title: 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.
authors.affiliation: Department of Molecular and Cellular Biology, University of California, Berkeley 94720.
authors.forename: J M
authors.initials: JM
authors.lastname: Goldberg
authors.forename: R V
authors.initials: RV
authors.lastname: Swanson
authors.forename: H S
authors.initials: HS
authors.lastname: Goodman
authors.forename: J F
authors.initials: JF
authors.lastname: Kirsch
mesh_terms: D001219:Aspartate Aminotransferases / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D006860:Hydrogen Bonding; D007700:Kinetics; D008433:Mathematics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010649:Phenylalanine; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011733:Pyridoxamine / Q000031:analogs & derivatives / Q000378:metabolism; D013053:Spectrophotometry; D014443:Tyrosine
chemical_list: D015345:Oligonucleotide Probes; D014443:Tyrosine; D010649:Phenylalanine; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D001219:Aspartate Aminotransferases; C010627:pyridoxamine phosphate