Hydrogen tunneling in the flavoenzyme monoamine oxidase B.

Competitive kH/kT and kD/kT kinetic isotope effects on p-methoxybenzylamine oxidation by the 8 alpha-S-cysteinyl flavin adenine dinucleotide (FAD)-dependent enzyme monoamine oxidase B (MAO-B) have been measured as a function of temperature. At pH 7.5, exponents relating observed kH/kT and kD/kT isotope effects indicate the presence of a temperature-dependent change in rate-limiting step. At lower temperature (e.g., 2 degrees C), the presence of multiple rate-limiting steps (commitments) is clearly indicated from the size of the exponent and individual isotope effects. Noncompetitive kH/kD isotope effect measurements indicate a trend in observed isotope effects between pH 9.0 and 6.0, with isotope effects increasing at lower pH. Primary and secondary kH/kT and kD/kT isotope effects were therefore measured as a function of temperature at pH 6.1. Exponents relating primary and secondary kH/kT and kD/kT in the 10-43 degrees C range are 3.13 +/- 0.04 and 2.36 +/- 0.13, respectively, and do not systematically change with temperature. These data indicate that commitments, if present, remain constant across this temperature range. The temperature dependence of the observed primary isotope effects gives values for the ratios of Arrhenius prefactors of 0.13 +/- 0.03 (AH/AT) and 0.52 +/- 0.05 (AD/AT). Both values are well below the lower limits predicted in the absence of tunneling contributions to the reaction coordinate, indicating that both deuterium and protium tunneling take place in this reaction. The presence of a temperature independent commitment contribution cannot be rigorously ruled out; however, the effect of such a commitment on the observed AH/AT and AD/AT values is shown to be quite small.