Inhibition kinetics of mushroom tyrosinase by copper-chelating ammonium tetrathiomolybdate.

With a strategy of chelating coppers at tyrosinase active site to detect an effective inhibitor, several copper-specific chelators were applied in this study. Ammonium tetrathiomolybdate (ATTM) among them, known as a drug for treating Wilson's disease, turned out to be a significant tyrosinase inhibitor. Treatment with ATTM on mushroom tyrosinase completely inactivated enzyme activity in a dose-dependent manner. Progress-of-substrate reaction kinetics using the two-step kinetic pathway and dilution of the ATTM revealed that ATTM is a tight-binding inhibitor and high dose of ATTM irreversibly inactivated tyrosinase. Progress-of-substrate reaction kinetics and activity restoration with a dilution of the ATTM indicated that the copper-chelating ATTM may bind slowly but reversibly to the active site without competition with substrate, and the enzyme-ATTM complex subsequently undergoes reversible conformational change, leading to complete inactivation of the tyrosinase activity. Thus, inhibition by ATTM on tyrosinase could be categorized as complexing type of inhibition with a slow and reversible binding. Detailed analysis of inhibition kinetics provided IC50 at the steady-state and inhibitor binding constant (K(I)) for ATTM as 1.0+/-0.2 microM and 10.65 microM, respectively. Our results may provide useful information regarding effective inhibitor of tyrosinase as whitening agents in the cosmetic industry.