Multisubstrate‐based system: a kinetic mechanism study of catechol‐ <i>O</i> ‐methyltransferase

Catechol-O-methyltransferase (COMT, EC 2.1.1.6) can transfer the methyl group from S-adenosyl-l-methionine (SAM) to one of the hydroxyl groups of a catechol substrate in the presence of Mg²⁺. However, there is no consensus view of the kinetic mechanism of COMT involving multiple substrates. Further progress requires the development of methods for determining enzyme kinetic behavior and the binding mode of ligands to the protein. Here, we establish a multisubstrate kinetic system covering the fluorescence and mass spectrometry techniques to quantify the products in a COMT-catalyzed reaction. The catechol substrate, 3-BTD, can be methylated by COMT to form a single product, 3-BTMD, with a sensitive fluorescence response and the conversion of SAM to S-adenosyl-l-homocysteine (SAH) was monitored by LC-MS/MS. The kinetic assays suggested that the reaction occurred via an ordered sequential mechanism, in which SAM first bound to COMT, followed by the addition of Mg²⁺ and 3-BTD. The chemical step involved a quaternary complex of COMT-SAM-Mg²⁺-3-BTD, followed by the ordered dissociation of 3-BTMD, Mg²⁺, and SAH. In cooperation with molecular dynamics simulation, the binding of COMT to Mg²⁺ induced a shape change in the catechol-binding site, which accommodated 3-BTD binding and facilitated catalysis. These findings provide new insights into the kinetic mechanism of COMT, contributing to the development of previously undescribed functional COMT ligands.