Kinetic Isotope Effects on the Catalytic Activity of Pig‐Plasma Benzylamine Oxidase

Abstract

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 [α, α‐2H]benzylamine as the substrate. Replacement of the α‐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. 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 α‐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. Copyright © 1976, Wiley Blackwell. All rights reserved