The effect of 7,8,4′-trihydroxyflavone on tyrosinase activity and conformation: Spectroscopy and docking studies

Tyrosinase is a ubiquitous enzyme that plays an essential role in the production of melanin. Effective inhibitors of tyrosinase have extensive applications in the medical, cosmetic and food industries. In this study, a combination of enzyme kinetics, ultraviolet (UV)-visible absorption, fluorescence spectroscopic techniques and a computational simulation method was used to characterize the inhibitory mechanism of 7,8,4-trihydroxyflavone on tyrosinase. 7,8,4 '-Trihydroxyflavone was found to strongly inhibit the oxidation of l-DOPA by tyrosinase with an IC50 value of 10.31 +/- 0.41 mu M. The inhibitory mechanism was determined to be reversible and non-competitive with a K-i of 9.50 +/- 0.40 mu M. The UV absorption spectra showed that 7,8,4''-trihydroxyflavone could chelate with copper ions and form a complex with tyrosinase. The intrinsic fluorescence of tyrosinase was quenched by 7,8,4-trihydroxyflavone through a static quenching mechanism. 7,8,4 '-Trihydroxyflavone was found to occupy a single binding site with a binding constant of 7.50 +/- 1.20 x 10(4)M(-1) at 298K. The conformation of tyrosinase changed, and the microenvironment became more hydrophilic after 7,8,4 '-trihydroxyflavone binding. Thermodynamics parameters indicated that the binding was a spontaneous process and involved hydrogen bonds and van der Waals forces. The binding distance was evaluated to be 4.54 +/- 0.05nm. Docking simulation analysis further authenticated that 7,8,4'-trihydroxyflavone could form hydrogen bonds with the residues His244 and Met280 within the tyrosinase active site. Our results will contribute to further understanding of the inhibitory mechanisms of 7,8,4'-trihydroxyflavone against tyrosinase and will facilitate future screening for tyrosinase inhibitors.