INHIBITION OF CATECHOL-O-METHYLTRANSFERASE BY 1-VINYL DERIVATIVES OF NITROCATECHOLS AND NITROGUAIACOLS - KINETICS OF THE IRREVERSIBLE INHIBITION BY 3-(3-HYDROXY-4-METHOXY-5-NITRO BENZYLIDENE)-2,4-PENTANEDIONE

lt is well known that activated alkene derivatives react with thiol gr oups according to a Michael's addition reaction. On the basis of the p resence of at least one thiol group essential for the activity of cate chol-O-methyltransferase (COMT), several 1-vinyl derivatives of nitroc atechol and nitroguaiacol were synthesized and tested as potential irr eversible active site-directed inhibitors of COMT. All the synthesized products were potent inhibitors of partially purified pig liver COMT. However, the inhibition was reversible in most cases, with the except ion of roxy-4-methoxy-5-nitrobenzylidene-2,4-pentanedione (compound 2) which inhibited COMT in an irreversible manner. When the inhibition o f COMT was measured as a function of the length of time of pre-incubat ion with 2, biphasic kinetics were observed, suggesting the modificati on of at least two thiol groups which are essential for COMT activity. The analysis of the two parts of the inhibition curve as a function o f the inhibitor concentration showed that compound 2 modified the more reactive group in a non-specific manner, while it behaved as an activ e site-directed inhibitor on the second slow-reacting thiol group. Imp ortantly, a saturating concentration of S-adenosyl-L-methionine (AdoMe t) in the pre-incubation mixture gave pseudo-first order kinetics. sug gesting total protection of one thiol group. Magnesium ions had no eff ect on the protection of COMT by AdoMet. In the presence of 3,5-dinitr ocatechol (DNC) slight protection of COMT was observed; when the inact ivation of both groups was analysed independently, protection of the s pecifically modified group was detected, while the reaction with the o ther group was faster in the presence of DNC. When both AdoMet and DNC were present, inactivation of COMT by 2 was not observed, suggesting that both reacting groups are located at or near the active site.