Voprosy meditsinskoi khimii (ISSN 0042-8809)

Characterization of the S1' subsite specificity of carboxypeptidase T Thermoactinomyces vulgaris by site-directed mutagenesis

   


1. Institute of Genetics and Selection of Industrial Microarganisms
PubMed Id: 12698557
Year: 2002 vol: 48  issue:6  pages: 577-585
Abstract: The site-directed mutagenesis in the S1'-pocket of Thermoactinomyces vulgaris carboxypeptidase T was performed and two variants containing double D253S,T255D and single T255D mutations were obtained. Precursors of the wild-type carboxypeptidase T and its mutant derivatives were expressed in Escherichia coli as inclusion bodies, refolded, activated by subtilisin, purified by gelfiltration on Superdex G-75. The catalytic activity with tripeptide substrates DNPAAR and ZAAL was analysed. The introduction of the aspartic residue in 255 position ( like to mammalian carboxypeptidase B), insigniticantly enzymatic activity of the double mutant towards both substrates, as measured by Km and kcat. An addition of the aspartic residue into S1'-binding pocket did not affect single mutant binding with the basic substrate while the Km value for the hydrophobic substrate increased approximately 40 times as compared with wild-type carboxypeptidase T and attained a level comparable with carboxypeptidase B
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Reference: Trachuk L.A., Bushueva A.M., Shevelev A.B., Novgorodova S.A., Akparov V.H., Chestukhina G.G., Characterization of the S1' subsite specificity of carboxypeptidase T Thermoactinomyces vulgaris by site-directed mutagenesis, Voprosy meditsinskoi khimii, 2002, vol: 48(6), 577-585.
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