C-13 and N-15 kinetic isotope effects on the reaction of aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant

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 amin otransferase and the tyrosine-225 to phenylalanine mutant of Escherichia co li aspartate aminotransferase. The effects of deuteration at C-2 of asparta te and of D2O on the observed heavy atom isotope effects were determined. T he 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 tyro sine-225 to phenylalanine mutant, we are able to conclude that ketimine hyd rolysis is the major rate-determining step.