The conjoint substitution of three active-site residues in aspartate aminotransferase (AspAT) of Escherichia coli (Y225R/R292K/R386A) increases the ratio of L-aspartate beta-decarboxylase activity to transaminase activity >25 million-fold. This result was achieved by combining an arginine shift mutation (Y225R/R386A) with a conservative substitution of a substrate-binding residue (R292K). In the wild-type enzyme, Arg(386) interacts with the alpha-carboxylate group of the substrate and is one of the four residues that are invariant in all aminotransferases; Tyr(225) is in its vicinity, forming a hydrogen bond with O-3' of the cofactor; and Arg(292) interacts with the distal carboxylate group of the substrate. In the triple-mutant enzyme, k(cat)' for beta-decarboxylation of L-aspartate was 0.08 s(-1), whereas k(cat)' for transamination was decreased to 0.01 s(-1). AspAT was thus converted into an L-aspartate beta-decarboxylase that catalyzes transamination as a side reaction. The major pathway of beta-decarboxylation directly produces L-alanine without intermediary formation of pyruvate. The various single- or double-mutant AspATs corresponding to the triple-mutant enzyme showed, with the exception of AspAT Y225R/R386A, no measurable or only very low beta-decarboxylase activity. The arginine shift mutation Y225R/R386A elicits beta-decarboxylase activity, whereas the R292K substitution suppresses transaminase activity. The reaction specificity of the triple-mutant enzyme is thus achieved in the same way as that of wild-type pyridoxal 5'-phosphate-dependent enzymes in general and possibly of many other enzymes, i.e. by accelerating the specific reaction and suppressing potential side reactions.