Tyrosine 70 fine-tunes the catalytic efficiency of aspartate aminotransferase.
The aspartate aminotransferase mutant Y70F exhibits kcat = 8% and kcat/KM = 2% of the wild type values for the transamination of aspartate and alpha-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 alpha-methylaspartate, which forms a stable, covalent external aldimine. The high population of the quinonoid intermediate formed in the reaction of the wild type with beta-hydroxyaspartate is more than 75% diminished by the mutation. The values of the Y70F C alpha-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 D(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 functionally of the deleted hydroxyl group is possibly replaced by a water molecule.
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
M D
MD
Toney
J F
JF
Kirsch
D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D004791:Enzyme Inhibitors; D004926:Escherichia coli / Q000201:enzymology*; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D007554:Isotopes; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008298:Maleates / Q000378:metabolism; D009154:Mutation; D016202:N-Methylaspartate / Q000378:metabolism; D013053:Spectrophotometry; D014443:Tyrosine / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005971:Glutamates; D007554:Isotopes; D007656:Ketoglutaric Acids; D008298:Maleates; D001224:Aspartic Acid; D018698:Glutamic Acid; D014443:Tyrosine; D016202:N-Methylaspartate; C030272:maleic acid; D001219:Aspartate Aminotransferases