THE TYROSINE-225 TO PHENYLALANINE MUTATION OF ESCHERICHIA-COLI ASPARTATE-AMINOTRANSFERASE RESULTS IN AN ALKALINE TRANSITION IN THE SPECTROPHOTOMETRIC AND KINETIC PKA VALUES AND REDUCED VALUES OF BOTH KCAT AND KM

Tyrosine-225 is hydrogen-bonded to the 3'-hydroxyl group of pyridoxal 5'-phosphate in the active site of aspartate aminotransferase. Replacement of this residue with phenylalanine (Y225F) results in a shift in the acidic limb of the pK(a) of the k(cat)/K(asp) vs pH profile from 7.1 (wild-type) to 8.4 (mutant). The change in the kinetic pK(a) is mirrored by a similar shift in the spectrophotometrically determined pK(a) of the protonated internal aldimine. Thus, a major role of tyrosine-225 is to provide a hydrogen bond that stabilizes the reactive unprotonated form of the internal aldimine in the neutral pH range. The K(m) value for L-aspartate and the dissociation constant for alpha-methyl-DL-aspartate are respectively 20- and 37-fold lower in the mutant than in the wild-type enzyme, while the dissociation constant for maleate is much less perturbed. These results are interpreted in terms of competition between the Tyr225 hydroxyl group and the substrate or quasi-substrate amino group for the coenzyme. The value of k(cat) in Y225F is 450-fold less than the corresponding rate constant in wild type. The increased affinity of the mutant enzyme for substrates, combined with the lack of discrimination against deuterium in the C(alpha) position of L-aspartate in Y225F-catalyzed transamination [Kirsch, J. F., Toney, M. D., & Goldberg, J. M. (1990) in Protein and Pharmaceutical Engineering (Craik, C. S., Fletterick, R., Matthews, C. R., & Wells, J., Eds.) pp 105-118, Wiley-Liss, New York], suggests that the rate-determining step in the mutant is hydrolysis of the ketimine intermediate rather than C(alpha)-H abstraction which is partially rate-determining in wild type.