title: 13C and (15)N kinetic isotope effects on the reaction of aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant.
abstract: Heavy atom isotope effects at C-2, C-3, and the amino nitrogen of aspartate were determined for the reaction of porcine heart cytosolic aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant of Escherichia coli aspartate aminotransferase. The effects of deuteration at C-2 of aspartate and of D(2)O on the observed heavy atom isotope effects were determined. The multiple isotope effects support the contribution of C(alpha)-H cleavage, ketimine hydrolysis, and oxaloacetate dissociation to the rate limitation with the wild-type enzyme. The existence of a quinonoid intermediate could not be determined due to the kinetic complexity of the enzyme. For the tyrosine-225 to phenylalanine mutant, we are able to conclude that ketimine hydrolysis is the major rate-determining step.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison 53705, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Rishavy
authors.forename: W W
authors.initials: WW
authors.lastname: Cleland
mesh_terms: D019943:Amino Acid Substitution; D001219:Aspartate Aminotransferases / Q000737:chemistry*; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D010649:Phenylalanine / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D014443:Tyrosine; D010649:Phenylalanine; D001219:Aspartate Aminotransferases