M. Miyazaki et al., CYSTEINE188 REVEALED AS BEING CRITICAL FOR THE ENZYME-ACTIVITY OF ARYLMALONATE DECARBOXYLASE BY SITE-DIRECTED MUTAGENESIS, Bulletin of the Chemical Society of Japan, 70(11), 1997, pp. 2765-2769
Arylmalonate decarboxylase (AMDase) catalyzes the asymmetric decarboxy
lation of alpha-aryl-alpha-methylmalonic acid. Since this enzyme is in
hibited by SH-reagents, a cysteine residue is supposed to be involved
at the catalytic site. Cloning of the gene which codes the enzyme reve
aled that this enzyme contains four cysteine residues. Titration of fr
ee SH groups by p-(chloromercurio)benzoate disclosed that all four Cys
are in the reduced form. In this study, four mutant enzymes (C1O1S, C
148S, C171S, and C188S) were prepared, in which one of four cysteines
was replaced by serine. The CD spectra indicated that the conformation
al differences of C1O1S and C188S compared to that of the native enzym
e are not so significant. The catalytic activities of the four mutants
were measured. Among these mutant enzymes, only C188S showed a drasti
c decrease in enzyme activity, indicating that cysteine(188) is locate
d at the active center of the enzyme. The catalytic activities of the
other mutants are also discussed.