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The roles of Glu93 and Tyr149 in astacin‐like zinc peptidases
Author(s) -
Yiallouros Irene,
Große Berkhoff Eva,
Stöcker Walter
Publication year - 2000
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(00)02163-3
Subject(s) - mutant , chemistry , biochemistry , tyrosine , escherichia coli , zinc , hydrolysis , substrate (aquarium) , microbiology and biotechnology , biology , gene , organic chemistry , ecology
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.