Molecular mechanisms controlling the rate and specificity of catechol O-methylation by human soluble catechol O-methyltransferase

Molecular mechanisms determining the turn-over rate and specificity of cate chol O-methylation were studied by combining enzyme kinetic measurements, c omputational modeling of substrate properties and fitting ligands in a 3D m odel of the active site of the enzyme. Enzyme kinetic measurements were car ried out for 46 compounds, including most clinically used catechol drugs, b y using recombinant human soluble catechol O-methyltransferase (COMT). The most important mechanism decreasing the turnover rate and increasing affini ty was the electron withdrawing effect of substituents. Several other mecha nisms by which substituents affected reactivity and affinity were identifie d. Highest turnover rates were determined for unsubstituted catechol and py rogallol. Pyrogallol derivatives generally seemed to be more specific subst rates than catechols. Catecholestrogens were the most specific endogenous s ubstrates, whereas catecholamines were rather poor substrates. Among the ca techol drugs used in the L-DOPA treatment of Parkinson's disease, the COMT inhibitors entacapone and tolcapone were not methylated, whereas the DOPA d ecarboxylase inhibitor benserazide was 15 times more specific substrate tha n L-DOPA, the target of COMT inhibition. The structure-activity relationshi ps found allow the prediction of reactivity, affinity, and specificity with useful accuracy for catechols with a wide range of structures and properti es. The knowledge can be used in the evaluation of metabolic interactions o f endogenous catechols, drugs and dietary catechols, and in the designing o f drugs with the catechol pharmacophore.