Phosphoenolpyruvate carboxylase of Escherichia coli. Affinity labeling with bromopyruvate.
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.
D000345:Affinity Labels / Q000494:pharmacology*; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000378:metabolism*; D003545:Cysteine / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism*; D011485:Protein Binding; D011773:Pyruvates / Q000494:pharmacology*
D000345:Affinity Labels; D011773:Pyruvates; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase; D003545:Cysteine