The copper amine oxidases (CAOs) have evolved to catalyze oxidative deamination of unbranchedprimary amines to aldehydes. We report that a branched primary amine bearing an aromatization-prone moiety, ethyl 4-amino-4,5-dihydrothiophene-2-carboxylate (1), is recognized enantioselectively (S >> R) by bovine plasma amine oxidase (BPAO) both as a temporary inactivator and as a substrate. Substrate activity results from an O(2)-dependent turnover of the covalently modified enzyme, with release of 4-aminothiophene-2-carboxylate (2) as ultimate product. Interaction of (S)-1 with BPAO occurs within the enzyme active site with a dissociation constant of 0.76 microM. Evidence from kinetic and spectroscopic studies, and HPLC analysis of stoichiometric reactions of BPAO with (S)-1, combined with a model study using a quinone cofactor mimic, establishes that the enzyme metabolizes 1 according to a transamination mechanism. Following the initial isomerization of substrate Schiff base to product Schiff base, a facile aromatization of the latter results in a metastable N-aryl derivative of the reduced cofactor aminoresorcinol, which is catalytically inactive. The latter derivative is then slowly oxidized by O(2), apparently facilitated partially by the active-site Cu(II), to form a quinonimine of the native cofactor that releases 2 upon hydrolysis or transimination with substrate amine. Preferential metabolism of (S)-1 is consistent with the preferential removal of the pro-Salpha-proton in metabolism of benzylamine by BPAO. This study represents the first report of product identification in metabolism of a branched primary amine by a copper amine oxidase and suggests a novel type of reversible mechanism-based (covalent) inhibition where inhibition lifetime can be fine-tuned independently of inhibition potency.