Abstract
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.
Highlights
In the engineering of protein catalysts with new functional properties, the modification of existing enzymes provides an alternative to the production of catalytic antibodies or, in a more distant future, the de novo design of enzymes
To determine the partition ratio of the two pathways, the consumption of oxalacetate and the production of pyruvate in the presence of L-aspartate and oxalacetate were followed in parallel with the -decarboxylation of L-aspartate (Table II). Both aspartate aminotransferase (AspAT) Y225R/R386A and AspAT Y225R/R292K/R386A produced pyruvate with a kcat of only 0.01 sϪ1, corresponding to a partition ratio (7 3 8 versus 7 3 9) of 8
In the wild-type enzyme, production of FIG. 2. -Decarboxylation of L-aspartate catalyzed by AspAT Y225R/R292K/R386A (q), AspAT Y225R/R386A (), and wild-type AspAT (f)
Summary
AspAT, aspartate aminotransferase; PLP, pyridoxal 5Ј-phosphate; B6 enzyme, PLP (vitamin B6)-dependent enzyme; PMP, pyridoxamine 5Ј-phosphate. Into AspAT Y225R/R386A, would decrease further transaminase activity without affecting -decarboxylase activity. The only mutation among many tested that brought about this effect was the replacement of the second active-site arginine residue, i.e. Arg292 (a residue of the adjacent subunit of the AspAT homodimer) with lysine. In the wild-type enzyme, Arg292 binds the distal carboxylate group of the substrate (Fig. 1). The single R292K mutation had been previously found to decrease transaminase activity to 0.2% of that of the wild-type enzyme [15]. In the triple-mutant enzyme, -decarboxylase activity exceeded transaminase activity by a factor of 8
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