Catechol O-methyltransferase. 11. Inactivation by 5-hydroxy-3-mercapto-4-methoxybenzoic acid

5-Hydroxy-3-mercapto-4-methoxybenzoic acid was synthesized as a potential affinity-labeling reagent for catechol O-methyltransferase (COMT, EC 2.1.1.6). This compound was shown to produce noncompetitive inhibition of COMT when assayed in the presence or absence of the reducing agent, dithiothreitol (DTT). If COMT was assayed in the absence of DTT, the compound was shown to be a more potent inhibitor (Kis = 59.9 +/= 15.9 mumol; Kii = 30.2 +/- 5.8 mumol) than if the assays were conducted in the presence of the reducing agent (Kis = 1140 +/- 233 mumol; Kii = 743 +/- 141 mumol). The potent inhibitory effects produced in the absence of DTT could be partially reversed by the addition of DTT to the incubation mixture or by dialysis of the modified enzyme against DTT -containing buffer. These data suggest that in the absence of DTT, 5-hydroxy-3-mercapto-4-methoxybenzoic acid serves as an affinity-labeling reagent for COMT by reaction of the 3-mercapto group of the ligand with an active-site sulfhydryl group. This ligand-protein disulfide bond can be reduced with DTT with subsequent reversal of the inhibitory effects.