| Abstract: | Phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli W was alkylated by incubation with bromopyruvate, substrate analog, leading to irreversible inactivation. The reaction followed pseudo-first-order kinetics. Mg2+, an essential cofactor for catalysis, enhanced the inactivation, and the enhancing effect increased as the pH increased. The inactivation rate showed a tendency to saturate with increasing concentrations of bromopyruvate, indicating that an enzyme-bromopyruvate complex was formed prior to the alkylation. DL-Phospholactate, a potent competitive inhibitor with respect to phosphoenolpyruvate, protected the enzyme from inactivation in a competitive manner. Examination of the acid hydrolysate of the enzyme modified with [14C]bromopyruvate by paper chromatography showed that radioactivity was solely incorporated into carboxyhydroxyethyl cysteine. In addition, determination of sulfhydryl groups of the native and modified enzymes with 5,5'-dithiobis(2-nitrobenzoate) showed that inactivation occurred concomitant with the modification of one cysteinyl residue per subunit. The results indicate that bromopyruvate reacted with the enzyme as an active-site-directed reagent. |
