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

Abstract

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.

Fig. 1. Molecular structures of TPs.

Fig. 2. Emission spectra of tyrosinase excited at 274 nm wavelength in the presence of different concentrations of catechin (A), ECG (B) and EGCG (C). Inset: Stern–Volmer plots describe the tyrosinase quenching caused by binding with TPs.

Fig. 3. Cyclic voltammetry (CV) curves of 20 μM catechin (A), ECG (B) and EGCG (C) measured on glassy carbon electrode modified by MWCNT and tyrosinase at 50 mV s⁻¹ in pH 6.5 phosphate buffer. The oxidation peaks are marked.

Fig. 4. The time-dependent curves of oxygen utilization in the oxidation of (A) TPs catalyzed by tyrosinase and (B) l-DOPA catalyzed by the remaining tyrosinase. The initial reaction velocities of tyrosinase in oxidizing l-DOPA are illustrated by the slope of oxygen utilization curves at the starting reaction time.

Fig. 5. Cresolase-like and diphenolase-like catalytic pathways of tyrosinase, which catalyze the monophenols (black) and diphenols (blue), respectively. Both catalytic mechanisms were reported by Solomon et al. and Land et al. and re-depicted here.

Fig. 6. The optimal binding structures of catechin (A), ECG (B) and EGCG (C) with tyrosinase clustered out from MD simulation trajectories. The dicopper ions in the catalytic center are represented as orange spheres, tyrosinase and the skeleton of TPs are shown using cartoon and ball-and-stick model, respectively. The included angles between the ring B of TPs and the connected line of two copper ions (illustrated in D) and the distances between the oxygen atoms of ring B and copper ions were labeled.