pH and kinetic isotope effects in D-amino acid oxidase catalysis - Evidence for a concerted mechanism in substrate dehydrogenation via hydride transfer

The effects of pH, solvent isotope, and primary isotope replacement on subs trate dehydrogenation by Rhodotorula gracilis D-amino acid oxidase, were in vestigated. The rate constant for enzyme-FAD reduction by D-alanine increas es approximate to fourfold with pH, reflecting apparent pK(a) values of app roximate to 6 and approximate to 8, and reaches plateaus at high and low pH . Such profiles are observed in all presteady-state and steady-state kineti c experiments, using both D-alanine, and D-asparagine as substrates, and ar e inconsistent with the operation of a base essential to catalysis. A solve nt deuterium isotope effect of 3.1 +/- 1.1 is observed on the reaction with D-alanine at pH 6; it decreases to 1.2 +/- 0.2 at pH 10. The primary subst rate, isotope effect on the reduction rate with [2-D]D-alanine is 9.1 +/- 1 .5 at low and 2.3 +/- 0.3 at high pH. At pH 6.0, the solvent isotope effect is 2.9 +/- 0.8 with, [2-D]D-alanine, and the primary isotope effect is 8.4 +/- 2.4 in D2O. Thus, primary and solvent kinetic isotope effects (KIEs) a re independent of the presence of the other isotope, i.e. the 'double' kine tic isotope effect is the product of the individual KIEs, consistent with a transition state in which rapture of the two bonds of the substrate to hyd rogen is concerted. These results support a hydride transfer mechanism for the dehydrogenation reaction in D-amino acid oxidase and argue against the occurrence of any intermediates in the process. A pK(a,app) of approximate to 8 is interpreted to arise from the microscopic ionization of the substra te amino acid alpha -amino group, but also includes contributions from kine tic parameters.