Affinity Labeling of Aspartate Aminotransferase Isozymes by Bromopyruvate

Incubation with bromopyruvate did not cause appreciable inactivation of either supernatant or mitochondrial isozyme of aspartate aminotransferase from pig heart. In the presence of either l-cysteine sulfinate or l-aspartate, however, bromopyruvate rapidly inactivated both isozymes. Bromopyruvate also acted as a keto acid substrate in the conversion of the pyridoxamine form of both enzymes to their pyridoxal forms. This finding, together with the demonstration of the equimolar formation of oxalacetate, pyruvate, and ammonia from l-aspartate and bromopyruvate, supported the following reaction sequences: [see PDF for equation] [see PDF for equation] where Em is the pyridoxamine form and El the pyridoxal form of these enzymes. A kinetic study of these reactions revealed that inactivation occurs during Reaction I. Spectral and chemical analyses showed that the coenzyme in both inactivated enzymes is present as pyridoxamine phosphate and the carbon 3 moiety of bromopyruvate is covalently bound to the enzyme active sites. This finding indicates that bromopyruvate is an efficient affinity label for the pyridoxamine form of each isozyme. Kinetics of the incorporation of bromo[2-14C]pyruvate and chemical analyses of modified amino acid residues demonstrated that inactivation of each isozyme resulted from alkylation of an essential cysteinyl residue in the active region by bromopyruvate.