tailieunhanh - Báo cáo khoa học: Escherichia coli cyclopropane fatty acid synthase Mechanistic and site-directed mutagenetic studies

Escherichia colifatty acid cyclopropane synthase (CFAS) was overproduced and purified as a His6 -tagged protein. This recombinant enzyme is as active as the native enzyme with aKmof 90lMforS-AdoMet and a specific activity of 5·10 )2 lmolÆmin )1 Æmg )1 . The enzyme is devoid of organic or metal cofactors and is unable to catalyze the wash-out of themethyl protons ofS-AdoMet to the solvent, data that do not support the ylidemechanism. Inactivationof the enzyme by 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB), a pseudo first-order process with a rate constant of )1 Æs )1 , is not protected by substrates. Graphical analysis of the inactiva-tion byDTNB revealed that only one cysteine is responsible for the inactivation of the enzyme. . | Eur. J. Biochem. 271 4769-4778 2004 FEBS 2004 doi Escherichia coli cyclopropane fatty acid synthase Mechanistic and site-directed mutagenetic studies Fabienne Courtois Christine Guerard Xavier Thomas and Olivier Ploux Laboratoire de Chimie Organique Biologique UMR7613 CNRS Universite Pierre et Marie Curie Paris France Escherichia coli fatty acid cyclopropane synthase CFAS was overproduced and purified as a His6-tagged protein. This recombinant enzyme is as active as the native enzyme with a Km of 90 M for S-AdoMet and a specific activity of 5 X 10 2 pmol-min-1-mg_1. The enzyme is devoid of organic or metal cofactors and is unable to catalyze the wash-out of the methyl protons of S-AdoMet to the solvent data that do not support the ylide mechanism. Inactivation of the enzyme by 5 5 -dithiobis- 2-nitrobenzoic acid DTNB a pseudo first-order process with a rate constant of M-1-s-1 is not protected by substrates. Graphical analysis of the inactivation by DTNB revealed that only one cysteine is responsible for the inactivation of the enzyme. The three strictly conserved Cys residues among cyclopropane synthases C139 C176 and C354 of the E. coli enzyme were mutated to serine. The relative catalytic efficiency of the mutants were 16 for C139S 150 for C176S and 63 for C354S. The three mutants were inactivated by DTNB at a rate comparable to the rate of inactivation of the His6-tagged wildtype enzyme indicating that the Cys responsible for the loss of activity is not one of the conserved residues. Therefore none of the conserved Cys residues is essential for catalysis and cannot be involved in covalent catalysis or general base catalysis. The inactivation is probably the result of steric hindrance a phenomenon irrelevant to catalysis. It is very likely that E. coli CFAS operates via a carbocation mechanism but the base and nucleophile remain to be identified. Keywords cyclopropane fatty acid synthase hydrogen isotope exchange enzymatic .

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