The effect of modifed tryptophan residues on kinetic, thermodynamic and struture of mashroom tyrosinase

Background: Mashroom Tyrosinase (MT) is a metalloenzyme and is a good model in mechanistic point of view for the study of tyrosinase as the key enzyme in melanogenesis. To recognizing mechanism of MT’s activity its inhibition, activation, mutation and modification are important methods of investigations.Objective: Here, the chemical modification of MT tryptophan residues have been done by N-bromosuccinimide (NBS) and the activity, stability and structure of native and modified enzyme were comprised.Material and methods: Chemical modification of MT tryptophan residues induced by enzyme incubation with different concentrations of NBS. The relative activity of native and modified MT were investigated through catecholase reaction of enzyme by L-Dopa as substrate. Thermodynamic parameters including: ΔG25°C and Tm obtained from thermal denaturation of the native and modified enzyme. The circular dichroism and intrinsic fluorescence techniques have been used for study of secondary and tertiary structure of MT respectively.Results: The relative activity reduced from 100% for native to 10%, 7.9% and 6.4% for modified MT with different 2, 10 and 20 mM concentrations of NBS, respectively. Thermal stability of modified enzyme were emphasized by decreasing in Tm and ΔG25°C values. In accordance with kinetic and thermodynamic studies the lower stability of modified MT was observed from the changes of its secondary and tertiary structures.Conclusions: Chemical modification of tryptophan residues with NBS reduces activity and stability of MT in line with its structural change. So this study emphasized to the crucial role of tryptophan residues in the structure-function relationship of the enzyme.

Keywords:

Chemical Modification , Mashroom Tyrosinase , N-Bromosuccinimide , Kinetic , Structure