Catechol O-methyltransferase. 7. Affinity labeling with the oxidation products of 6-aminodopamine

Abstract

6-Aminodopamine (6-NH2DA) and various analogs of 6-NH2DA were evaluated for their ability to inactivate purified catechol O-methyltransferase (COMT) in vitro. The inactivation of COMT by these agents could be prevented by including an antioxidant in the preincubation mixture or by excluding O2; catalase did not protect the enzyme from inactivation. Substrate protection studies and kinetic studies suggested that the loss of enzyme activity resulted from the alkylation of an amino acid residue at the active site of COMT by the quinoid type products which were generated upon air oxidation of 6-NH2DA. The reactivity toward COMT of specific intermediates in the oxidation pathway of 6-NH2DA was studied by using various 6-NH2DA analogs. 6-Aminodopamine-p-quinone (6-NH2DAQ) is perhaps the most toxic species toward COMT. The aminochromes which are formed from 6-NH2DAQ are also effective in inactivating COMT. A useful model system is provided for observing the interaction of 6-NH2DA and its oxidation products with proteins; additional insight is also provided into the topography of the active site of COMT.