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

Abstract

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 pKa of the kcat/KAsp vs pH profile from 7.1 (wild-type) to 8.4 (mutant). The change in the kinetic pKa is mirrored by a similar shift in the spectrophotometrically determined pKa 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 Km 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 kcat 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.

The Roles of Tyr70 and Tyr225 in Aspartate Aminotransferase Assessed by Analysing the Effects of Mutations on the Multiple Reactions of the Substrate Analogue Serine O‐Sulphate

“…of AspAT Y225F on viscosity [26] indicated that the dissociation of the 0x0 acid product is not rate determining for the mutant. It was concluded that, in the AspAT Y225F-catalysed transamination of normal substrates, ketimine hydrolysis had become rate determining [22]. The results of the present work support this conclusion.…”

“…The enzyme preparations were honiogeneous as assessed by SDS/PAGE. The enzyme subunit concentration was determined by using c2xo = 44000 M ' cm ' and 40000 M-' cm-' for the wild-type enzyme and AspAT Y22SF, respectively [22]. In the case of the Y70M mutant, c,,,, = 46000 M-' c m -' was used for the pyridoxal phosphate form Kinetic characterization with L-serine 0-sulphate.…”

Histidine 282 in 5-Aminolevulinate Synthase Affects Substrate Binding and Catalysis

“…In aspartate aminotransferase and 1-aminocyclopropane-1-carboxylate synthase, the phenolic oxygen interacts with a tyrosine residue (14,15). The deletion of the hydrogen bond through the replacement of the active site tyrosine with phenylalanine reveals a different function in the kinetic properties of each enzyme.…”

Redesign of the substrate specificity ofescherichia coliaspartate aminotransferase to that ofescherichia colityrosine aminotransferase by homology modeling and site-directed mutagenesis

“…The acidic form of the aldimine has an absorbance maximum of 430 nm, whereas the deprotonated aldimine absorbs maximally near 360 nm. Thus, the association of these inhibitors can be monitored by either the disappearance of the deprotonated form (360 nm) or the appearance of the protonated aldimine (430 nm) (Jenkins & D'Ari, 1966;Goldberg et al, 1991). The a-methyl amino acids (a-MeAsp, a-MePhe, a-MeTyr) form an equilibrium mixture of the Michaelis complex and a Schiff base linkage with the PLP cofactor (Kintanar et al, 1991;Goldberg et al, 1993).…”

“…Transamination is precluded by the Ca-methyl group. Their association with the enzyme can be followed in a manner similar to that employed with Hca and maleate because the external aldimine formed between these inhibitors and the PLP cofactor has a higher pK, than the internal aldimine present in unliganded enzyme (Goldberg et al, 1991). The aromatic inhibitors Hca, a-MePhe, and a-MeTyr have a very weak …”