The roles of Glu93 and Tyr149 in astacin‐like zinc peptidases

FEBS Letters

Stöcker, Walter¹  Yiallouros, Irene¹  Große Berkhoff, Eva¹ 

[1] Institute of Zoophysiology, University of Münster, Hindenburgplatz 55, D-48143 Münster, Germany

关键词: Astacin;    Astacin family;    Metzincin;    Catalytic mechanism;    Site-directed mutagenesis;    Dns;    5-(dimethylamino)-naphthalene-1-sulfonyl;    NTA;    nitrilotriacetic acid;    IPTG;    isopropyl β-D-thiogalactopyranoside;    GuHCl;    guanidine hydrochloride;    PAGE;    polyacrylamide gel electrophoresis;    Ψ;    indicates replacement of the peptide bond by the group given in parentheses;   

DOI  :  10.1016/S0014-5793(00)02163-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The catalytic zinc of astacin, a prototype of the astacin family and the metzincin superfamily of metalloproteinases is coordinated by three histidines, a glutamate bound water and a tyrosine. In order to assess the roles of active site key residues, two mutants, Glu93Ala-astacin and Tyr149Phe-astacin, were expressed in Escherichia coli, affinity-purified and renatured. While the Glu93Ala mutant was inactive, the Tyr149Phe mutant retained about 2.5% residual activity toward Dns-Pro-Lys-Arg*Ala-Pro-Trp-Val, based on the k _cat/K _m value for recombinant wild-type astacin. These results support a model in which Glu93 is the general base in substrate hydrolysis, whereas Tyr149 contributes to transition state binding.

【 授权许可】

Unknown