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1、Biochimie93(2011)1424e1433ContentslistsavailableatScienceDirectBiochimiejournalhomepage:www.elsevier.com/locate/biochiResearchpaperRoleofhydrationonthefunctionalityofaproteolyticenzymea-chymotrypsinundercrowdedenvironmentPramodKumarVerma,SurajitRakshit,RajibKumarMitra**
2、,SamirKumarPal*UnitforNanoScience&Technology,DepartmentofChemical,Biological&MacromolecularSciences,S.N.BoseNationalCentreforBasicSciences,BlockJD,SectorIII,SaltLake,Kolkata700098,IndiaarticleinfoabstractArticlehistory:Enzymesandotherbio-macromoleculesarenotonlysensitiv
3、etophysicalparameterssuchaspH,Received21October2010temperatureandsolutecompositionbutalsotowateractivity.AuniversallyinstructivewaytovaryAccepted20April2011wateractivityistheadditionofosmoticallyactivebutotherwiseinertsolventswhichalsomimictheAvailableonline29April2011c
4、onditionofanintercellularmilieu.Inthepresentcontribution,theroleofhydrationonthefunctionalityofaproteolyticenzymea-chymotrypsin(CHT)isinvestigatedbymodulatingthewateractivitywiththeKeywords:additionofpolyethyleneglycols(PEGwithanaveragemolecularweightof400).Theadditiono
5、fPEGEnzymecatalysisincreasestheaffinityoftheenzymetoitssubstrate,however,followedbyadecreaseintheturnoverWateractivitynumber(kcat).Energeticcalculationsshowthatentrancepathforthesubstrateisfavoured,whereastheApparentspecificvolumeAdiabaticcompressibilityexitchannelisrestr
6、ictedwithincreasingconcentrationofthecrowdingagent.ThisdecreaseisattributedTime-resolvedfluorescencespectroscopytothethinningofthehydrationshelloftheenzymeduetothelossofcriticalwaterresiduesfromtheWaterrelaxationdynamicshydrationsurfaceoftheenzymeasevidencedfromvolumetri
7、candcompressibilitymeasurements.TheoverallsecondaryandtertiarystructuresofCHTdeterminedfromfar-UVandnear-UVcirculardichroism(CD)measurementsshownoconsiderablechangeinthestudiedosmoticstressrange.Fromkineticandequilibriumdata,wecalculate11530numbersofwatermoleculestobea
8、lteredduringtheenzymaticcatalysisofCHT.SpectroscopicobservationofwaterrelaxationandrotationaldynamicsofANSeCHT
