Evidence for sulfhydryl groups at the active site of catechol-O-methyltransferase

Earlier studies using affinity labeling reagents have suggested the existence of two nucleophilic groups at the active site of catechol-O-methyltransferase (S-adenosyl-L-methionine:catechol O-methyltransferase, EC 2.1.1.6). Both nucleophilic residues are critical for catalytic activity. In an effort to elucidate the nature of these residues and to further characterize the relationship between the chemical structure and the catalytic function of this enzyme, inactivation studies using N-ethylmaleimide were undertaken. Inactivation of the enzyme by N-ethylmaleimide under pseudo first-order conditions exhibited a non-linear relationship between the log of the fraction of enzyme activity remaining and preincubation time. Kinetic analysis of this inactivation process suggested the modification by N-ethylmaleimide of two residues at the active site of the enzyme, both crucial for catalytic activity. Detailed analysis of the inactivation process including substrate protection studies, pH profiles of inactivation, and incorporation studies using N-ethyl[2,3-14C2]maleimide provided additional evidence to support this conclusion.