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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Trachuk L.A., Bushueva A.M., Shevelev A.B., Novgorodova S.A., Akparov V.H., Chestukhina G.G. (2002) Characterization of the S1' subsite specificity of carboxypeptidase T Thermoactinomyces vulgaris by site-directed mutagenesis. Voprosy Meditsinskoi Khimii, 48(6), 577-585.
Trachuk L.A. et al. Characterization of the S1' subsite specificity of carboxypeptidase T Thermoactinomyces vulgaris by site-directed mutagenesis // Voprosy Meditsinskoi Khimii. - 2002. - V. 48. -N 6. - P. 577-585.
Trachuk L.A. et al., "Characterization of the S1' subsite specificity of carboxypeptidase T Thermoactinomyces vulgaris by site-directed mutagenesis." Voprosy Meditsinskoi Khimii 48.6 (2002): 577-585.
Trachuk, L. A., Bushueva, A. M., Shevelev, A. B., Novgorodova, S. A., Akparov, V. H., Chestukhina, G. G. (2002). Characterization of the S1' subsite specificity of carboxypeptidase T Thermoactinomyces vulgaris by site-directed mutagenesis. Voprosy Meditsinskoi Khimii, 48(6), 577-585.
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