MECHANISM-BASED INACTIVATION OF PORCINE KIDNEY DIAMINE OXIDASE BY 1,4-DIAMINO-2-BUTENE

Cis- and trans-1,4-diamino-2-butene are substrates and potent inactiva tors of porcine kidney diamine oxidase. Evidence from absorption and N MR spectra indicates that both are oxidized to pyrrole. Both substrate s are irreversible mechanism-based inactivators of the enzyme, althoug h the trans isomer is more potent and results in complete inactivation in a reaction which follows pseudo-first-order kinetics with an appar ent K-i of 0.34 mM and a second-order inactivation constant of 500 M(- 1) s(-1). Under the same conditions, 46% of the activity remains when the enzyme is reacted with cis-l,4-diamino-2-butene. Trans-4-amino-2-b utenal, the product of oxidation of the trans diamine, has been synthe sized and shown to undergo cyclization to pyrrole in a concentration-d ependent manner, approaching second-order at low concentrations. Trans -4-amino-2-butenal is itself a potent irreversible inhibitor with IC50 of 2.5 mu M. We propose that the irreversible inactivation by both ci s- and trans-1,4-diamino-2-butene involves attack by a protein-based n ucleophilic residue on the unsaturated aminoenal products of the enzym atic reactions, resulting in a covalent adduct. Cyclization of the cis -aminoenal to pyrrole is much more rapid than in the trans case, thus it is less available for inhibitory reaction with the protein.