Catechol O-methyltransferase. 9. Mechanism of inactivation by 6-hydroxydopamine

A series of methylated analogues of 6-hydroxydopamine (6-OHDA) has been synthesized and evaluated as irreversible inhibitors of catechol O-methyltransferase (COMT). These analogues have been prepared in an effort to elucidate the mechanism involved in the inactivation of this enzyme by 6-OHDA. The analogues prepared had methyl groups incorporated in the 2 and/or 5 positions of 6-OHDA so as to block nucleophilic attakc at these positions in the corresponding oxidation products [6-hydroxydopamine-p-quinone (6-OHDAQ), aminochromes I and II]. Such 2- and/or 5-methylated 6-OHDA analogues were found to be inhibitors of COMT with the inactivation apparently resulting from modification of an essential amino acid residue at the active site of the enzyme. The activity of these analogues as inhibitors of COMT argues against a mechanism involving a 1,4 Michael addition reaction by a protein nucleophile at the 2 or 5 positions on 6-OHDAQ or on the corresponding aminochromes. Instead, an alternative mechanism is proposed to explain these data, which involves attack of a protein nucleophile at the carbonyl group in the 6 position of 6-OHDAQ or at the imine functionality on aminochromes I and II. The results of the present experiments have provided insight into the mechanism involved in inactivation of COMT by 6-OHDA. In addition, this study has provided considerable insight into the chemical reactivity of the electrophilic species generated after oxidation of 6-OHDA.