Kinetic inactivation study of mushroom tyrosinase: intermediate detection by denaturants.
The unfolding and inhibition study of mushroom tyrosinase have been studied in the presence of different denaturants such as sodium dodecyl sulfate (SDS), guanidine hydrochloride (GdnHCl), and urea. The kinetic two-phase rate constants were commonly measured from semilogarithmic plots of the activity versus time, which resolved into two straight lines, indicating that the inactivation process consisted of fast and slow phases as a first-order reaction. This result also implied that transient partially folded intermediate existed during tyrosinase unfolding pathway. Mushroom tyrosinase had different behaviors to denaturants in regard with: noncooperative binding manner by SDS while cooperative interactions by GdnHCl and urea; in equilibrium state, SDS-micelle never completely inactivated enzyme activity while GdnHCl has single step denaturation and urea induced a typical transition-like process. Various kinetic parameters for each denaturant were calculated and the possible unfolding pathway scheme was discussed.
Park
Yong-Doo
YD
Clinical Research Center, Samsung Biomedical Research Institute, Seoul 135-710, Korea.
Jung
Jae-Yong
JY
Kim
Do-Won
DW
Kim
Won-Serk
WS
Hahn
Myong-Joon
MJ
Yang
Jun-Mo
JM
D000364:Agaricus / Q000201:enzymology*; D002384:Catalysis / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D012967:Sodium Dodecyl Sulfate; D014508:Urea; D014442:Monophenol Monooxygenase; D019791:Guanidine