Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/62047
DC FieldValueLanguage
dc.contributor.authorLee, L.C.en_US
dc.contributor.author蕭介夫zh_TW
dc.contributor.authorLee, Y.L.en_US
dc.contributor.authorLeu, R.J.en_US
dc.contributor.authorShaw, J.F.en_US
dc.date2006zh_TW
dc.date.accessioned2014-06-09T06:26:19Z-
dc.date.available2014-06-09T06:26:19Z-
dc.identifier.issn0264-6021zh_TW
dc.identifier.urihttp://hdl.handle.net/11455/62047-
dc.description.abstractEscherichia coli TAP (thioesterase 1, EC 3.1.2.2) is a multifunctional enzyme with thioesterase, esterase, arylesterase, protease and lysophospholipase activities. Previous crystal structural analyses identified its essential amino acid residues as those that form a catalytic triad (Ser(10)-Asp(154)-His(157)) and those involved in forming an oxyanion hole (Ser(10)-Gly(44)-Asn(73)). To gain an insight into the biochemical roles of each residue, site-directed mutagenesis was employed to mutate these residues to alanine, and enzyme kinetic studies were conducted using esterase, thioesterase and amino- acid-derived substrates. Of the residues, His(157) is the most important, as it plays a vital role in the catalytic triad, and may also play a role in stabilizing oxyanion conformation. Ser(10) also plays a very important role, although the small residual activity of the S10A variant suggests that a water molecule may act as a poor substitute. The water molecule could poss-ibly be endowed with the nucleophilic-attacking character by His(157) hydrogen-bonding. Asp(154) is not as essential compared with the other two residues in the triad. It is close to the entrance of the substrate tunnel, therefore it predominantly affects substrate accessibility. Gly(44) plays a role in stabilizing the oxyanion intermediate and additionally in acyl-enzyme-intermediate transformation. N73A had the highest residual enzyme activity among all the mutants, which indicates that Asn(73) is not as essential as the other mutated residues. The role of Asn(73) is proposed to be involved in a loop(75-80) switch-move motion, which is essential for the accommodation of substrates with longer acyl-chain lengths.en_US
dc.language.isoen_USzh_TW
dc.relationBiochemical Journalen_US
dc.relation.ispartofseriesBiochemical Journal, Volume 397, Page(s) 69-76.en_US
dc.relation.urihttp://dx.doi.org/10.1042/bj20051645en_US
dc.subjectcatalytic triaden_US
dc.subjectenzyme kineticsen_US
dc.subjectesteraseen_US
dc.subjectoxyanion holeen_US
dc.subjectsite-directeden_US
dc.subjectmutagenesisen_US
dc.subjectthioesterase I (TAP)en_US
dc.subjectsubstrate-specificityen_US
dc.subjectmolecular-cloningen_US
dc.subjectlipolytic enzymesen_US
dc.subjectcrystal-structureen_US
dc.subjectvibrio-mimicusen_US
dc.subjectarylesteraseen_US
dc.subjectfamilyen_US
dc.subjectgeneen_US
dc.subjectlysophospholipaseen_US
dc.subjecthydrolasesen_US
dc.titleFunctional role of catalytic triad and oxyanion hole-forming residues on enzyme activity of Escherichia coli thioesterase I/protease I/phospholipase L-1en_US
dc.typeJournal Articlezh_TW
dc.identifier.doi10.1042/bj20051645zh_TW
item.openairecristypehttp://purl.org/coar/resource_type/c_18cf-
item.openairetypeJournal Article-
item.cerifentitytypePublications-
item.fulltextno fulltext-
item.languageiso639-1en_US-
item.grantfulltextnone-
Appears in Collections:食品暨應用生物科技學系
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