A critical arginine residue in cytosolic aspartate aminotransferase from pig heart.

Abstract

Reaction of the pyridoxal form of cytosolic aspartate aminotransferase from pig heart with 1,2-cyclohexanedione or other alpha-dicarbonyls led to a progressive decrease in the enzymic activity toward natural dicarboxylic substrates. The inactivation was prevented by the presence of dicarboxylic substrate analogs. The dependence of the inactivation rate on the cyclohexanedione concentration indicated that the modifying reagent forms a dissociable complex with the enzyme prior to the inactivation. These saturation kinetics were observed also with other alpha-dicarbonyls tested. The inactivation was fully accounted for by the modification of a single arginine residue per monomeric unit of the enzyme. Activities for alpha, beta-elimination reaction with 3-chloro-L-alanine and transamination with L-alanine did not decrease but appeared to increase considerably with the progress of the arginine modification. In these aberrant reactions, affinity for the monocarboxylic substrates was higher with the modified enzyme than with the native unmodified enzyme. Glutamate or aspartate was still capable of reacting with the pyridoxal form of the extensively modified enzyme to produce the pyridoxamine form at a rate comparable to that of the reaction with 3-chloro-L-alanine or L-alanine. Succinate, glutarate, maleate, 2-methylaspartate or erythro-3-hydroxy-aspartate which bind strongly to the native enzyme and thus acts as potent inhibitors in the reactions with monocarboxylic substrates did not exhibit any appreciable inhibitory effect on these reactions catalyzed by the arginine-modified enzyme. Proton NMR spectroscopy demonstrated that succinate strongly interacts with the native enzyme to generate substantial changes in the enzyme spectra whereas there was no such evidence for the specific interaction with this dicarboxylate with the arginine-modified enzyme.