2H, 13C, and 15N kinetic isotope effects on the reaction of the ammonia-rescued K258A mutant of aspartate aminotransferase

Deuterium isotope effects at C2 of aspartate and heavy atom isotope effects at C2, C3, and the amino group of aspartate were determined for the reaction of the lysine-258 to alanine mutant of Escherichia coli rescued with exogenous ammonia. We were able to calculate an N-15 intrinsic isotope effect of 1.034. The intrinsic C-13 isotope effect at C3 is 1.0060, and the C-13 isotope effect at C2 is 1.0016. These isotope effects reveal that collapse of the carbinolamine (or gem-diamine) to give the final product is the rate-determining step in this system. Furthermore, these results indicate that lysine-258 is critical to the catalysis of the final breakdown to give product, and in fact this step is more strongly affected by mutation of lysine-258 than the deprotonation of the external aldimine.