Abstract

Various polyphenols, including 6-hydroxydopamine, 6-hydroxydopa, 5-hydroxydopamine, N¹-DL-seryl-N²(2,3,4,-trihydroxybenzyl)hydrazine (RO4-4602), pyrogallol, and 5,6-dihydroxyindole, have been found to inactivate the enzyme catechol O-methyltransferase irreversibly. This inactivation could be prevented by including an antioxidant in the preliminary incubation mixture or by excluding oxygen. However, catalase did not protect the enzyme from inactivation. The mechanism of 6-hydroxydopamine inactivation was investigated, and it was observed that 6-hydroxydopamine p-quinone the initial oxidation product of 6-hydroxydopamine, was as effective as 6-hydroxydopamine in inactivating the enzyme. However, 2-hydroxy-5-methyl-1,4-benzoquinone, which is not able to undergo a cyclization reaction to the aminochrome system as is 6-hydroxydopamine p-quinone, was not effective in inactivating this enzyme. This latter observation, as well as data from kinetic experiments, suggested that the aminochrome product resulting from cyclization of 6-hydroxydopamine p-quinone was probably the species reacting with the enzyme. This was further substantiated by the observation that 5,6-dihydroxyindole, which when air-oxidized forms a similar aminochrome system, also produced complete inactivation of the enzyme. Kinetic experiments, using adrenochrome as a model aminochrome compound, suggested that enzyme inactivation occurred by a unimolecular reaction within a dissociable complex rather than by a nonspecific bimolecular reaction. Substrate protection studies suggested that one of the active sites of the enzyme was involved in this inactivation. The results of the present experiments provide a useful model system for studying the interaction of 6-hydroxydopamine with proteins.