A mechanism previously proposed for inactivation of monoamine oxidase (MAO) by N-cyclopropylbenzylamine (N-CBA) [Silverman, R. B., & Hoffman, S. J. (1980) J. Am. Chem. Soc. 102, 884-886] is revised. Inactivation of MAO by N-[1-3H]CBA results in incorporation of about 3 equiv of tritium into the enzyme and release of [3H]acrolein. Treatment of inactivated enzyme with benzylamine, a reactivator for N-CBA-inactivated MAO, releases only 1 equiv of tritium as [3H]acrolein concomitant with reactivation of the enzyme. Even after MAO is inactivated by N-[1-3H]CBA, the reaction continues. At pH 7.2, a linear release of [3H]acrolein is observed for 70 h, which produces 55 equiv of [3H]acrolein while 2.3 equiv of tritium is incorporated into the enzyme. At pH 9, only 3.5 equiv of [3H]acrolein is detected in solution after 96 h, but 40 equiv of tritium is incorporated into the enzyme, presumably as a result of greater ionization of protein nucleophiles at the higher pH. N-[1-3H]Cyclopropyl-α-methylbenzylamine (N-CaMBA) produces the same adduct as N-CBA but gives only 1-1.35 equiv of tritium bound after inactivation of the enzyme. Denaturation of labeled enzyme results in reoxidation of the flavin without release of tritium, indicating attachment is not to the flavin but rather to an amino acid residue. Enzyme inactivated with N-[l-3H]CaMBA is reactivated by benzylamine with the release of 1 equiv of [3H]acrolein, which must have come from an adduct attached to an active site amino acid residue. About half of the tritium remains bound to the enzyme if the N-[l-3H]CaMBA-inactivated enzyme is treated with a low concentration of sodium borohydride prior to benzylamine treatment. The adducts formed when N-CBA, N-CαMBA, and N-cyclopropyl-N-methylbenzylamine inactivate MAO appear to be identical. They have identical rates of reactivation by benzylamine at pH 7.2 and 9. These results suggest that N-CBA is oxidized by one electron to the amine radical cation followed by homolytic cyclopropyl ring cleavage and attachment to an active site radical, producing a 3-(amino acid residue)propanal adduct.
ASJC Scopus subject areas