PH dependence of deuterium isotope effects and tritium exchange in the bovine plasma amine oxidase reaction: a role for single-base catalysis in amine oxidation and imine exchange

The pH dependence of steady-state parameters for [1,1-1H2]- and [1,1-2H2]benzylamine oxidation and of tritium exchange from [2-3H]dopamine has been measured in the bovine plasma amine oxidase reaction. Deuterium isotope effects on kcat/Km for benzylamine are observed to be constant, near the intrinsic value of 13.5, over the experimental pH range, indicating that C-H bond cleavage is fully rate limiting for this parameter. As a consequence, pKa values derived from kcat/Km profiles, 8.0 +/- 0.1 (pK1) and 9.0 +/- 0.16 (pKs), can be ascribed to microscopic pKa values for the ionization of an essential active site residue (EB1) and substrate, respectively. Profiles for kcat and Dkcat show that EB1 undergoes a perturbation from 8.0 to 5.6 +/- 0.3 (pK1') in the presence of substrate; additionally, a second ionization, pK2 = 7.25 +/- 0.25, is observed to mediate but not be essential for enzyme reoxidation. The pH dependence of the ratio of tritium exchange to product formation for dopamine also indicates base catalysis with a pKexch = 5.5 +/- 0.01, which is within experimental error of pK1'. We conclude that the data presented herein support a single residue catalyzing both substrate oxidation and exchange, consistent with recent stereochemical results that implicate a syn relationship between these processes [Farnum, M., and Klinman, J.P. (1985) Fed. Proc., Fed. Am. Soc. Exp. Biol. 44, 1055]. This conclusion contrasts with earlier kinetic data in support of a large rate differential for the exchange of hydrogen from C-1 vs. C-2 of phenethylamine derivatives