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 mas 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. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B,V, All rights reserved.