Mechanism-based inactivation of mitochondrial monoamine oxidase by N-(1-methylcyclopropyl)benzylamine.

Three different radioactively labeled N-(1-methylcyclopropyl)benzylamines [N-(1-Me)CBA] were synthesized and used to show which atoms of the inactivator remain bound to monoamine oxidase (MAO) after inactivation. Organic chemical reactions were employed to elucidate the structure of the enzyme adduct and clarify the mechanism of inactivation. Following inactivation and dialysis, the benzyl substituent is lost, but the methyl group and cyclopropyl carbons remain attached to the enzyme even after further dialysis against solutions containing 1 mM benzylamine or 8 M urea. Treatment of inactivated enzyme with sodium cyanoborohydride prior to dialysis results in the retention of the benzyl group, suggesting an imine linkage. One hydride from sodium boro[3H]hydride is incorporated into the dialyzed inactivated enzyme consistent with a ketone functional group. When Pronase-digested N-(1-Me)CBA-inactivated MAO is treated with basic potassium triiodide, iodoform is isolated, indicating the presence of a methyl ketone. During inactivation, the optical spectrum of the covalently bound active site flavin changes from that of oxidized to reduced flavin. After urea denaturation, the flavin remains reduced, suggesting covalent linkage of the inactivator to the cofactor. On the basis of previous results [Silverman, R. B., Hoffman, S. J., & Catus, W. B., III (1980) J. Am. Chem. Soc. 102, 7126-7128], it is proposed that the mechanism of inactivation involves transfer of one electron from N-(1-Me)CBA to the flavin, resulting in an amine radical cation and a flavin radical. Then, either the cyclopropyl ring is attacked by the flavin radical or the cyclopropyl ring opens, and the radical generated is captured by the flavin radical. The product of this mechanism is the imine of benzylamine and 4-flavinyl-2-butanone, the proposed enzyme-inactivator adduct.