SPECTRAL AND KINETIC-STUDIES OF IMINE PRODUCT FORMATION IN THE OXIDATION OF P-(N,N-DIMETHYLAMINO)BENZYLAMINE ANALOGS BY MONOAMINE OXIDASE-B

The oxidative deamination of p-(N,N-dimethylamino)benzylamine and N-methyl-p-(N,N-dimethylamino)benzylamine by bovine liver monoamine oxidase B has been investigated by absorption spectral, steady-state, and stopped-flow kinetic studies. An absorbing intermediate with a maximum at 390 nm is observed with either analogue in turnover experiments at neutral pH and is identified as due to the formation of protonated imine as the initial product. p-(N,N-Dimethylamino)benzaldehyde is the final product formed from either substrate analogue. Anaerobic stopped-flow measurements show N-methyl-p-(N,N-dimethylamino)benzylamine to reduce enzyme-bound flavin with a limiting rate of 1.8 s-1 concurrent with the appearance of a 390-nm absorption due to protonated imine product with a limiting rate of 1.7 s-1. Both observed rates are somewhat faster than catalytic turnover (1.5 s-1). Under anaerobic conditions, the decay of protonated N-methyl-p-(N,N-dimethylamino)benzenimine is much slower than turnover (k = 4.8 x 10(4) s-1). p-(N,N-Dimethylamino)benzylamine reduces the enzyme with a limiting rate of 2.1 s-1, which is faster than catalytic turnover (1.2 s-1). Protonated imine formation is also observed with this substrate with an apparent limiting rate of 1.3 s-1. The decay of the protonated p-(N,N-dimethylamino) benzenimine absorbance is slower than catalytic turnover but faster than the rate of aldehyde formation under anaerobic conditions. Deuterium kinetic isotope effect values of approximately 10 are observed both for flavin reduction and for protonated imine formation. No isotope effect is observed for the rate of imine decay. These results demonstrate that the protonated form of the imine is the initial product in the oxidation of benzylamines by monoamine oxidase B. The reduced enzyme-imine complex is kinetically stable to dissociation, but reacts with O2 at rates consistent with catalysis. Imine hydrolysis occurs nonenzymatically after release from the oxidized form of the enzyme.