Active-site Arg → Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase

Abstract

Arg386 and Arg292 of aspartate aminotransferase bind the α and the distal carboxylate group, respectively, of dicarboxylic substrates. Their substitution with lysine residues markedly decreased aminotransferase activity. The k(cat) values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25 °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 Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged. Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates. All three mutant enzymes catalyzed as side reactions the β-decarboxylation of L- aspartate and the racemization of amino acids at faster rates than the wild- type enzyme. The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L- alanine 15 times faster (k(cat) = 0.002 s-1) than the wild-type enzyme. The rates 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 aminotransferase decrease aminotransferase activity but increase other pyridoxal 5'-phosphate- dependent catalytic activities. Apparently, the reaction specificity of pyridoxal 5'-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct.