Stereochemistry of Benzylamine Oxidation by Copper Amine Oxidases

Copper amine oxidases (EC 1.4.3.6) exhibit stereochemical heterogeneity in their reaction specificities. Enzymes isolated from different sources have previously been shown to catalyze the deamination of tyramine and dopamine with abstraction of the pro-R hydrogen at C-1, the pro-S hydrogen, and net nonstereospecific proton abstraction. In this study we report on the stereochemical course of proton removal from benzylamine for six copper amine oxidases using stereospecifically deuterated benzylamines prepared by a combined chemical-enzymatic method. The product benzaldehydes from the amine oxidase reactions were reduced in situ with alcohol dehydrogenase and NADH providing benzyl alcohols which were analyzed by 1H NMR spectroscopy. The amine oxidases isolated from pea seedlings and bovine, horse, porcine, rabbit, and sheep plasma all react with abstraction of the pro-S hydrogen of benzylamine, irrespective of the stereochemical course of the oxidation of tyramine or dopamine. We also report that the enzymes isolated from horse and rabbit plasma contain 2,4,5-trihydroxyphenylalanine (topaquinone) as an organic cofactor based on visible absorbance spectroscopy of p-nitrophenylhydrazine derivatized enzymes.