Kinetic isotope effects on the catalytic activity of pig-plasma benzylamine oxidase.

1. Isotope effects on the catalytic activity of benzylamine oxidase at pH 7 and 9 have been studied by steady-state and transient-state kinetics methods, using [alpha,alpha-2H]benzylamine as the substrate. 2. Replacement of the alpha-hydrogen atoms in benzylamine by deuterium has no significant effect on substrate-binding to benzylamine oxidase, neither does it affect the rate of reoxidation of the reduced form of the enzyme. Conversion of the primarily formed enzyme-substrate complex into the reduced enzyme species, however, exhibits an isotope effect of about 3. 3. The data obtained are consistent with a mechanism in which reduction of benzylamine oxidase takes place by a rapid pre-equilibration between enzyme and substrate to form an amine-pyridoxal Schiff-base, which is then tautomerized by a comparatively slow prototropic shift to an amino aldehyde-pyridoxamine Schiff-base from which there is a rapid hydrolytic release of the aldehyde product corresponding to the amine substrate. Proton abstraction from the alpha-carbon of the amine moiety in the primary Schiff-base appears to be at least partially rate-limiting for the tautomerization step, and hence for the entire process of enzyme reduction.