ALTERATION OF THE SPECIFICITY OF ECOTIN, AN ESCHERICHIA-COLI SERINE PROTEINASE-INHIBITOR, BY SITE-DIRECTED MUTAGENESIS

The gene of ecotin, an E. call proteinase inhibitor, was cloned, and by site-directed mutagenesis the active site residue of the protein, Met(84), was mutated to Lys, Arg and Leu. The recombinant wild-type and mutant inhibitors were overexpressed in E. coli, purified to homogeneity and their inhibitory effects on trypsin, chymotrypsin and elastase were compared. Of these serine proteinases trypsin is the most strongly inhibited by wild type ecotin and its mutants. According to our results the character of residue 84 of ecotin significantly but not dramatically modifies the specificity of the inhibitor.