Abstract
Mitochondrial monoamine oxidase was inactivated by o-mercaptobenzylamine ( 1) and o- ( 2) and p-methylthiobenzylamine ( 5). Experiments were carried out to provide evidence for possible mechanisms of inactivation. The corresponding o- ( 3) and p-hydroxybenzylamine ( 4) are not inactivators. Four radiolabeled analogues of 2 and 5, having radioactivity at either the methyl or benzyl groups, were synthesized, and all were shown to incorporate multiple equivalents of radioactivity into the enzyme. Inactivation in the presence of an electrophile scavenger decreased the number of molecules incorporated, but still multiple molecules became incorporated; catalase did not further reduce the number of inactivator molecules bound. Two inactivation mechanisms are proposed, one involving a nucleophilic aromatic substitution (S NAr) mechanism and the other a dealkylation mechanism. Evidence for both mechanisms is that inactivation leads to reduction of the flavin (oxidation of the inactivator), but upon denaturation the flavin is reoxidized, indicating that attachment is not at the flavin. A cysteine titration indicates the loss of four cysteines after inactivation and denaturation. Support for the S NAr mechanism was obtained by showing that o- and p-chlorobenzylamine also inactivate MAO. Chemical model studies were carried out that also support both S NAr and dealkylation mechanisms.
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