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首页 镍离子与酵母乙醇脱氢酶的相互作用(英文).doc

镍离子与酵母乙醇脱氢酶的相互作用(英文).doc

镍离子与酵母乙醇脱氢酶的相互作用(英文).doc

上传者: brant晓珲 2017-11-11 评分 4.5 0 79 11 359 暂无简介 简介 举报

简介:本文档为《镍离子与酵母乙醇脱氢酶的相互作用(英文)doc》,可适用于高等教育领域,主题内容包含镍离子与酵母乙醇脱氢酶的相互作用(英文)镍离子与酵母乙醇脱氢酶的相互作用(英文)April物理化学(WuliHuaxueXuebao)Acta尸s一符等。

镍离子与酵母乙醇脱氢酶的相互作用(英文)镍离子与酵母乙醇脱氢酶的相互作用(英文)April物理化学(WuliHuaxueXuebao)Acta尸s一ChimSin,,():Article】镍离子与酵母乙醇脱氢酶的相互作用尹国维尉薇徐佳wwwwhxbpkueducn李芝芬王保怀杜为红,(中国人民大学化学系,北京北京大学化学与分子工程学院,物理化学研究所,北京)摘要:不同体系中,金属离子与蛋白以不同的结合方式相互作用酵母乙醇脱氢酶是一个含锌金属酶,它可催化乙醇脱氢为乙醛的反应本文应用紫外一可见光谱,荧光光谱,差示扫描量热法等技术研究了二价镍离子与酵母乙醇脱氢酶的相互作用镍离子与酶结合后在nm出现了紫外吸收带,同时荧光光谱反映了酶的构象变化,紫外与荧光光谱均展现了结合过程的双相动力学镍离子与酶的相互作用导致了酶由四聚体向二聚体的解离在酶热变性过程中,镍离子增加了乙醇脱氢酶的变性温度和变性焓研究工作揭示了镍离子与酶相互作用复杂和深层的作用机制关键词:构象变化镍离子酵母乙醇脱氢酶相互作用中图分类号:OInteractionofNickel(II)withYeastAlcoholDehydrgenaseYINGuoWeiWEIWeiXUJiaLIZhiFenWANGBaoHuaiDUWeiHong'(DepaetmentofChemist~,RenminUniversityofChina,Beijing,PRChinaInstituteofPsicalChemist~,CollegeofChemist~andMolecularEngineeringPekingUniversity,BeijingPRChina,Abstract:ThebindingmodeofmetalionswithproteinsiSusuallydifferentindifferentsystemsYeastalcoholdehydrogenase(YADHiSazinccontainingmetalloenzymethatcatalyzesthefermentationreactionofalcoholtoacetaldehydeUVVisspectroscopy,fluorescencespectroscopy,anddifferentialscanningcalorimetry(DSC)wereusedtoinvestigatetheinteractionofnickel(II)withYeastalcoholdehydrogenaseTllebindingofNi(II)toYeastalcoholdehydrogenaseshowsaamUVabsorbancebandandtheenzymeconformationalchangeisreflectedinthefluorescencedataBothUVVisandfluorescencespectraexhibitbiphasickineficsfor血ebindingprocessTheinteractionofNi(II)wimleeastalcoholdehydrogenasecausestheenzymetotransformfromatetramertoadimerTheconformationalchangeoftheYeast~coholdehydrogenaseresultsinanincreaseinthedenaturationtemperatureandinamolarenthalpychangeduringtheDSCprocessThisstudyrevealsacomplexbutdeepseatedmechanismfortheinteractionofNi(II)withYADHKeyWords:ConformationalchangeNickel(II)YeastalcoholdehydrogenaseInteractionMetalionsareindispensableinkindsofbiomacromolecules,especiallyinmetalloproteinsTheyactsascofactororinhibitorinvariousproteinsandinevenengineeredproteinsYeastalcoholdehydrogenase(YADH)isametalloenzymewhichcatalyzethefermentationreactionofalcoholtoacetaldehyde【JThisfermentationhasbeenwidelystudiedforitsimplicationsinwineandbeerproduction田andanincreasinginterestinitsapplicationforbiotechnologicalprocessesofbioconversionofdifferentorganicwastesintoethanoltobeusedassolventorfuelt一YADHisatetramer(kDa)withfoursubunitsheldtogethertgReceived:October,Revised:December,PublishedonWeb:FebruaryCorrespondingauthorEmail:whduchemmeeducnTel:lOTheprojectwassupposedbytheNationalKeyBasicResearchProgramofChina()(CB),NationalNaturalScienceFoundationofChina(),andKeyProjectoftheMinistryofEducationofChina()国家重点基础研究发展规划项~()(CB),国家自然科学基金()及教育部科技重点项目()资助杜为红,年月至年月在北京大学化学与分子工程学院攻读博士学位E~tofialofficeofActaPhysicoChimicaSinicaActaPs一ChimSin,VOEachsubunitcontainstwozincionswithonezincionlocatedattheactivesite(catalyticzinc)andboundtotwocysteines,onehistidineandawatermoleculeTheotherzincionboundtofourcysteinesandmaintainsthetertiarystructureoftheenzymes(structuralzinc)TheZn(n)sitesareconservedamongADHsfromdifferentspecies~ThestructureactivityrelationshipsofADHsarewidelyconcemedinrecentyearsEvaluationofHofmeistereffectsonADHandotherproteinsindicatedthattheproteinkineticsstabilitycouldbeinfluencedbysaltspeciesandtheirconcentration,andthethermodynamicparameterswerealsoaffectedbysomesmalmoleculestqToimprovetheactivityofADHsomeworksfocusedonthetransitionmetalsubstitutionforzincionsandpossiblesubstratesandinhibitorswerestudiedt】MoreoverthequantummechanicsmethodwasusedtoshowthekineticisotopeeffectsandenzymemotionTheinteractionofmithramycinandchromomycinwitllADHwereperformedtocheckthebindingaffmityofthetwoanficancerantibioticstobivalentcationsiezincionsinstructurasiteorcatalyticsite"ThismeansthattheinhibitorsofADHarewidelydistributedThevincludenotonlytheclassicalreagentmethylpyrazoleandtheanticancercompoundsbutalsodifferentmetaionssuchascopperwandbismuthAmongthebivalentcations,Ni(II)isreportedtoinhibitADHinamodeofmixedtypemechanisminpreviousstudy圆butlittleisknownaboutthedetailedinformationonNi(II)YADHinteractionInthisPaper,wehavecharacterizedtheinteractionandinhibitionofNi(II)toYADHUVVisspectroscopyandfluorenscencespectroscopywereusedtoinvestigatethebindingprocessofNi(II)andYADHEllmanmethod~wascarriedouttodeterminethethiolategroupbindingtoNi(II)AndtheinhibitionmechanismwasstudiedbyenzymaticreactionFurthermore,thedifferentialscanningcalorimetry(DSC)andfastproteinliquidchromatography(FPLC)wereperformedtoevaluatethethermodynamicstabilityofproteinMaterialsandmethodsSamplesYADHandnicotinamideadeninedinucleotidfNADwerepurchasedfromSigmaAldrichCorUSA)TheenzymewasusedwithourtherpurificaftonNickelousacetatetetrahydrate,trihydroxymethylaminomethane(Tris),and,一dithiobis(一nitrobenzoicacid)(DTNB)werepurchased丘omBBIcompany(USA)AllotherreagentswereofanalyticalgradeUVVisspectroscopyUVVisspectroscopywasusedtostudythebindingprocessofNi(andYADHTheyophilizedpowderofYADHwasdissolvedinmolL,TrisHCbu艉ratpHEnzymeconcentrationwasdeterminedfromtheUVabsorbanceatnmwithanabsorptioncoe衔cient(s)ofxmolLcm嘲ThespectrumwidthwasfromtonrnfoldsofNi(II)fxltoolL,wasaddedtoxmoLYADHinxl~molLTrisHCbufferatpHKThecourseofthereactionwasmonitoreduptominAUUVvjsspectrawererecordedonaCaryspectrometer(Varian,USAwiththermostatholdersatKFluorescencespectroscopyTheexperimentwascarriedoutonaPerkinElmerL$fluorescencespectrometerAsolutionofxlmolLYADHreactedwitllfoldsofNi(II)inxl~molL,TrisHClbufferatpHKTheexcitationwavelengthwasatnrnand血eexitslitwassettonrnThechangesinemissionintensitieswereobtainedatregulartimeintervalsEachspectrumwascorrectedbyblanksubtractionusing,xl~toolL,TrisHClbufferatpHEnzymecatalysisreactionTheconcentrationsofNADandNADHweredeterminedusingtheextinctioncoefficientsofxmolLcnlatnmEnzymeactivitywasdeterminedbythechangesofinitialrateofabsorbanceatnmcorrespondingtothereductionofNADtoNADHaspreviouslyreportedf】ThesolutionofYADHwasincubatedinthepresenceofexcessofNi(II)forminAnaliquotwaswithdrawnandaddedtoasolutioncontainingxtoolLNADandmolLEtOHThefinalenzyl~econcentrationwasx~mo卜L'insolutionTheinitialrates(rnofreactionwererecordedatdifferentconcentrationsofethanolrangingfromxltoxlmolLTheMichaelisconstant(andthemaximalreactionvelocity(rmx)forinhibitedreactionsandcontrolwereobtainedfromLineweav~一Burkplotsts器():监士()Sr眦ThiolategroupanalysisofYADHEllmansmethodwasutilizedtodeterminethefreethiolatecontent(SH)ofYADHbeforeandafterreactingwithNi(II)YADHwasincubatedwimfoldsofNi(II)atKinxmolLTrisHClbuffer,pHAsolutionofDTNBrxmolL,wasaddedtothemixtureThefinasolutioncontainedxlmolL『YADHOxlOmolLNi(II)andxlmolLDTNBThereactionsolutionwasincubatedforhuntiltheabsorbanceatamdidnotchangeTheamountofgeneratedPnitrothiolatewasdeterminedusingtheextinctioncoefficient()ofxmol~LcmDifferentialscanningcalorimetryDifferentialscanningcalorimetryexperimentswereperformedwithaSetaram(LyonsFrance)MicroDSCIIIcalorimeterThemixtureoffoldsofx~molLNi(II)andmolLYADHwasincubatedforhinxlmolLTrisHClbufferatpHO,KThenitwasmeasuredusmgthesc砌ingmteofOK'minTheexperimen诅ltemperamrerangewasfromtoKTem口eraturecoITectionandbaselinecorrecdonhadbeendnebeforeprceedingwiththeexperimentThesamplevOlumewasOmLTrisHCbufferwasusedasmereferenceinalthethreerepeatexperimentsNoYINGuoWeieta:InteractionofNickel(II)withYeastAlcoholDehydrogenaseFastproteinliquidchromatographyAsolutionofixmoLYADHwasincubatedwimfoldsofNi(II)inlmmolLTrisHClatpHKAfterminthemixnrewasinjectedtofastproteiniquidchromatography(FPLC)systemeTrisHCbuffer(mmol'LpH,wasusedasanelutionsolventChromatogramswererecordedbymonitoringtheabsorbaliceatamwithaUVdetectorControlexperimentswereperformedintheabsenceofNi(II)Themolecularmasscalibrationcurvefor也ecolumnwasobtainedusingbovineserumalbumin(kDaandcytochromeCfkDaasstandardsResultsBindingofNi(II)andYADHTheinteractionofNi(II)withYADHleadstoanewUVVisabsorptionband(FigA)WitlthemixtureoffoldsofNi(IItYADHsolutiontheabsorbalicecenteredatnmincreasedgraduallyThisbandwasassignedasSNi(II,ligandtometalchargetransfer(LMCT)transitionsduetoNi(II)bindingtothethiolateligandItcouldbeusedtomonitortheprogressofthereactionbetweenNi(II)andYADHKineticsofthereactionwasdescribedbythedependenceofabsorptionspectrumontime(FigIB)ItwascharacterizedbyaninitialrapidincreaseinabsorbantethenaprogressiveincreaseforthedurationThetwo一|nmrminFig(A)Timescaleofabsorptionspectrum,(B)kineticsofthereactionofNi(II)toYADHatnnlsolutioncontainingYADH(xlOmolL)andfoldsofNi(n)inxltoolTrisHClatpH,K:thebroadbandcenteredatoininFigAindicatingformationofNi)S(thiolate)bonds,reactiontimefrombottomtotop:,,,,,,,,,o,,millkineticstepscouldberesolved,whichobeyfirstorderkineticsandfittoabiexponentialgrowthusingthenonlinearleastsquaremethod:A(=Aexp(一klA~exp(一)y()wherekandkaretherateconstantsofthetwokineticphases,AandAarethecorrespondingamplitudesthatshowthecontributionoftheindividualkineticphasestotheobservedchangeinabsorbanceTherateconstantkl,wasmeasuredtobemin,,andcontributedtothewholereactiontAndtherateconstantkhadavalueofxmin一representingtherestofthereactionConformationalchangeinYADHduetothebindingofNi(II)FluorescencespectroscopyiswidelyusedinproteinconformationalinvestigationsincethetryptophanandtyrosineresiduescanproduceintrinsicfluorescenceoYADHhasfivetryptophanresiduesineachsubunitTheseresiduesproduceanintrinsicfluorescenceforYADHatamWiththemixtureoffoldsofNi(II)toYADHsolution,thefluorescenceemissionintensitydecreasedobviouslyfFigA)ItrevealedthatconformationalchangesoccurredinYADHuponNiI)bindingThedecreaseofYADHintensityveFsll$timewasalsoinabiphasicprocessandcouldbefittedbyatwoexponentialfunctionasusedinUVdataprocessingrFigB)erateconstantkxforthefaststepwasmeasured至童号OOnmFig(A)Timescaleoffluorescenceemissionspectratllanexcitationwavelengthofnnl(B)kineticsofthereactionofNi(II)toYADHatnnlemissionintensitysolutioncontainingYADHfxlmolLandfoldsofNi(II)inxlmolL,TrisHClatpHK:reactiontimefromtoptoboaom:,l,,,,,,,,,,,,,,,,,rainllActaPhys一ChimSin,VO一I,一r'IOOOOEtOH(Lmol)FigLineweaverBurkplotsoftheenzymecatalysisreactionThesolutioniscomposedofxlmolLYADHandxlmoNADatK,xlmolLTrisHC,pHforcon~olreaction()wasfoundtobexmolLThevalueisinagreementwiththatofcon~olinthepresenceof()and()foldsofNi)tobemin】whichcontributedtoofthereaction,whiletherateconstantfortheslowstephadavalueofxminmatrepresentedtherestofthereactionTheratesareslightlyhigherthanthecorrespondingvaluesobtainedfromUVvisspectroscopyInhibitionofNi(II)toYADHactivityBasedontheinteractionstudyofYADHandNi(II)wemeasuredtherateofethanoloxidationcatalyzedbyYADHatdifferentsubstrateconcentrationsinthepresenceofNi(II)ThekineticsofenzymecatalysisreactioncanbedescribedbyaMichaelisMentenmodeInthepresentwork,theandrnmwerecalculatedtobexmolLandODsfD:opticadensity)respectivelyfortheuninhibitedcontrolreaction,whichwereacceptableforf~erenzymaticinhibitionanalysisrFigAndtheKmvaluesobtainedinthepresenceoffoldsandfoldsofNi(II)werealmostthesameasthatintheabsenceofNi(II)However,therrrmdecreasedsignificantlyduetotheincreaseofNi(II)TheLineweaverBurkplotsshowedatypicalmodeofnoncompetitiveinhibitionl~ThiolategroupanalysisofYADHThefreethiolatecontentsweredeterminedusingDTNBbyElmansmetodsoastoinvestigatewhetherNifII)bindstofleelKFigDSCcurvesforpureYADHinthepresenceandabsenceofNi(II)IntheabsenceofNi~,theonsettemperatureismeasuredat()K(cRrvea),thecorrespondingvalueinthepresenceoffoldsofNiDismeasuredat(i)K(curveb)CysresiduesofYADHTheamountofgeneratedPnitrothiolatewasdeterminedusingtheabsorbanceatnlnasdescribedintheexperimentalsectionTotallyfreeCysresiduesarefoundineachYADHsubunitaftertreatmentwithdithiolthreitol(DTT)freeSHgroupsinthenativeenzymeweredeterminedinthepresentworkAfterincubationwithfodsofNi(II)forhatpHTrisHCbuffer,K,thenumberoffreethiolategroupswasdeterminedtobelThereforethereisonefreethiolgrouplossineachsubunitofYADHcomparedtoitsintactformThermaldenaturationofYADHuponbindingofNi(II)TheDSCmethodprovidedsignificantinformationaboutthethermodynamicpropertiesofproteinmolecules,andtheinfluenceofmolecularinteractionsoffthestabilityofproteinsandnucleicacidsDThedenaturationexperimentofYADHbvDSCstartedfromtoKandretttrnedfromtoKYADHshowedanirreversibledenaturationprocess(Fig)Therewasanexothermicpeakat()K(onsetpoint),whichwasverysimilarasreportedThemolarenthalpychangeofdenaturationoff一)xlOkJmolwastoolargeforconformationalchangeandpossiblyduetoproteinsedimentaRelativeretentionvolumeRelativeretentionvolumeFigFPLCprofilesofYADHafterincubationwithNi(II)(A)nativeYADH,)YADHwithfoldsofNi(II)incubationafterlminasolutionofcaIxmolLYADHwasincubatedwithfoldsofNi(H)inmmol'LTrisHCpHEc苗ls口uluu^I苗一加舳加ONOYNGuoWeieta:InteractionofNickel(II)withYeastAlcololDehydrogenaselltionAdditionofNi(II)resultedintheincreaseofmolarenthalpychangeanddenaturationtemperatureto(o)xkJmol一and()K(onsetpoint),respectivelyFastproteinliquidchromatographystudyMetalionsareknowntoaffectsignaltransductionandproteinproteininteractionWecarriedout血isexperimentinordertodemonstratewhetherNi(II)interfereswiththequaternarystructureofthenativeYADHAfterloadingYADHsolutionintothecolumn,therelativeretentionvolumevaluewasobservedatandrespectivelyPeakainFigAcorrespondstoakDaspeciesbasedonthemasscalibrationcurvewhilepeakbcorrespondstoacomponentwithamolecularmassaboutkDaTherefore,theycanbeassignedtothetetrameranddimerOfYADHrespectivelyTheexistenceofdimermightbefromconformationaequilibriumofADHinsolutionAfterincubationwimNi(II)thepeakatdecreasedinitsrelativeintensityobviouslyrFigB)ilethepeakatin

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