title: 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.
abstract: 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.
authors.affiliation: Department of Molecular & Cell Biology, University of California, Berkely 94720-3206.
authors.forename: J M
authors.initials: JM
authors.lastname: Goldberg
authors.forename: J F
authors.initials: JF
authors.lastname: Kirsch
mesh_terms: D001219:Aspartate Aminotransferases / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D005075:Biological Evolution; D002384:Catalysis; D003198:Computer Simulation; D003903:Deuterium; D004058:Diffusion; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007097:Imines; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D010071:Oxaloacetates / Q000378:metabolism; D012997:Solvents; D013053:Spectrophotometry; D013816:Thermodynamics; D000637:Transaminases / Q000378:metabolism; D014783:Viscosity
chemical_list: D007097:Imines; D010071:Oxaloacetates; D012997:Solvents; D001224:Aspartic Acid; D003903:Deuterium; D000637:Transaminases; D001219:Aspartate Aminotransferases