The roles of Glu93 and Tyr149 in astacin-like zinc peptidases.

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.