Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase 英文参考文献.docVIP

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Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase 英文参考文献.doc

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Pyridoxal 5′-Phosphate Is a Slow Tight Binding Inhibitor of E. coli Pyridoxal Kinase 英文参考文献

Pyridoxal59-PhosphateIsaSlowTightBindingInhibitor ofE.coliPyridoxalKinase MohiniS.Ghatge1.,RobertoContestabile2.,MartinoL.diSalvo2,JigarV.Desai1,AmitK.Gandhi1, ChristinaM.Camara1,RitaFlorio2,IsabelN.Gonza′lez3,4,AlessiaParroni2,VerneSchirch1,MartinK.Safo1* 1Department of Medicinal Chemistry, Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia, United States of America,2IstitutoPasteur-FondazioneCenciBolognettiandDipartimentodiScienzeBiochimiche,SapienzaUniversita` diRoma,Roma,Italy,3ConsiglioNazionaledelle Ricerche,IstitutodiBiologiaAgroambientaleeForestale,MonterotondoScalo,Roma,Italy,4InstituteofBiocomputationandPhysicsofComplexSystems,Universidadde Zaragoza,Zaragoza,Spain Abstract Pyridoxal59-phosphate(PLP)isacofactorfordozensofB6requiringenzymes.PLPreactswithapo-B6enzymesbyforming an aldimine linkage withthe e-amino groupof an active site lysine residue,thus yieldingthe catalytically active holo-B6 enzyme.Duringproteinturnover,thePLPissalvagedbyfirstconvertingittopyridoxalbyaphosphataseandthenbackto PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 49-aldehyde moiety forms covalent adducts with other compounds and non-B6 proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complexwiththeproductPLPtoformaninactiveenzymecomplex.Evidenceispresentedthat,intheinhibitedcomplex, PLPhasformedanaldiminebondwithanactivesitelysineresidueduringcatalyticturnover.TherateofdissociationofPLP fromthecomplexisveryslow,beingonlypartiallyreleasedaftera2-hourincubationwithPLPphosphatase.Interestingly, theinactivepyridoxalkinaseNPLPcomplexcanbepartiallyreactivatedbytransferringthetightlyboundPLPtoanapo-B6 enzyme.Theseresultsopennewperspec