ACTIVE-SITE ARG-]LYS SUBSTITUTIONS ALTER REACTION AND SUBSTRATE-SPECIFICITY OF ASPARTATE-AMINOTRANSFERASE

Arg(386) and Arg(292) of aspartate aminotransferase bind the alpha and the distal carboxylate group, respectively, of dicarboxylic substrate s. Their substitution with lysine residues markedly decreased aminotra nsferase activity. The k(cat) values with L-aspartate and 2-oxoglutara te as substrates under steady-state conditions at 25 degrees C were 0. 5, 2.0, and 0.03 s(-1) for the R292K, R386K, and R292K/R386K mutations , respectively, k(cat) of the wild-type enzyme being 220 s(-1). Longer dicarboxylic substrates did not compensate for the shorter side chain of the lysine residues. Consistent with the different roles of Arg(29 2) and Arg(386);, substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocap roic acid) and aromatic substrates diverged. Whereas the R292K mutatio n did not impair the aminotransferase activity toward these substrates , the effect of the R386K substitution was similar to that on the acti vity toward dicarboxylic substrates. All three mutant enzymes catalyze d as side reactions the beta-decarboxylation of L-aspartate and the ra cemization of amino acids at faster rates than the wild-type enzyme. T he changes in reaction specificity were most pronounced in aspartate a minotransferase R292K, which decarboxylated L-aspartate to L-alanine 1 5 times faster (k(cat) = 0.002 s(-1)) than the wild-type enzyme. The r ates of racemization of L-aspartate, L-glutamate, and L-alanine were 3 , 5, and 2 times, respectively, faster than with the wild-type enzyme. Thus, Arg --> Lys substitutions in the active site of aspartate amino transferase decrease aminotransferase activity but increase other pyri doxal 5'-phosphate-dependent catalytic activities. Apparently, the rea ction specificity of pyridoxal 5'-phosphate-dependent enzymes is not o nly achieved by accelerating the specific reaction but also by prevent ing potential side reactions of the coenzyme substrate adduct.