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Conversion of Aspartate Aminotransferase into anl-Aspartate β-Decarboxylase by a Triple Active-site Mutation

1999; Elsevier BV; Volume: 274; Issue: 44 Linguagem: Inglês

10.1074/jbc.274.44.31203

1083-351X

Rachel Graber, Patrik Kasper, V.N. Malashkevich, Pavel Strop, Heinz Gehring, Johan N. Jansonius, Philipp Christen,

Enzyme Structure and Function

The conjoint substitution of three active-site residues in aspartate aminotransferase (AspAT) of Escherichia coli (Y225R/R292K/R386A) increases the ratio ofl-aspartate β-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 α-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 β-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 β-decarboxylase that catalyzes transamination as a side reaction. The major pathway of β-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 β-decarboxylase activity. The arginine shift mutation Y225R/R386A elicits β-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.