Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation.

Abstract

Site-directed mutagenesis can be employed to alter activity critical residues in proteins which are susceptible to chemical oxidation. Previous studies have implicated methionine 222 as a primary site for oxidative inactivation of subtilisin (Stauffer, C. E., and Etson, D. (1969) J. Biol. Chem. 244, 5333-5338). Because of uncertainties in predicting which amino acid would be the optimal substitute for methionine 222, we prepared all 19 amino acid substitutions at this site in the cloned subtilisin gene using a cassette mutagenesis method (Wells, J. A., Vasser, M., and Powers, D. P. (1985) Gene (Amst.), in press). Mutant enzymes were expressed in Bacillus subtilis and were found to vary widely in specific activity. Mutants containing nonoxidizable amino acids (i.e. Ser, Ala, and Leu) were resistant to inactivation by 1 M H2O2, whereas methionine and cysteine enzymes were rapidly inactivated. These studies demonstrate the feasibility of improving oxidative stability in proteins by site-directed mutagenesis.