The inhibition kinetics of rat liver and duodenum soluble catechol-O-methyltransferase (COMT) with a disubstituted catechol OR-462 was studied. After preincubation of the enzyme and inhibitor in the presence of magnesium and S-adenyosylmethionine, an inhibition about thirty times greater than that without preincubation was observed. Reversible tight-binding inhibition was demonstrated with Ki values of 0.7 nM and 1.0 nM for liver and duodenum enzyme, respectively. Km values of 53.4 microM and 56.9 microM for substrate 3,4-dihydroxybenzoic acid and 23.0 microM and 17.5 microM for S-adenosylmethionine were calculated for liver and duodenum enzyme, respectively. A catalytic number of 24/min for liver soluble COMT was calculated.