Interaction of the D-isomer of gamma-methylene glutamate with an active site thiol of gamma-glutamylcysteine synthetase.

Abstract

gamma-Glutamylcysteine synthetase has a thiol group in the vicinity of its glutamate-binding site. During efforts to find a covalently bound inhibitor, interaction of the enzyme with gamma-methylene glutamate was examined because this analog of glutamate, which has an alpha,beta-unsaturated moiety, would be expected to bind at the glutamate site and might react with an active site thiol. gamma-Methylene glutamate, which is not a significant substrate, inhibits the enzyme competitively toward glutamate. Preincubation of the enzyme with gamma-methylene DL-glutamate led to substantial inactivation which was dependent upon the presence of Mg2+ or Mn2+; glutamate protected against inactivation. Inactivation was observed with the D-isomer of gamma-methylene glutamate, but not with the corresponding L-isomer. The inactivated enzyme contains close to 1 mol of gamma-methylene glutamate/mol of enzyme. Studies in which enzyme inactivated by treatment with [14C]gamma-methylene glutamate was hydrolyzed indicate that gamma-methylene glutamate reacts with an active site thiol.