Evidence that both protium and deuterium undergo significant tunneling in the reaction catalyzed by bovine serum amine oxidase

Abstract

The magnitudes of primary and secondary H/T and D/T kinetic isotope effects have been measured in the bovine serum amine oxidase catalyzed oxidation of benzylamine from 0 to 45.degree. C. Secondary H/T and D/T kinetic effects are small and in the range anticipated from equilibrium isotope effects; Arrhenius preexponential factors (AH/AT and AD/AT) determined from the temperature dependence of isotope effects also indicate semiclassical behavior. By contrast, primary H/T and D/T isotope effects, 35.2 .+-. 0.8 and 3.07 .+-. 0.07, respectively, at 25.degree. C, are larger than semisclassical values and given anomalously low preexponential factor ratios, AH/AT = 0.12 .+-. 0.04 and AD/AT = 0.51 .+-. 0.10. Stopped-flow studies indicate similar isotope effect on cofactor reduction as seen in the steady state, consistent with a single rate-limiting C-H bond cleavage step for Vmax/Km. The comparison of primary and secondary isotope effects allows us to rule out appreciable couplingn between the primary and seocndary hydrogens at C-1 of the substrate. From the properties of primary isotope effects, we conclude that both protium and deuterium undergo significant tunneling in the course of substrate oxidation. These findings represent the first example of quantum mechanical effects in an enzyme-catalyzed proton abstraction reaction.