Effect of thiohydroxyl compounds on tyrosinase: inactivation and reactivation study.
An unusual thioether bridge (Cys-His) has been detected at the active site of mushroom tyrosinase, and the effects of thiohydroxyl compounds such as dithiothreitol (DTT) and beta-mercaptoethanol (beta-ME) on Cu2+ at the active site have been elucidated. Treatment with DTT and beta-ME on mushroom tyrosinase completely inactivated 3,4-dihydroxyphenylalanine oxidase activity in a dose-dependent manner. Sequential kinetic studies revealed that DTT and beta-ME caused different mixed-type inhibition mechanisms: the slope-parabolic competitive inhibition (Ki = 0.143 mM) by DTT and slope-hyperbolic noncompetitive inhibition (Ki = 0.0128 mM) by beta-ME, respectively. Kinetic Scatchard analysis consistently showed that mushroom tyrosinase had multiple binding sites for DTT and beta-ME with different affinities. Reactivation study of inactivated enzyme by addition of Cu2+ confirmed that DTT and beta-ME directly bound with Cu2+ at the active site. Our results may provide useful information regarding interactions of tyrosinase inhibitor for designing an effective whitening agent targeted to the tyrosinase active site.
Park
Yong-Doo
YD
Clinical Research Center, Samsung Biomedical Research Institute, Seoul 135-710, Korea.
Lee
Su-Jin
SJ
Park
Kyung-Hee
KH
Kim
So-yeon
SY
Hahn
Myong-Joon
MJ
Yang
Jun-Mo
JM
D000363:Agaricales / Q000201:enzymology; D002413:Cations, Divalent; D003300:Copper / Q000737:chemistry / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000737:chemistry / Q000494:pharmacology; D004229:Dithiothreitol / Q000737:chemistry / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004793:Enzyme Reactivators / Q000737:chemistry; D007700:Kinetics; D008623:Mercaptoethanol / Q000737:chemistry / Q000494:pharmacology*; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013440:Sulfides / Q000737:chemistry
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D004793:Enzyme Reactivators; D013440:Sulfides; D008623:Mercaptoethanol; D003300:Copper; D004228:Dithionitrobenzoic Acid; D014442:Monophenol Monooxygenase; D004229:Dithiothreitol