Understanding the inhibitory mechanism of tea polyphenols against tyrosinase using fluorescence spectroscopy, cyclic voltammetry, oximetry, and molecular simulations.

Inhibiting the activity of tyrosinase is a very effective and safe way to prevent enzymatic browning in food and to resist pests in agriculture. Tea polyphenols (TPs), regarded as safe and non-toxic food additives, have been reported due to their potential inhibitory capability against tyrosinase, but their ambiguous inhibitory mechanisms have severely limited their application. In the present work, fluorescence spectroscopy, cyclic voltammetry (CV), oximetry and molecular simulation approaches were employed to shed light on the underlying inhibitory mechanisms of TPs with different structures including (+)-catechin, (-)-epicatechin gallate (ECG) and (-)-epigallocatechin gallate (EGCG) against tyrosinase. Fluorescence spectra show that the three TPs are capable of binding tyrosinase with a molar proportion of 1 : 1. The analysis of CV curves and oxygen utilization suggests that these three TPs can be oxidized by tyrosinase, revealing that these three TPs are suicide inhibitors of tyrosinase. Furthermore, ECG and catechin make tyrosinase irreversibly inactivated due to their catechol group (ring B) being catalyzed by tyrosinase through a cresolase-like pathway, while EGCG inhibits the activity of tyrosinase by competing with or delaying the oxidation of substrate. Molecular simulations further confirm that ring B of ECG and catechin makes a significant contribution to tyrosinase inhibitory activities, and has a direct interaction with the coupled binuclear copper ions in the optimal orientation required by the cresolase-like pathway.