生物化学英文版课件：chapter 22 biosynthesis of amino acids and nucleotides

Chapter22

Biosynthesisofaminoacidsandnucleotides1.Sourcesofnitrogen,carbonskeletonsand sulfur.2.Thedenovoandsalvagepathways.3.Waystobalancethesynthesisofeach buildingblocks.ForDec.25and27,2012(byProfessorZengyiChang)Keyissues:BiosynthesisofAminoacidsandnucleotidesarecloselyrelated

Nitrogenarisesfromcommonbiologicalsources(N2fixation).Thetwosetsofpathwaysareextensivelyintertwined(withsharedintermediates).Muchcommonchemistryarefoundinbothpathways:transferofnitrogen(oftenfromGln)orone-carbonunits(carriedontetrahydrofolate).Thenitrogeninaminoacids,purines,pyrimidines,andotherbiomoleculesultimatelycomesfromatmosphericnitrogen,N2(namedas“azote,”meaning“withoutlife”,byLavoisier)

N2

→NH3

TheamountofN2newlyfixedperyear:

Bydiazotrophic(nitrogen-fixing) microorganisms,60%(1011kg);

ByLightningandultravioletradiation,15%;

Byindustrialprocesses,25%UnveilingthepuzzleofnitrogenfixationJeanL.Boussingault(1802-1887)Someplantsraisethenitrogencontentofthesoil(1838)HermannHellriegel(1831-1895)andMartinusBeijerinckLeguminousplantsfixnitrogengasbyitssymbiosiswithRhizobiumintherootnodules(1880s)JamesCarnahanandleonardMortenson:cell-freeextractsactiveinnitrogenfixationwereobtainedandnitrogenaseisolated(1960s)RobertH.Burris(1914-2010)15NlabeledN2

entersNH3andthenGluandAsp

(1940s)ArtturiVirtanen(1895-1973)NobelPrize1945N2fixationisthermodynamicallyfavorable,butkineticallyextremelyslowHasabondenergyof930kJ/mol(whilethatforaC-Ois350kJ/mol)Thechemistryofnitrogenfixationreaction

Industrial(ironcatalyst,500°C,300atm):ActualbiologicalN2fixationindiazotrophs:Requiredreaction:ATPhydrolysisisessentialtoreducetheheightsofactivationbarriersalongthereactionpathway,thusmakingitkineticallyfeasible.

Oneofthemostremarkable(expensive)reactionsinbiology!Nitrogenfixationiscatalyzedbythenitrogenasecomplex,presentonlyincertainbacteria(diazotrophslikecyanobacteriaandrhizobia)andenergeticallycostly.

CyanobacteriaRhizobiaThenitrogenasecomplex

Nitrogenase(MoFeProtein)Nitrogenasereductase(FeProtein)ElectronDonors:ferredoxinorflavodoxinATPbindingandhydrolysisisthoughttobothdrivethereductionoftheP-cluster&totriggeraconformationalchangeinthereductasethatcausesittodissociatetransientlyfromthenitrogenase,assuringunidirectionalelectronflow.PclusterMoFecofactor[4Fe-4S]clusterN2N2FerredoxinwasfoundtobearequiredforN2fixation(1962)MORTENSON,L.E.(1964).Ferredoxinrequirement

forN2-fixationbyextractsofclostridiumpasteurianum.Biochim.Biophys.

Acta81:473-477.Browncolored,mostreducingagentAbsolutely(togetherwithATP)requiredforN2-fixationbyex-tractsofC.pasteurianum(freedfromferredoxinbyDEAE-cellulosetreatment)whenpyruvateservingastheelectrondonor.Alsofunctioninphotosynthesis!ThenitrogenasecomplexisextremelylabiletoO2andvariousprotectivemechanismshaveevolved:livinganaerobically,formingthickwalls,uncouplinge-transportfromATPsynthesis(enteringO2isusedimmediately)orbeingprotectedbyO2-bindingproteins(e.g.,leghemoglobin)..

heterocystCyanobacteriaRhizobiaO2isbelievedtodamagetheFe-ScentersAmmoniacanbeincorporatedintobiomoleculesorconvertedtonitriteandnitrate,andbacktoN2bycertainbacteria(revealedinthe1980s)SubstituteO2asterminalelectronacceptorReducednitrogenintheformofNH4+isfirstassimilatedintoGluandGln(NH4+istoxicathighlevelstohumanbeingsandothermammals)Glnsynthetase:incorporatesNH4+intoL-GlutoformL-Gln,TheKMforNH4+islowbutconsumesATP

Glusynthase:Aniron-sulfideflavoprotein,catalyzesthereductive

aminationofα-ketoglutaratewiththe

useofglutamineasthenitrogendonor

Thesum:netsynthesisofGlufroma-ketoglutarateandNH4+!Occursonlyinbacteriaandplants.ContainingFAD,FMNandIron-sulfurcenters.GlnsynthetaseGlusynthaseGlnsynthetase-Glusynthasepathwayofammoniaassimilationa-ketoglutarate+GluTheGlutaminesynthetaseisaprimaryregulatorypointinnitrogenmetabolism:beingregulatedbyatleasteightallostericeffectorsandreversibleadenylylationinprokaryotes.

Theglutaminesynthesisisconstantlytailoredtocellularneeds!TheE.coli

glutaminesynthetase:12subunits(dodecamers)ActivesitesatinterfacesMg2+Mn2+MnTworingsofhexamersMg2+Mn2+AbifunnelactivesitecontainigtwobivalentmetalionsTheglutaminesynthetase

iscumulativelyinhibitedbyatleast8allostericeffectors,mostlyendproductsofglutaminemetabolism.Eachofthe50kDasubunitcontainsbindingsitesforallthe8allostericeffectorsinadditiontotheactivesites!AstrikingdemonstrationofcumulativefeedbackinhibitionandofcontrolbyacascadeofreversiblecovalentmodificationsAspecificTyrinbacterialglutaminesynthetase

isreversiblyadenylylatedbythecatalysisofadenylyltransferase,whichisinturniscontrolledbyreversibleuridylylation.Theanimalenzymeseemstoberegulatedbychangingitsoligomericstatus(octamerictotetrameric).

TheinactiveformAdenylylationincreasesthesensitivityofeachsubunittothe8allostericinhibitors.

AMPTyr397TheamideaminogroupofGlnsuppliesnitrogentomanyother“acceptor”compoundsviathecatalyzesofglutamine

amidotransferases:ammoniaisgeneratedfirstandthenaddedtotheacceptors

Aproposedgeneralactionmechanismforglutamineamidotransferases.Two-domainenzymesHighlyconservedVariesL-Aminoacidbiosynthesis:thecarbonskeletonsarederivedmainlyfromintermediatesofglycolysis,citricacidcycle,andpentosephosphatepathway;

nitrogenfromGlnandGlu.

Thestereochemistryattheα-carbonatomisestablishedbyatransaminationreactioninvolvingpyridoxalphosphateandaconservedfamilyoftransaminases.Thebiosynthesesofthe20aminoacidsuseintermediatesoftheglycolyticcycle,citricacidcycleandthepentosephosphatepathways

Sixgroupscanbecategorized123456Essential(indispensable)aminoacids(WilliamRose,1950s)Cannotbesynthesizeddenovothusmustbesuppliedinthediet(humans).Mnemonic:VITALpHTML(asdesignedbyZhileiZhao)Adeficiencyofevenoneaminoacidwillleadtonegativenitrogenbalance(moreproteinbeingdegradedthansynthesized)Nutritionalqualityofproteinsforhumans:Mammals>fish&poultry>fruits&plants.(1887-1985)RemovalofThr,butnotHiscausedpronouncednegativenitrogenbalance.(Hisfoundtobeessentiallater)Pathwaysforsynthesizingthe“essential”aminoacidsareusuallycomplex,involving5-16steps.

Transaminases(pyridoxalphosphate,PLP)

iskeyforaminoacidbiosynthesisaminoacida-ketoacidaminoacida-ketoacidAping-pongmechanismALT(SGPT)AST(SGOT)LevelofASTandALTarecommonlymeasuredinbloodtestreflectingliverorheartdamagesStereospecificityofthetransaminases:additionofaprotonfromtheLysresiduetoonesideofthequinonoidintermediatedeterminestheLconfiguration.

Structuresoftransaminasesarehighlyconserved.AlexanderBraunstein(1902-1986)ARussianscientistDiscoveryofthetransaminationreaction(1937)EsmondE.Snell1914-2003)Discoveryoftheroleofpyridoxal(vitaminB6)intransaminases(1945)Discoverydatesofthevitamins1913VitaminA(Retinol:retinal)

1910VitaminB1(Thiamine;Thiaminepyrophosphate)1920VitaminC(Ascorbicacid)

1920VitaminD(Calciferol)

1920VitaminB2(Riboflavin;FAD,FMN)1922(VitaminE)(Tocopherol)

1926VitaminB12(Cobalamins;cobalamins)1929VitaminK1(Phylloquinone)

1931VitaminB5(Pantothenicacid;CoenzymeA)1931VitaminB7(Biotin;biotin)1934VitaminB6(Pyridoxine;Pyridoxalphosphate)1936VitaminB3(Niacin;NAD,NADP)

1941VitaminB9(Folicacid;tetrahydrofolate)

1.Asp,Asn,Met,Thr,andLys(alsoIle)arederivedfromOxaloacetate

2steps8steps2steps4stepsCysOnefourthofthebuildingblockaminoacidsofproteinsaremadehere!

AspartatepyruvateTetrahydrofolate(H4folate)isahighlyversatilecarrierofactivatedone-carbonunitsTheone-carbongroup,atthreeoxidationstates,iscarriedatN-5orN-10nitrogenatom(denotedasN5andN10)ortoboth.H4folate510Requiredforthedenovosynthesisofpurines,dTMP,andcertainaminoacids.2.

Ala,

Ile,Val,Leu(and

partiallyLys)

arederivedfrompyruvate4steps3steps1step4stepsThenitrogengroupsareallprovidedbyGlu!DehydrataseΔPLPandTPParetwocofactorsusedhere;ΔThelastfourstepsofIleandValbiosynthesisarecatalyzedbythesamefourenzymes;ΔThesethreeaminoacidsseemtoserveonlyasbuildingblocksofproteins;ΔTheconceptofallostericcontrolwaslargelydevelopedhere.ThrAla3.

Glu,ProandArgarederivedfroma-ketoglutarate8steps4stepsGlu,Carbamoylphosphate

(alsoforpyrimidinebiosynthesis)AspInmammals,Argcanbederivedviatheureacycle,thusnotneededinadults(butessentialforinfants)(thea-aminogroupisprotectedbyacetylationforArgsynthesis)GluAspGlu4.Ser,GlyandCysarederivedfrom3-phosphoglycerate3steps1step2stepsH4folate,PLPInmammals(MetprovidesSulfur)PLPInbacteriaandPlantsPLPb-sythaseg-lyaseSulfurassimilation,comparabletoglutaminesynthesisinnitrogenassimilation!5.Trp,Phe,andTyrarederivedfromerythrose4-phosphateandphosphoenolpyruvate(PEP)6steps5steps2steps2steps+PEP+Gln,SerPRPP+Glu+GluIsozymes;Feedbackinhibitionof1stcommittedstep;Operon;Branchingpoints.PEP

BranchingpointSerPRPP

BranchingpointPEPChorismatemutaseGlnTheaandbsubunitsoftryptophansynthasehavedifferentenzymaticactivities,substratechannelingoccursinthisenzyme.aabba2b2PLP6.Hisisderivedfromribose5-PandATP8steps+Gln+GluPurinenucleotidebiosynthesisInherentlylinkedtothepathwaysofnucleotideformation;ThebeginningofRNAtoProteinWorldswitch?PRPPATPAminoacidsaremainlyderivedfromintermediatesofglycolysis,thecitricacidcycle,andthepentosephosphatepathway.VITALpHTML

(asinventedbyZhileiZhao)NH3(Gln)GluGluGlnNH3NH3GlnS2-CysSourcesofN,SintheaminoacidsAspGlnTherateofthesebiosyntheticpathwaysareoftenregulatedviaallostericfeedbackinhibition(andatthegenelevel).

AsparingeffectofIleonThrobservedforathreoninelessmutantofE.coli;Ilefoundtobe100timesInhibitorythanLeutoinhibitThrdeamination.EndproductThefirstcatalyticenzymeofIlesynthesisfromThr.Thefirstcaseofallostericfeedbackinhibition:ThrdehydrataseinhibitedbyIle

Umbarger,H.E.(1956)“Evidenceforanegative-feedbackmechanisminthebiosynthesisofisoleucine”,Science123,848.ThreecommonpatternsoffeedbackinhibitioninbranchingbiosyntheticpathwaysConcertedFeedbackInhibition:

BothIandFrequiredtoinhibita.CooperativeInhibition:EitherIorFweaklyinhibitsa;

CumulativeInhibition:IandFeachinhibitsatoagivendegree.

Sequentialfeedbackinhibition.FandIinhibitenzymesdandg,Cinhibitsenzymea

EnzymemultiplicityasensitivetoI

a’sensitivetoF.1.EnzymeMultiplicity2.Concertedinhibition(bothLysandThrrequired)A:aspartokinaseisozymes(notpresentinanimals!)BranchingbiosyntheticpathwaysoftencoordinatedviamultilayersoffeedbackinhibitionsB:HomoserinedehydrogenaseC:ThrdehydrataseManyaminoacidsactinregulatingthegenesoftheirbiosynthesis:trp

andhis

operonsextensivelystudied.Aminoacidsareprecursorsofhormones,coenzymes,nucleotides,alkaloids,cellwallpolymers,porphyrins(hemes),antibiotics,pigments,andneurotransmitters.

Glutathione

GlutathioneS-transferase,GST,oftenusedasaproteintagforaffinitypurificationArg,Gly,Met,Glu,Cys

usedhere.Messenger15Nand14Clabeling(1940sDavidShemin

)NOsynthase(fivecofactors)Synthesizedinliverandtransportedtomuscle.AntioxidantMelatonin(aneurohormone)“脑白金”Tyr,Glu,His,Trp

serveasprecursorsofneurotransmitters,hormones,vasodilators.

NAD+NeurotransmitterVasodilatorNeurotransmittersandhormones

5-hydroxyltryptamine(5-HT)Inhibitoryneurotrasmitter5-methoxyN-acetyltryptamineNucleotidebiosynthesisThesebasesarenotintermediates!Anamplesupplyofnucleotidesisessentialformanylifeprocesses.NTPareprecursorsofnucleicacidsynthesis.Sourceofenergy(ATP,GTP).Nucleotidederivativesparticipateinbiosyntheticprocesses(e.g.,UDP-glucoseintheformationofglycogen).Secondmessengers(cAMPandcGMP).Donorofphosphorylgroups(ATP,byproteinkinases).Moietyofcofactors,NAD/NADP,FAD/FMN,CoA(ATP).Etc.Nucleotidesaresynthesizedviaeitherthedenovoorsalvagepathways.ThebaseissynthesizedfromsimplerstartingmaterialsAbaseisreattachedtoaribose(ribonucleotides)

(ribonucleotides)

Isotopetracerexperiments(mainlyusing13C-,14C-labeledprecursors)inpigeons(andlateritscellfreeliverextract)revealedtheoriginsoftheatomsinthepurineandpyrimidinerings(JohnBuchananandRobertGreenberg,1946-51).

Approach:Fedradiolabeledprecursorstopigeons,andchemicallydegradedtheuricacidtheyexcreted.

Buchanan(1917-2007)Greenberg(1918-2005)Thepurineringisassembledonribose-P

fromsimple

precursors.(H4folate)(H4folate)123456789Thepyrimidineringisassembledfromsimpleprecursorsbeforeattachedtoribose-P

(orGln)Denovopurinenucleotidesynthesis:thebaseassemblesontheribose-P;IMPisthefirstnucleotidesynthesized.CommittingstepNonsequentialsteps1,3,5arecatalyzedbyonemultifunctionalproteinineukaryotes!(Unstable)1stringclosureStepwiseassemblyofthepurineringoccursonribosephosphateRibose5-Pa(remnantofATPreleasedduringHisbiosynthesis)Steps10and11arecatalyzedbyoneprotein.2ndringclosureAspdonatesanaminogroupinasimilarfashionasitdoesintheureacycle.Biotinnotusedhere!BiosynthesisofAMP&GMPfromIMP.GTPasanenergysource!BalancedsynthesisofAMPandGMP:AMPformationrequiresGTP&GMPformationrequiresATP!ThebiosynthesisofAMPandGMPisregulatedmainlybysequentialfeedbackinhibition(nocovalentregulation)

Astrategyascommonlyusedinregulatingaminoacidbiosynthesis.ThecommittingstepAMPandGMPactsynergistically.SynthesesofAMP&GMParebalancedviafeedbackinhibition.ATP++GTPThedenovobiosynthesisofpyrimidinenucleotides:thebasering(asorotate)isfirstassembledbeforebeingattachedtothephosphoribosylgrouptoformorotidylate.CarbamoylphosphatesynthetaseII(cytosol)Asptrans-carbamoylaseDihydroorotaseDihydroorotatedehydrogenaseOrotatephosphoribosyltransferaseOrotidylate

decarboxylaseThedenovobiosynthesisofUTPandCTP(dTMPderivedfromUMP)Rateenhancement:1017-foldCAD:Carbamoyl-phosphatesynthetase2Aspartatetranscarbamylase,DihydroorotaseCarbamoylphosphatesynthetaseIIcontainsthreeactivesitesviausingtwosubstratechannels.Gln

HCO3-(ATP)ATPNH4+Pyrimidinenucleotidebiosynthesisinbacteriaisregulatedattheaspartatetranscarbamoylase(ATCase),of12subunits,alsobyusingthefeedbackinhibition(byCTP)strategy.TwocatalytictrimersCTPATPActive(“relaxed”)Inhibited(“tight”)FeedbackinhibitionbyCTPThreeregulatorydimersRegulationofbacterialATCase

Archetypalexampleofallostericmodulation

(apurinenucleotide)(apyrimidenucleotide)BasespecificNMPkinasestogetherwithanonspecificNDPkinaseconvertsNMPstoNTPsEachofthefivenucleosidemonophosphate(NMP)kinasesconvertsthecorrespondingNMP/dNMPstoNDP/dNDPsusingATP.Asinglenucleosidediphosphate(NDP)kinaseconvertsallNDP/dNDPstoNTP/dNTPsusingATPorotherNTPsTransphosphorylationreactionsAlldNDPs(includingdUDP,butexceptdTDP)arederivedfromNDPsviathecatalysisofastrictlyconserved

ribonucleotidereductase,viaafreeradicalmechanism.

ThisisconsistentwiththenotionthatRNAprecededDNAinthecourseofevolution!Ribonucleotidereductase

containstwotypesofsubunits.

Bothactivity&Specificitymodulated:BalancedsynthesisofdNTPs.Substratespecificitysite:ThebindingofonedNTPwillpromotethesynthesisoftheothers

notTDP!A=dATPorATPA=UDPandCDPProposedcatalyticmechanismforribonucleotidereductase.Radicalat3`-CRadicalcationDonorsofone-carbonunits&electronsReductivemethylation.ThesoledenovopathwayforproductingdTMP:goodtargetforcancerdrug.

dUMPRaltitrexed(Tomudex)dTMPissynthesizedfromdUMP!DHFRdUMPisderivedfromdUTP,whichisinturn

derivedfrom

dCTP

or

dUDP.DNAXdUTPase(ordUTPpyrophosphatase):EssentialforlimitingintracellularpoolsofdUTPandthuspreventingtheproductionofUMP-containingDNA;

ThereactionscatalyzedbyribonucleotidereductaseandthymidylatesynthaseareprobablykeyforthetransitionfromanRNAworldtooneinwhichDNAstoresgeneticinformation.Purineandpyrimidinebasescanbereconvertedtoribonucleotidesviathesalvagepathway.SalvagepathwayofpurinenucleotidebiosynthesisDefectofHGPRTcausesLesch-Nyhansyndrome.Note:thisisnotforthebiosynthesisofdeoxynucleotidesDr.MichaelLeschDr.BillNyhan

LNSpatient:Self-mutilatingandgoutUricacidistheexcretedendproductofpurinenucleotidecatabolisminhumansandmanyotheranimals.purinenucleosidephosphorylasepurinenucleosidephosphorylaseXanthineoxidaseXanthineoxidasenucleotidasenucleotidaseAdenosineDeaminaseADA)guaninedeaminaseAMPdeminaseExcessuricacid,willdepositinthejointsorkidney,causinggout(痛风)orkidneystone.OxidationofGMPandAMPtoproduceuricacidThedeficiencyofADAwillcauseseverecombinedimmunodefficiency(SCID)inhuman.BaseBaseNucleosideNucleosideNucleotideLowwatersolubilityMocofactorFeS-clusterFAD

(antioxidant)

Harmfulorhelpful?

Allopurinolwasdesignedtobeasuicideinhibitorofxanthineoxidasetotreatgout(ElionandHitchingssharedtheNobelPrizein1988fortheirdiscoveriesofimportantprinciplesforsuchdrugdesign).别嘌呤醇

Uricacidisfurtherdegradedinmanyanimals.ofteninvolvingC-NbondcleavageIncreasedwatersolubilityAmidotransferases,thymidylatesynthasea