THE MECHANISM OF INHIBITION OF BENZYLAMINE OXIDASE BY 3,5-DIETHOXY-4-AMINOMETHYLPYRIDINE (B-24)

B-24, 3,5-diethoxy-4-aminomethylpyridine, is a specific inhibitor of t he semicarbazide-sensitive amine oxidase with high affinity for benzyl amine (BnNH2.SSAO). It is a site-directed inhibitor of pig plasma benz ylamine 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 an d 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 preincubatio n lime. This decrease is dependent on the gradual release of free enzy me which reacts with the substrate, giving [ES] without any interferin g free B-24 When the B-24/BAO molar ratio is higher than 1, the free e nzyme released by the oxidative deamination of B-24 reacts with the su bstrate, but the free B-24 present competitively inhibits the formatio n of [ES] and the affinity of benzylamine is therefore reduced. This i s the reason why B-24, in the kinetic experiments in which the inhibit or is not preincubated with the enzyme, may appear to be a competitive inhibitor or a mixed inhibitor, mainly competitive. When B-24 is prei ncubated with the enzyme and the initial rate of benzylamine oxidation is measured, it appears as a non-competitive inhibitor becoming a mix ed one only when the B-24/BAO molar ratio is high and the incubation t ime is long.