The mechanism of inhibition of benzylamine oxidase by 3,5-diethoxy-4-aminomethylpyridine (B24)

B-24, 3,5-diethoxy-4-aminomethylpyridine, is a specific inhibitor of the semicarbazide-sensitive amine oxidase with high affinity for benzylamine (BnNH2.SSAO). It is a site-directed inhibitor of pig plasma benzylamine oxidase (BAO) with an affinity for the enzyme much higher than that for benzylamine. B-24 inhibition is dependent on the molar ratio B-24/BAO because the inhibitor reacts mole to mole with the enzyme and benzylamine appears to be ineffective in removing the inhibitor from the adduct [EI]. B-24 is a weak substrate of BAO and for this reason the degree of inhibition (when the molar ratio B-24/BAO is lower than 1) decreases with the incubation time as well as with the preincubation lime. This decrease is dependent on the gradual release of free enzyme which reacts with the substrate, giving [ES] without any interfering free B-24 When the B-24/BAO molar ratio is higher than 1, the free enzyme released by the oxidative deamination of B-24 reacts with the substrate, but the free B-24 present competitively inhibits the formation of [ES] and the affinity of benzylamine is therefore reduced. This is the reason why B-24, in the kinetic experiments in which the inhibitor is not preincubated with the enzyme, may appear to be a competitive inhibitor or a mixed inhibitor, mainly competitive. When B-24 is preincubated with the enzyme and the initial rate of benzylamine oxidation is measured, it appears as a non-competitive inhibitor becoming a mixed one only when the B-24/BAO molar ratio is high and the incubation time is long.