Chemical modification of a functional arginyl residue (Arg 292) of mitochondrial aspartate aminotransferase. Identification as the binding site for the distal carboxylate group of the substrate.
Mitochondrial aspartate aminotransferase is inactivated by dicarbonyl reagents selectively modifying arginyl residues. Treatment with phenylglyoxal inactivates the enzyme with concomitant modification of 2.7 mol of arginyl residues/mol of subunit. If the reaction is performed in the presence of the transaminating substrate pair aspartate/oxalacetate, only 1.3 mol of arginyl residues/mol of subunit are labeled and the enzymic activity remains at 75% of the original value. One particular residue, identified by peptide analysis as Arg 292, is completely protected against modification in the presence of the substrate pair, indicating a role of its guanidinium group in substrate binding. On the basis of x-ray crystallographic studies of the complex of apoenzyme with N-(5'-phosphopyridoxyl)-aspartate (minus pyridoxal form of the enzyme), Arg 292 has been proposed as the binding site of the distal carboxylate group (Ford, G. C., Eichele, G., and Jansonius, J. N. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 2559-2563). The enzyme with blocked Arg 292 is not completely inactive, and its molecular activity toward dicarboxylic substrates is of the same order of magnitude as that of the native enzyme toward alanine, which is 10(5) times lower than that toward dicarboxylic substrates. The activity toward alanine is unchanged but the rate-enhancing effect of formate on the transamination of alanine is impaired. Formate is assumed to occupy the binding site of the distal carboxylate group (Morino, Y., Osman, A. M., and Okamoto, M. (1974) J. Biol. Chem. 249, 6684-6692). Apparently, the interaction of the distal carboxylate group of the substrate with Arg 292 underlies not only the binding specificity but also the kinetic specificity of aspartate aminotransferase for dicarboxylic substrates.
D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D001120:Arginine; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001665:Binding Sites; D002645:Chickens; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D010658:Phenylglyoxal / Q000494:pharmacology*; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D000447:Aldehydes; D001120:Arginine; D001219:Aspartate Aminotransferases; D010658:Phenylglyoxal