Inactivation of Escherichia coli 2-amino-3-ketobutyrate CoA ligase by phenylglyoxal and identification of an active-site arginine peptide.
Treatment of homogeneous preparations of 2-amino-3-ketobutyrate CoA ligase from Escherichia coli, a pyridoxal 5'-phosphate-dependent enzyme, with phenylglyoxal, 4-(oxyacetyl)phenoxyacetic acid, 2,3-butanedione, or 1,2-cyclohexanedione results in a time- and concentration-dependent loss of enzymatic activity. Phenylglyoxal in 50 mM phosphate buffer (pH 7.0) is the most effective modifier, causing > 95% inactivation within 20 min at 25 degrees C. Controls establish that this inactivation is not due to modifier-induced dissociation or photoinduced nonspecific alteration of the ligase. The substrate, acetyl CoA, or the coenzyme, pyridoxal 5'-phosphate, gives > 50% protection against inactivation. Enzyme partially inactivated by phenylglyoxal has the same Km value for glycine but the Vmax decreases in proportion to the observed level of inactivation. Whereas the native apoligase shows good recovery of activity with time in parallel with an increase in 428-nm absorptivity when incubated with pyridoxal 5'-phosphate, no such effects are seen with the phenylglyoxal-modified apoligase. Reaction of the enzyme with [14C]phenylglyoxal allowed for the isolation of a peptide which, by amino acid composition and sequencing data, was found to correspond to residues 349-378 in the intact enzyme. These results indicate that arginine residue-366 and/or residue-368 in the primary structure of E. coli 2-amino-3-ketobutyrate ligase is at the active site.
Mukherjee
J J
JJ
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
Dekker
E E
EE
D000123:Acetyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D001051:Apoenzymes / Q000378:metabolism; D001120:Arginine; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D010449:Peptide Mapping; D010658:Phenylglyoxal / Q000494:pharmacology*; D011732:Pyridoxal Phosphate / Q000494:pharmacology
D000596:Amino Acids; D001051:Apoenzymes; D011732:Pyridoxal Phosphate; D001120:Arginine; D000123:Acetyltransferases; C020679:glycine acetyltransferase; D010658:Phenylglyoxal