoodstuffsarestoredasNADH(aswellasFADH2).,ElectronsinNADHhaveenergy(Eoisameasureofenergypotential)butarestabletooxygen.,F.ExperimentalMethodsforStudyingMetabolism,Addlabeledsubstratetotissues,cells,andfollowemergenceofintermediatesUsesensitiveisotopictracers(3H,14Cetc)VerifypathwaystepsinvitrobyusingisolatedenzymesandsubstratesUsemetabolicinhibitorstoidentifyindividualstepsandsequenceofenzymesinapathway,10-27,Pathwayswereinitiallyprobedwithradiolabeledfoodstuffs.,Eventuallyenzymeswereisolatedandpathwaysverified.,Naturalinhibitorscanblockapathway,increasingintermediatesbeforetheblock.,Chapter11-Glycolysis,Forcenturies,bakeriesandbrewerieshaveexploitedtheconversionofglucosetoethanolandCO2byglycolysisinyeast,11-28,Theanswerisbeer,wine,brandyandbread.Thequestionis:,Not“Whatgoeswithcheese?”but“whatisproducedbyyeastfermentation?”,Yeastproducealcoholsothecolonycangrowandreproduceintheabsenceofoxygen.,Theimportantpathwayisglycolysis.,A.GlycolysisIsaUbiquitousPathway,11-29,Glycolysisproduces2C3pyruvatefromoneC6glucose(nocarbonislost),TwoATPcomefromeachroundofglycolysis,Thefateofpyruvateinananimalcelldependsonthepresenceofoxygen.Inanaerobicmetabolism,pyruvatelactate,Inthepresenceofoxygen,pyruvateacetylCoA.,MostoftheenergyinglucoseisreleasedattheacetylCoAstageinTCAcycle.,Netreactionofglycolysis,TwomoleculesofATPareproducedTwomoleculesofNAD+arereducedtoNADH,Glucose+2ADP+2NAD+2Pi2Pyruvate+2ATP+2NADH+2H+2H2O,11-30,GlycolysisisusefulbecauseitgeneratestwonetmoleculesofATP,aswellasNADH.,NotetheglycolysisrequirestwoNAD+(oxidizedform).,Overview:Thehexosestage,11-31,Thehexosephaseinvolvesfour6-carbonsugars.,Instep1glucoseisphosphorylatedatC6toglucose-6-phosphate.,Instep2,G-6-Pisisomerizedtotheketosesugarfructose-6-phosphate,Notethatstep1isirreversible(energyreleased),Whilestep2atequilibriumiscloseto50%product/substrate.,Movementintothetriosestage,Phosphofructokinase-1,11-32,Instep3F-6-PisphosphorylatedwithATPatC1toformfructose-1,6-bisphosphate.,Thisisalsoirreversible,andusesasecondATP.Thusweinputtwohighenergybondstoinitate.,Instep4,wecleavethe6Csugartogivetwo3carbonsugarphosphates.,OneC3,dihydroxyacetonephosphate,hasaketone.,Thesecond,glyceraldehyde-3-phosphate,hasanaldenhyde.,Thetriosesugarstage,11-33,Thetriosephaseinvolvesboth3carbonunits.,G-3-Pisoxidizedto1,3-bis-phosphoglycerate,whichcontainsamixedanhydride(highenergy).,ThereleasedreducingequivalentsarestoredasNADH.,TheoxidationofG-3-PinducesDHAPtoformG-3-P,whichcanthenbeoxidized.,ThisequilibriumallowsbothC3unitstobemetabolizedusethesametriosestage.,Glycolysisthenproducestwo1,3-BPG,andtwoNADH.,AtthisstagethenetATPiszero(-2fromhexosestage,+2from1,3-BPGstage).,Thustheenergyreleasedinaldehydeoxidationiscapturedinthemixedanhydride,ThemixedanhydrideisusedtomakeATPfromADP,withreleaseof3-phosphoglycerate(3-PG),3-PGbecomesalowenergypointforglycolysis.,11-34,3-PGisisomerizedto2-PG.,And2-PGisconvertedtothehighenergyPEPbyeliminationofwater.,Thisisacleverreactionsincewaterelimination/additionisenergyneutral(50%atequilibrium),Buttheeliminationproductishighenergyduetoamixedanhydride.,ThehighenergybondofPEPistransferredtoADPtogiveATPandpyruvate.,BecausetwoPEPformfromeachglucose,thisstepgives2ATP,foranettotalof2ATP/glucose.,1.Hexokinasereaction(Fig11.3),11-35,Enzymesarenamedbasedonsubstrateorproductandreactiontype.,Hexoisshortforhexose,thesixcarbonsugar,whichisphosphorylated.,Thephosphorylistransferredtothe6-positionofthesugar.,TheoxygenattacksthegammapositionofMg-ATP.,TheproductsareMg-ADPand.,thesugar-6-phosphate,glucose-6-phosphateinthiscase.,Thisreactionisessentiallyirreversiblesinceabout-4kcal/molisreleased.,Kinasereferstotransferofahighenergyphoshphoryl.,Isozymesofhexokinase,11-36,Isozymesaredifferentproteinsthatcatalyzethesameorsimilarreactionsbutwithdifferentcharacteristics.,Thehexokinasescatalyzeshexose6-phosphorylationwithhexosesinmMconcentrations.,TheglucokinasesarespecificforglucosebutareactiveonlyathighmMsubstrateconcentrations.,Wellseelaterthathexokinaseisanaturalcontrolpointforsugaractivation.,2.G-6-Pisomerase:ConversionofG6PtoF6P(Fig11.4),11-37,Isomerasesinterconvertstructuralisomers.Thesubstrateisglucose-6-phosphate.,Theisomerizationisbestseen,andrequires,thelinearform.,Thebeta-hydroxyaldehydeinglucoseisconvetedto,Thebeta-hydroxyketoneinfructose-6-phosphate(F-6-P).,Thetransitionalintermediateisthesymmetricalene-diol:,G-6-Prequiresisomerizationbecauseweneedtoincorporatea2ndphosphateatthe1position.,3.Phosphofructokinase:TheReaction,11-38,PFK-1ismetabolicallyirreversibleandacriticalregulatorypointforglycolysisinmostcells(PFK-1isthefirstcommittedstepofglycolysis)Asecondphosphofructokinase(PFK-2)synthesizesfructose2,6-bisphosphate(F2,6BP),aPFK-1controlmolecule,PFKisakinase,sotransfersaphosphorylgroup.,PFKusesATPandF-6-P,AndtransfersthegammaphosphateofATPtothe1positionofF-6-PtogiveF-1,6-BP.,ThesecondproductisADP.,RecentlyasecondPFKthatphosphorylatesF-6-Patthe2positionwasidentifiedasacontrolenzyme.,PFK-1istheenzymeusedinglycolysistoproduceF-1,6-BP.,WellseelaterthatPFK-2producesF-2,6-BP,acontrolmolecule.,4.TheAldolaseReaction,11-39,Thethirdenzymemovesustothetriosephase.,Theending“ase”referstoabreakdown.,Aldol-asemeansthebreakdownofanaldolproduct.Aldolisthecondensationproductbetweenanaldehydeandketone(or2aldehydes).,Lookfirstinthereversedirection(itisreversible).,Here,the3positionofDHAPisdeprotonatedtogiveacarbonanion.,Theanionaddstothealdehyde(1-positionofG-3-P).,AbondformsbetweenC3andC4toformF-1,6-BP.,Inthefor