Conversion of aspartate aminotransferase into an L-aspartate beta-decarboxylase by a triple active-site mutation

The conjoint substitution of three active-site residues in aspartate aminot ransferase (AspAT) of Escherichia coli (Y225R/R292K/R386A) increases the ra tio of L-aspartate P-decarboxylase activity to transaminase activity >25 mi llion-fold. This result was achieved by combining an arginine shift mutatio n (Y225R/R386A) with a conservative substitution of a substrate-binding res idue (R292K), In the wild-type enzyme, Arg(386) interacts with the cw-carbo xylate group of the substrate and is one of the four residues that are inva riant in all aminotransferases; Tyr(225) is in its vicinity, forming a hydr ogen 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)' f or 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 a n L-aspartate beta-decarboxylase that catalyzes transamination as a side re action. The major pathway of beta-decarboxylation directly produces L-alani ne without intermediary formation of pyruvate, The various single- or doubl e-mutant AspATs corresponding to the triple-mutant enzyme showed, with the exception of AspAT Y225R/R386A, no measurable or only very law beta-decarbo xylase activity. The arginine shift mutation Y225R/R386A elicits beta-decar boxylase activity, whereas the R292K substitution suppresses transaminase a ctivity, The reaction specificity of the triple-mutant enzyme is thus achie ved in the same way as that of wild-tape pyridoxal 5'-phosphate-dependent e nzymes in general and possibly of many other enzymes, i.e. by accelerating the specific reaction and suppressing potential side reactions.