The inhibition kinetics and thermodynamic changes of tyrosinase via the zinc ion.
We found that Zn(2+) conspicuously inactivated tyrosinase in a mixed-type inhibition manner: the final level of residual activity was abolished at the equilibrium state with concentration of 0.25 mM Zn(2+). Changes of both K(m) and V(max) by various concentrations of Zn(2+) in Lineweaver-Burk plot were observed. To see whether Zn(2+) also induced conformational change of tyrosinase and how thermodynamical changes by ligand binding were occurred, the intrinsic fluorescence studies as well as calorimetric measurements were conducted. The results showed that the Zn(2+) binding to tyrosinase directly induced conformational change of tyrosinase, and the changes of thermodynamic parameters such as enthalpy (DeltaH), Gibbs free-energy (DeltaG), and entropy (DeltaS) were obtained as 60+/-7.0 kJ/mol, -14.54 kJ/mol and 248.53 J/(K mol), respectively. The inactivating effect of Zn(2+) on tyrosinase was completely prevented by incubation with bovine serum albumin, which has a Zn(2+) binding motif in its structure. We suggested that Zn(2+) ligand-binding affected the substrate's accessibility due to the conformational changes and thus, the complex type of inhibition has occurred with the calorimetric changes.
Han
Hong-Yan
HY
Department of Biology, College of Life Sciences, Soochow University, Suzhou 215123, PR China.
Zou
He-Chang
HC
Jeon
Jun-Yeong
JY
Wang
Ye-Jing
YJ
Xu
Wei-An
WA
Yang
Jun-Mo
JM
Park
Yong-Doo
YD
D000818:Animals; D002417:Cattle; D003300:Copper / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D007477:Ions; D007700:Kinetics; D008543:Melanins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008968:Molecular Conformation; D014442:Monophenol Monooxygenase / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D013816:Thermodynamics; D015032:Zinc / Q000737:chemistry*; D005719:gamma-Globulins / Q000737:chemistry
D007477:Ions; D008543:Melanins; D005719:gamma-Globulins; D003300:Copper; D014442:Monophenol Monooxygenase; D015032:Zinc