The reaction catalyzed by Escherichia coli aspartate aminotransferase has multiple partially rate-determining steps, while that catalyzed by the Y225F mutant is dominated by ketimine hydrolysis

The mechanism of transamination catalyzed by Escherichia coli wild-type aspartate aminotransferase (AATase) and the mutant AATase in which Tyr-225 is converted to Phe (Y225F) was investigated. The absorbance spectrum of wild-type AATase in the presence of excess L-Asp and oxalacetate is dominated by species absorbing near 330 nm. The primary C-alpha H-2-Asp kinetic isotope effects (KIEs) on reactions catalyzed by wild-type AATase at pH 8.9 and 7.5 on k(cat)/K-M(Asp) are approximately 2, and the KIEs on k(cat) are 1.9 (pH 8.9) and 1.4 (pH 7.5). The C-alpha H-2-Asp KIEs on reactions catalyzed by Y225F are near unity at both pH values, The solvent deuterium KIEs (SKIEs) on k(cat) for reactions with L-Asp catalyzed by wild-type AATase and Y225F at their pH/pD maxima approximate to 2, and the SKIE on k(cat)/K-M(Asp) is increased from 1.3 to 2.3 by the mutation. The C-4' (S)-H-2-pyridoxamine 5'-phosphate KIE values on reactions of a-ketoacids with both enzymes are near unity. The viscosity effects on k(cat)/K-M(Asp) and k(cat) for wild-type AATase at pH 9 are 0.10 and 0.31, respectively, indicating that the reaction is partially diffusion limited. The viscosity effects on k(cat)/K-M(Asp) and k(cat) for Y225F are reduced to -0.02 and 0.06, respectively, indicating that the mutant catalyzed reaction is almost fully chemistry-limited. A free-energy profile for the L-Asp-to-oxalacetate half-reaction was constructed for wild-type AATase, C-alpha H abstraction, ketimine hydrolysis, and oxalacetate dissociation are all partially rate-determining. Ketimine hydrolysis is the sole rate-determining step for the corresponding Y225F-catalyzed reaction.