[Characterization of S1' subsite specificity of Thermoactinomyces vulgaris carboxypeptidase T by site-directed mutagenesis].
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 gel efiltration 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.
Trachuk
L A
LA
Institute of Genetics and Selection of Industrial Microarganisms, Maskow, Russia.
Bushueva
A M
AM
Shevelev
A B
AB
Novgorodova
S A
SA
Akparov
V Kh
VKh
Chestukhina
G G
GG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins; D002268:Carboxypeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008848:Micromonosporaceae / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002268:Carboxypeptidases; C076614:CPT protein, Thermoactinomyces vulgaris
Izuchenie S1'-karmana pervichnoĭ spetsifichnosti karboksipeptidazy T Thermoactinomyces vulgaris metodom napravlennogo mutageneza.