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 2H-Asp kinetic isotope effects (KIEs) on reactions catalyzed by wild-type AAtase at pH 8.9 and 7.5 on kcat/KMAsp are approximately 2, and the KIEs on kcat are 1.9 (pH 8.9) and 1.4 (pH 7.5). The C alpha 2H-Asp KIEs on reactions catalyzed by Y225F are near unity at both pH values. The solvent deuterium KIEs (SKIEs) on kcat for reactions with L-Asp catalyzed by wild-type AATase and Y225F at their pH/pD maxima approximately 2, and the SKIE on kcat/kMAsp is increased from 1.3 to 2.3 by the mutation. The C4' (S)-2H-pyridoxamine 5'-phosphate KIE values on reactions of alpha-ketoacids with both enzymes are near unity. The viscosity effects on kcat/KMAsp and kcat 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 kcat/KMAsp and kcat 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 partially rate-determining. Ketimine hydrolysis is the sole rate-determining step for the corresponding Y225F- catalyzed reaction.