Fasolotoxyna, Publikacje naukowe
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//-->Biochem.J.(1985)228,347-352PrintedinGreatBritain347Theinactivation ofornithinetranscarbamoylase byN-(N-sulpho-diaminophosphinyl)-L-ornithineMatthewD.TEMPLETON,*§RobinE.MITCHELL,IPatrickA.SULLIVAN*andMaxwellG.SHEPHERDll*Department ofBiochemistry andtExperimentalOralBiologyUnit,University ofOtago,Dunedin,NewZealand,andtDivisionof HorticultureandProcessing,DepartmentofScientificandIndustrialResearch,Auckland,New Zealand(Received8October1984/31December1984;accepted18January1985)Phaseolotoxin,atripeptideinhibitor ofornithinetranscarbamoylase,is aphytotoxinproducedbyPseudomonassyringae pv.phaseolicola,the causalagentofhalo-blight inbeans.Invivothe toxin is cleavedtoreleaseN6-(N'-sulpho-diaminophosphinyl)-L-ornithine,themajortoxicchemicalspecies presentindiseasedleaftissue.This paperreportsontheinteractionbetweenN8-(N'-sulpho-diaminophosphinyl)-L-ornithineand ornithine transcarbamoylase.N6-(N'-Sulpho-diaminophosphinyl)-L-ornithinewasfoundtobeapotentinactivatorof the enzyme, incontrastwithphaseolotoxin,whichpreviouslyhasbeenreported toinhibit theenzymereversibly.InactivationbyN6-(N'-[35S]sulpho-diaminophosphinyl)-L-ornithineresulted in theincorporationof35Sintoethanol-precipitated protein.Thestoicheiometryof35Sincorporationwasapproximately1mol/molofactivesites. Inactivationwassecond-orderanda rateconstantof106M-1 .-1at0°Cin50mM-Tris/HCl,pH9.0,wasobtained.Carbamoylphosphate,asubstrateofornithinetranscarbamoylase, protectedtheenzymefrominactivation.Adissociationconstantof3pMfor theenzyme-carbamoyl phosphatecomplexwascalculated.L-Ornithine,the second substrate forornithinetrans-carbamoylase,protectedtheenzymeonlyathighconcentrations.The resultsare con-sistentwithN6-(N'-sulpho-diaminophosphinyl)-L-ornithinebeingapotentaffinitylabel that binds via thecarbamoylphosphate-bindingsite of ornithinetrans-carbamoylase. CleavageofphaseolotoxintoN,-(N'-sulpho-diaminophosphinyl)-L-ornithineinvivoappearstobeanimportantfunctioninthephysiologyofthedisease.Phaseolotoxinisachlorosis-inducingtripeptidephytotoxin produced byPseudomonassyringaepv.phaseolicola,thecausalagentofhalo-blightofbeans(Phaseolus vulgaris L.)(Mitchell,1976;Mooreetal.,1984).An extensivekineticanalysisoftheinhibitionof ornithinetranscarbamoylase(OTCase)fromEscherichia coliWby purifiedphaseolotoxinhasbeenperformed (Templetonetal., 1984).PhaseolotoxinwasfoundtobeaAbbreviationsused:OTCase, ornithinetranscarbam-oylase; PSorn,N&-(N'-sulpho-diaminophosphinyl)-L-ornithine.§Presentaddress:Plant DiseasesDivision,Depart-mentofScientificand IndustrialResearch,Auckland,New Zealand.1Towhomrequestsforreprintsshouldbeaddressed.reversibleinhibitorof OTCaseanddisplayedmixed inhibitionwithrespecttocarbamoylphos-phatewithanapparentKiandapparentK,'of0.2pMand10pMrespectively.Inhibitionwas non-competitivewithrespecttoL-ornithinewithanapparentK,of 0.9gm.Theseresultssuggestedthatphaseolotoxinwasbinding primarilytothe carb-amoylphosphate-bindingsite ofOTCasebut thatit could alsobindtotheOTCase-carbamoylphos-phate complex,althoughthiseventwaskineticallylesssignificant(Templetonetal.,1984).Although phaseolotoxinisproducedinliquidculturesofPs.syringaepv.phaseolicola (Mitchell,1976),there isstrongevidencethatthe toxiniscleavedinvivobyplant peptidases (Mitchell&Bieleski, 1977).Onthe basis of therecentstruc-tural revision ofphaseolotoxin (Mooreetal., 1984),Vol. 228348M.D.Templeton,R. E.Mitchell,P. A. Sullivan and M. G.Shepherdments enzymeconcentrationwascalculatedwithrespecttothesubunitconcentration,i.e.theactive-site concentration.Incorporationof radioactivityintoethanol-precipit-ableproteinEnzyme(5.2pM)and[35S]PSorn(0-6.25pM)were incubated in 50l of50mM-Tris/HCl,pH 9.0,at0°Cfor30 min.Portions(5p1)wereremovedandassayedfor enzymeactivity.Bovineserumalbu-min(50lof a 10mg/mlsolution)wasaddedtoeachremaining45jlsampleas acarrier. Proteinwasprecipitatedwith900 4u1of95% (v/v)ethanol,andthemixturewaskeptonice for 30min.Pre-cipitatedmaterialwascollectedonWhatman2.1cm GF/F glass-fibrefiltersthat had been pre-soakedin ethanol. The filterswerewashed(1x15ml)with aq.50% (v/v)ethanol solutioncon-taining10mM-K2HPO4and10mM-ornithinetoremovenon-specificallybound[35S]PSorn.Omis-sion of thisstepresultedinnon-specific bindingofapprox.10%oftheoriginal radioactivity.Filterswerewashedwith95%(v/v)ethanol(2x15ml)anddriedinan oven at80°Cfor3 h.Theradioactivityprecipitatedonfilterswasmeasuredin10mlofTriton/toluenescintillant.thepeptidase productwouldbeN6-(N'-sulpho-diaminophosphinyl)-L-ornithine(PSorn),with thestructure,asproposed byMooreetal.(1984),shownin Scheme 1. Beanplantsweregrownin thepresenceof35SO42-andinfectedwithPs.syringaepv.phaseolicola.Extractsofthechlorotictissuewerefoundtocontainup to 1.5ygofPSorn/gfreshwt.,buttheconcentrationsofphaseolotoxinwereapproximately100-fold less(Mitchell&Bieleski,1977).Phaseolotoxinhasbeenshowntobeeasilycleavedby peptidases (Mitchell, 1976)in vitro.ItwasthereforeimportanttoinvestigatewhetherthecleavageofphaseolotoxintoPSornaffectedtheinhibitionofOTCase.Previouslypublished datasuggestthat PSornmayinhibitOTCase in amannerdifferent fromphaseolotoxin.Kwok et al.(1979)reported that achemically unidentified toxin preparationisolatedfromchloroticleaves, whichpossiblycontainedsignificantquantitiesofPSorn,was anaffinitylabel forOTCase.Inaddition,Kwok &Patil(1982) showedthatphaseolotoxintreated withleucine aminopeptidasewas amorepotent inhibi-torof OTCaseunderstandardassayconditions.Thepresent paper reports theresultsof experi-ments onthemode ofaction ofpurifiedPSorn onOTCasefromE.coli W.ExperimentalMaterialsCarbamoylphosphate(disodium salt),L-citrul-line, L-ornithine,Tris, leucine aminopeptidaseandbovineserumalbuminwerefromtheSigmaChemicalCo.,St.Louis, MO, U.S.A.OTCase waspurified fromE.coliWasdescribedby Templetonetal.(1984).[35S]PhaseolotoxinwaspreparedfromaliquidcultureofPs. syringae pv. phaseolicola(strainPlantDiseasesDivisionCultureCollection4419)supplied with35SO42-(AmershamInter-national,Amersham, Bucks.,U.K.),as describedbyMitchell & Bieleski(1977).[35S]PSorn(92.464Ci/pmol)wasprepared bytreatmentof[35S]-phaseolotoxinwith leucineaminopeptidaseasdescribedbyMitchell&Bieleski(1977)for thepreparationofnon-radioactively-labelledPSorn.ThepurifiedPSornwasradiochemicallypureon atwo-dimensionalt.l.e/t.l.c.system(Mitchell, 1976).SephadexG-50wasobtainedfromPharmacia,Uppsala,Sweden.Enzymeandprotein assayOTCasewasassayedasdescribedbyTempletonetal.(1984)andhadaspecificactivityof 3000units/mgofprotein.ProteinwasestimatedbyassumingAIjo=9.56(Templetonetal.,1984)orbythe methodofPeterson(1977)withcrystallinebovineserumalbuminas astandard.Inallexperi-Gel-permeationchromatography ofinactivatedOTCaseOTCase,inactivated with[35S]PSornasde-scribedabove,waschromatographedon aSepha-dex G-50column(1cmx14cm;V0=6ml)equili-bratedin0.1%sodiumdodecyl sulphate.Fractions(1ml)werecollecteddirectlyinto scintillationvials,and measured forradioactivityinBray's(1960)scintillationfluid.Inseparateexperiments,inactivatedenzyme(501u)wastreatedseparatelywith either0.1%sodium dodecyl sulphate,5M-ureaor 5M-guanidin-ium chloride(final concentrations)at37°Cfor30min. Each enzymepreparation(100pl)was thenchromatographedonSephadexG-50asdescribedabove.InactivationofOTCasebyPSornOTCase(O0nM)andPSorn(I0nM)wereincubat-edat0°Cin1mlof50mM-Tris/HCI,pH9.0. Thetemperaturewasmaintainedat0°Cwithanice/waterbath.Portions(10,ul)wereremovedattheappropriatetimesandassayedforOTCaseactivity.Theconcentrationofactiveenzyme(e)attimetwascalculated fromthefollowingformula:e=eo(vlvo)whereeois theinitial concentration ofenzymeandv0andv aretheenzyme velocitiesinitiallyandattimetrespectively.Experimentswereperformedin the presence of substrateinasimilar fashion.1985Inactivation ofornithinetranscarbamoylase349with[35S]PSornandchromatographedona col-umnofSephadexG-50equilibratedin50mM-Tris/HCl,pH8.0, containing 0.1%sodiumdodecylsulphate,theproteinpeakwas35S-labelledandmostof theradioactivitywaselutedinthe voidvolume(Fig.2b).Similar results wereobtainedwheninactivated enzymewastreated with5M-urea or 5M-guanidinium chloride beforegel-permeationchromatography.Kineticsofinactivation ofOTCase by PSornCarbamoylphosphateorL-orMithinewasmixedwith enzymeinavolumeof990landthereactionwas started by the additionof10ulofPSorn.ResultsanddiscussionAnexperimentwasdesigned todeterminewhether35Sremained bound to OTCase inactiva-ted with[35S]PSomafterprecipitationwitheth-anol. The proportionofenzyme activityremaining(e/eo)wasplotted againsttheinitialmolar ratio of[35S]PSornto enzyme([[35S]PSorn]/e0)(Fig.1).Theslopeofthislineindicatedthat 0.95mol ofPSornwasrequiredtoinactivate1 molofactivesites.Aplotof nmol of35Sincorporatedagainsttheinitialmolarratioof[35S]PSornto enzyme waslinearand thestoicheiometryof35Sincorporationwas0.85mol permol of activesites.These datashow that approx.1molof[35S]PSornwasboundpermol ofinactivatedenzyme.Co-migrationof35Sandproteinduringgel-filtrationchromatographyprovided furtherevi-dence thattherewasacovalent linkbetween[35S]PSornandOTCase. OTCaseand[35S]PSorn(run separately)partitioneddistinctly,with elutionvolumes of6.5mland13 mlrespectively(Fig.2a).Incontrast, when enzyme(5.2.uM)wasinactivatedUnderpseudo-first-orderconditions(i>e),en-zymeinactivationoccurredtoorapidlytobemeasured accurately. The inactivation ofOTCasewas thereforemeasured undersecond-ordercondi-tions(Fig.3).Therateconstantof106M-1-S-1ishigh,but lowerthan thatexpectedforasimplediffusionprocess(109-111 M-1*s-)(Fersht,1977). Second-order kinetics indicated thattheconcentrationof aMichaelis-type bindingcom-plexwasnegligible under these conditions.Similarconclusionshave madeby Knott-Hunzikeretal.(1980)andArisawa & Then(1983)forthe0.20.6I.Za9._A0.500.75[[5SIPSornI/eoFig.1.Stoicheiometry ofinactivation ofOTCaseby[35S]PSornEnzyme(5.2pM)andvariousamounts(0-6.25pM)of[35S]PSornwereincubatedat0°Cin50mM-Tris/HCl,pH9.0.Thereactionvolumewas50pl.After30min5*lsampleswereremovedandassayedforOTCaseactivity.The remainderwasusedforthemeasurementofethanol-precipitableradio-activityasdescribedintheExperimentalsection.The resultswereplottedas(0)e/eOagainst[PSorn]/eo,whereeoandearetheenzymeactivityinitiallyandafter30minrespectively,and(-)nmolof35Sincorporatedagainst[35S]PSorn/eo.Fractionno.Fig.2.Gel-permeationchromatographyofOTCaseafterinactivationwith[35S]PSornOTCasewasinactivatedwith[35]PSornasde-scribedin theExperimentalsection. Chromato-graphywasperformedonSephadexG-50equili-brated in50mM-Tris/HCI,pH8.0,containing0.1%sodiumdodecylsulphate.Fractions(Iml)werecollecteddirectlyinto scintillation vialsandtheradioactivitywasmeasured.(a)*,OTCase(A280);0,[35S]PSorn,runseparately.(b)0,Inactivatedenzymesamples.Vol.228350M. D.Templeton,R. E.Mitchell,P. A.SullivanandM.G. Shepherd0.1501001502CTime(s)Fig.3. InactivationofOTCasebyPSornInactivationofOTCasebyPSornwasperformedasdescribedintheExperimentalsection. Theresultswereplottedasl/e-lieoagainsttime, whereeoande aretheconcentrationsofactiveenzymeinitiallyandattimetrespectively.1234Time(min)inactivationofP-lactamaseby6-p-bromopenicil-lanicacidand6-acetylmethylenepenicillanicacidrespectively.ProtectionofinactivationbysubstrateThestructuralhomologybetweenPSornand thesubstratesofOTCasesuggested that it wasactingatthe activesite.Moreconclusive evidencecan beobtained if therateofinactivationis decreasedinthepresenceofsubstrate.OTCasehasacompul-sory-ordermechanismwithcarbamoylphosphatethe firstsubstratetobind(Legrain&Stalon, 1976).Ratesofinactivationweremeasured in thepresence ofvarious concentrationsofcarbamoylphosphate(Fig.4).Lowconcentrationsof carb-amoylphosphatesignificantlydecreasedthe rateofinactivation.Thedissocationconstantfor the enzyme-carbamoyl phosphatecomplexcanbecalculatedfrom thesedataby usingthe equations derivedfromthefollowingexpressions:Ki,kEAE+AE+I-+E-1whereArepresentscarbamoyl phosphate,Irepre-sentsPSorn,E-Iis theinactivatedcomplexandEis freeenzyme.Thederivation isessentiallythatdescribedbyDixon& Webb(1979),modifiedslightlybecauseofthe order ofthereaction.Fromthepreviousexpressions:e=ef+x+zx=efaIKiwhereeand efaretheconcentrationsof totalandFig.4.InactivationofOTCasebyPSornin thepresenceofcarbamoylphosphateInactivationofOTCasebyPSorninthe presence ofcarbamoylphosphatewasperformedasdescribedintheExperimentalsection. The concentrationsofcarbamoylphosphatewere:0,OM;A,2.5uM;*,5.0gM;0,10gM;E,15gM.765.4-k1023251015[Carbamoylphosphate](#M)Fig.5.Calculation ofthedissociationconstantfortheOTCase-carbamoylphosphatecomplexThereciprocalsofthe apparentrateconstantsobtainedfrom thedataofFig.4wereplottedagainsttheconcentrationof carbamoyl phosphate.Avalueof3gmwasobtained forthe dissociationconstantfor theOTCase-carbamoylphosphatecomplex.1985Inactivationofornithinetranscarbamoylase351NH3+HNH,freeenzymeandxandzaretheconcentrationsofEA andElrespectively. Solvingfor ef:ef=(e-z)Kia/(Kia+a)AssumingPSornbindsonlytofreeenzyme:-def/dt=kef(i-z)CH-[CH2-3-N-P-NH-S03H2KwBEnzymeHwherei isthe concentrationof PSorn. In thepresenceofcarbamoyl phosphate,substitutiongivestheexpression:-def/dt=[kKia!(Kia+a)](e-z)(i-z)and thus:-def/dt=kapp.(e-z)(i-z)Thisis the form ofasecond-orderreaction,andthusatequimolarconcentrations of PSorn andenzymeit willintegrateto:l/e-l/eo=kapptInreciprocalforml/kapp.isdefinedas:1/kapp.=1/k+a/KiakThereforeaplotof 1 /kapp.against[carbamoylphos-phate]willhaveaninterceptof 1/konthel/kapp.axis,aslopeof1/Kiakandaninterceptof-Kiaonthe substrate concentration axis.The results fromFig.4werereplottedinthismanner(Fig. 5).Theplotwaslinear andadis-sociationconstantforcarbamoylphosphateof3/IMwasobtained. Thisvalue is lower than that of15gLMobtainedfromasteady-statekineticanalysisatpH 8.0byLegrain& Stalon(1976),but thismaybe duetothedifferent pH.Therateof OTCaseinactivationwasmeasuredin thepresenceof 3mM- andIOmM-L-ornithine.Therateconstantkwasnotaffectedby3mM-L-ornithine;however,10 mM-L-ornithinedecreasedtherateof inactivationby37%,suggestingthatL-ornithinewasbindingtofreeenzymeathighcon-centrations.Legrain& Stalon(1976)reportedthatthespeciesofornithine withzeronetcharge(PKa=8.88)wasabletobindtofreeenzymeinsuchawayastoprecludethebindingofcarbamoylphosphate.AtpH9.0therearesignificantconcen-trationsof thisspecies,and thismayexplainthelimitedprotectionofOTCaseinactivationbyornithine.Apossiblemechanismfor theinactivationofOTCasebyPSornisoutlinedinScheme1.Fromtheproposedmechanismwepredictthatfreeornithinewill be released intothemediumandanamino acid residueonthe enzyme will bephos-phorylatedwiththephosphinylsubstituentofPSorn. The observation thatphaseolotoxinisunabletoinactivate OTCase(Templetonetal.,1984)suggeststhatafreecarboxygroupontheL-ornithineresidue isrequiredforcompletebindingNH3+NH2CH-[CH2]3-NH3++0=P-NH-SO3-CO-BEnzymeScheme1.ProposedmechanismofinactivationofOTCasebyPSornNucleophilicattackof PSornbyabasic residue(B)of theenzyme(OTCase)occursasshown,with thereleaseofornithineandirreversibleinactivationofOTCase.toand henceinactivation ofOTCase. Alter-natively,theadditionalbulk ofthetwoextraamino acidsmaybeaffectingthebindingof thetoxininsomeway.Mitchell&Bieleski(1977)found thatPSornwasthemajortoxicproductfoundininfectedbeanleaves,implying thatphaseolotoxinwascleavedtoPSornbytheplantinvivo.Thesignificance of thisobservationisnowapparent.Theconversionofphaseolotoxin,areversibleinhibitor,intoPSorn,apotentinactivatorofOTCase,isanimportantpartofthediseaseprocess.The conversionofabacterialphytotoxinintoa morepotentformhasbeenreportedinthecaseoftabtoxin,whichiscleavedtotabtoxinine-fJ-lactam,apotent inactiva-torofglutaminesynthetase(Uchytil&Durbin,1980; Thomasetal.,1983).Ferguson& Johnson(1980)andStaskawiczetal.(1980)reportedthatpre-incubationof purifiedphaseolotoxinwithcrudeextractsof OTCasesignificantlyincreasedenzymeinhibition.Itispossiblethatduringthepre-incubationperiodpep-tidasespresentintheOTCasepreparation degrad-edphaseolotoxintoPSorn.Inaddition,it wouldappearthat the toxinpreparationusedbyKwoketal.(1979)containedsignificantquantitiesofPSorn,and this wouldexplainthe inactivationkineticstheyobservedfor OTCase.Theirconclu-sion that the toxin formed'alooseMichaelis-type'complexbeforeinactivationwas notborneoutbythe presentstudy.Fromthese data it is clear that PSorn isapotentinactivatorofOTCasefromE.coliW,and indeedto ourknowledgeisoneof themostpotentnaturallyoccurringenzymeinactivatorsreported.Itappearslikelythat OTCase isanimportant,ifVol. 228
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