Catechol O-methyltransferase. 6. Affinity labeling with N-haloacetyl-3,5-dimethoxy-4-hydroxyphenylalkylamines

Several N-acyl-3,5-dimethoxy-4-hydroxyphenylalkylamines have been synthesized and evaluated for their ability to inactive catechol 9-methyltransferase (COMT). N-iodoacetyl-3,5-dimethoxy-4-hydroxyphenylethylamine was found to rapidly and irreversibly inactivate this enzyme. The corresponding N-bromoacetyl derivative also produced inactivation of COMT but at a slower rate than the N-iodoacetyl derivative. The N-acetyl and N-fumaryl derivatives were completely inactive. The inactivation of COMT by these reagents appears to proceed by a unimolecular reaction within a dissociable complex rather than by a nonspecific bimolecular reaction. The proximity of the amino acid residue being modified relative to the site which binds the aromatic portion of these inhibitors was determined using N-iodoacetylphenylakylamines of varying chain length. The number of methylene carbons separating the aromatic ring and the iodoacetamide moiety in these inhibitors did not greatly influence the binding to COMT nor did it affect how rapidly the enzyme was inactivated. From these observations it was concluded that the amino acid moiety being modified by this class of affinity labeling reagents must be relatively close to or part of the site which binds the aromatic region of these inhibitors.