Replacement of an Interdomain Residue Va139 of Escherichia coli Aspartate Aminotransferase Affects the Catalytic Competence without Altering the Substrate Specificity of the Enzyme

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Three mutant Escherichip coli aspartate aminotransferases in which Va139 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest K_m values for dicarboxylic substrates. The K_m values of the A1a39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Va139) enzyme. These two mutant enzymes showed essentially the same k_cat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/K_m) and catalytic ability (k_cat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Va139 with other residues altered both the k_cat and K_m values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the k_cat/K_m value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransfer-ases [Seville, M., Vincent M. G., & Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates.