Kinetic, Thermodynamic and Structural Studies of Native and N-Bromosuccinimide-Modified Mushroom Tyrosinase

Mushroom tyrosinase (MT) as a metalloenzyme is a good model for mechanistic studies of melanogenesis. To recognize the mechanism of MT action, it is important to investigate its inhibition, activation, mutation, and modification properties. In this study, the chemical modification of MT tryptophan residues was carried out by using N-bromosuccinimide (NBS) and then, the activity, stability, and structure of the native and modified enzymes were compared. Chemical modification of MT tryptophan residues was accomplished by enzyme incubation with different concentrations of NBS. The relative activity of native and modified MT was investigated through catecholase enzyme reaction in presence of dihydroxyphenylalanine (L-Dopa) as substrate. Thermodynamic parameters including standard Gibbs free energy change (∆G25°C) and Melting temperature (Tm) were obtained from thermal denaturation of the native and modified enzymes. The circular dichroism and intrinsic fluorescence techniques were used to study secondary and tertiary structure of MT, respectively. All experiments were conducted in 2015 in biophysical laboratory of Qazvin University of Medical Sciences and Islamic Azad University, Science and Research Branch, Tehran. The relative activity reduced from 100% for native enzyme to 10%, 7.9%, and 6.4% for modified MT with different NBS of concentrations 2, 10, and 20 mM, respectively. Thermal instability of modified enzyme was confirmed by decreased Tm and ∆G25°C values after modification. In accordance with kinetic and thermodynamic results, the lower stability of modified MT was observed from the changes occurred on its secondary and tertiary structures. Chemical modification of tryptophan residues with NBS reduces the activity and stability of MT simultaneously with its structural change. Thus, this study emphasizes the crucial role of tryptophan residues in the structure-function relationship of MT enzyme.