Bromopyruvate inactivation of glutamate apodecarboxylase. Kinetics and specificity.
A number of halo carboxylic and dicarboxylic acids were substrate-competitive inhibitors of glutamate decarboxylase, with bromosuccinate, 3-bromopropionate, and iodoacetate having the highest affinity for the enzyme. Some of the halo acids also inactivated the apoenzyme. Bromopyruvate at relatively low concentrations inactivated the apoenzyme irreversibly. The rate of the inactivation of the apodecarboxylase was proportional to bromopyruvate at low concentration and approached a constant rate of inactivation at high bromopyruvate concentration. These data are consistent with a two-step inactivation process in which an enzyme-bromopyruvate complex is formed followed by inactivation. The concentration of bromopyruvate giving the half-maximum rate of inactivation was 6.9 mM, and the maximum rate of inactivation was 1.75 min-1 at pH 4.6 and 23 degrees. Much faster rates of inactivation were obtained at pH 5.96 and 6.44. Phosphate, an inhibitor of pyrisoxal-P binding to the apoenzyme, competitively inhibited the inactivation of the apoenzyme by bromopyruvate. In addition, bromopyruvate inhibited the rate of pyridoxal-P binding to the apoenzyme. Kinetics of the incorporation of bromo[2-14C]pyruvate indicated that complete inactivation was obtained when 1.2 mol of radioactive residue were covalently bound per subunit of apoenzyme. Amino acid analyses demonstrated that a cysteinyl residue was alkylated by the bromopyruvate. The bromopyruvate was evidently interacting nincovalently with a cationic group at or near the pyridoxal-P-binding site, and then was alkylating a nearby cysteinyl residue.
D001051:Apoenzymes / Q000037:antagonists & inhibitors*; D001059:Apoproteins / Q000037:antagonists & inhibitors*; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors*; D005968:Glutamate Decarboxylase / Q000037:antagonists & inhibitors*; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D008433:Mathematics; D011422:Propionates / Q000494:pharmacology; D011773:Pyruvates / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013386:Succinates / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D001051:Apoenzymes; D001059:Apoproteins; D007461:Iodoacetates; D011422:Propionates; D011773:Pyruvates; D013386:Succinates; D013439:Sulfhydryl Reagents; D002262:Carboxy-Lyases; D005968:Glutamate Decarboxylase