The aspartate aminotransferase mutant Y70F exhibits kcat = 8% and kcat/KM = 2% of the wild type values for the transamination of aspartate and α-ketoglutarate. The affinity of the enzyme for the noncovalently bound inhibitor maleate is reduced 17-fold by the mutation, while only a 2.5-fold reduction is observed for α-methylaspartate, which forms a stable, covalent external aldimine. The high population of the quinonoid intermediate formed in the reaction of the wild type with β-hydroxyaspartate is more than 75% diminished by the mutation. The values of the Y70F Cα-H kinetic isotope effects for the aspartate reaction are larger than those of wild type (DV = 2.4 vs 1.52; D(V/K) = 2.5 vs 1.7). Conversely, the Y70F value of Q(V/K) for the glutamate reaction is decreased compared to wild type (1.75 vs 2.5). These results, combined with previous studies of Lys258 mutants, eliminate Tyr70 as an essential component of the catalytic apparatus, with the caveat that the functionality of the deleted hydroxyl group is possibly replaced by a water molecule.
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