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.
Park
Kyung-Hee
KH
Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.
Park
Yong-Doo
YD
Lee
Jae-Rin
JR
Hahn
Hwa-Sun
HS
Lee
Sang-Jin
SJ
Bae
Chang-Dae
CD
Yang
Jun-Mo
JM
Kim
Dong-Eun
DE
Hahn
Myong-Joon
MJ
D000363:Agaricales / Q000201:enzymology*; D002614:Chelating Agents / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008982:Molybdenum / Q000494:pharmacology*; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011485:Protein Binding; D013329:Structure-Activity Relationship
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D003300:Copper; D008982:Molybdenum; C020809:tetrathiomolybdate; D014442:Monophenol Monooxygenase