Integrated study of the mechanism of tyrosinase inhibition by baicalein using kinetic, multispectroscopic and computational simulation analyses.

Controlling the activity of tyrosinase, which plays an important role in melanin synthesis, is essential endeavor in the field of medicine, food, cosmetics, and agriculture. Baicalein is a widely available inhibitor of tyrosinase. The mechanism of this inhibition was studied through an integrated approach coupling enzyme kinetics, multiple types of spectroscopy and computational simulation. Baicalein conspicuously inhibited the diphenolase activity with an IC value of 0.11 mM. The inhibtion kinetics indicated that baicalein was a mix-type tight binding inhibitor with a K of 0.17 mM and an α value of 0.56. UV absorption spectra showed that a complex formed. Baicalein quenched the fluorescence via both static and dynamic quenching though a single binding site with a binding constant of 278,000 M. Thermodynamic parameters suggested spontaneous binding through hydrogen bonding and van der Waals forces. The binding distance was estimated at 3.41 nm. Phase diagram analysis suggested baicalein induced at least two structurally distinct intermediates. Synchronous fluorescence and three-dimensional fluorescence spectra showed blue shift of the maximum emission wavelength. The content of α-helix decreased from 32.67% to 29.00% according to circular dichroism spectra. It was revealed that baicalein induced conformational changes in tyrosinase and enhanced the hydrophobicity of the microenvironment around Trp residues. Docking simulations predicted that baicalein mainly bound tyrosinase via its Met280 residue. This research will provide reliable basis concerning the inhibitory mechanism of baicalein against tyrosianse, and therefore eventually contribute to development of anti-tyrosinase agents.