Cysteine188 revealed as being critical for the enzyme activity of arylmalonate decarboxylase by site-directed mutagenesis

Arylmalonate decarboxylase (AMDase) catalyzes the asymmetric decarboxylation of alpha-aryl-alpha-methylmalonic acid. Since this enzyme is inhibited by SH-reagents, a cysteine residue is supposed to be involved at the catalytic site. Cloning of the gene which codes the enzyme revealed that this enzyme contains four cysteine residues. Titration of free SH groups by p-(chloromercurio)benzoate disclosed that all four Cys are in the reduced form. In this study, four mutant enzymes (C1O1S, C148S, C171S, and C188S) were prepared, in which one of four cysteines was replaced by serine. The CD spectra indicated that the conformational differences of C1O1S and C188S compared to that of the native enzyme are not so significant. The catalytic activities of the four mutants were measured. Among these mutant enzymes, only C188S showed a drastic decrease in enzyme activity, indicating that cysteine(188) is located at the active center of the enzyme. The catalytic activities of the other mutants are also discussed.