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

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Abstract
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 [α, α-2H]benzylamine as the substrate. 2 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. 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.