CYSTEINE188 REVEALED AS BEING CRITICAL FOR THE ENZYME-ACTIVITY OF ARYLMALONATE DECARBOXYLASE BY SITE-DIRECTED MUTAGENESIS

Citation
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
Citations number
13
ISSN journal
00092673
Volume
70
Issue
11
Year of publication
1997
Pages
2765 - 2769
Database
ISI
SICI code
0009-2673(1997)70:11<2765:CRABCF>2.0.ZU;2-L
Abstract
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.