FEBS Letters | |
The roles of Glu93 and Tyr149 in astacin‐like zinc peptidases | |
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
【 预 览 】
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