The unravelling of the complex pattern of tyrosinase inhibition

Abstract

Tyrosinases are responsible for melanin formation in all life domains. Tyrosinase inhibitors are used for the prevention of severe skin diseases, in skin-whitening creams and to avoid fruit browning, however continued use of many such inhibitors is considered unsafe. In this study we provide conclusive evidence of the inhibition mechanism of two well studied tyrosinase inhibitors, KA (kojic acid) and HQ (hydroquinone), which are extensively used in hyperpigmentation treatment. KA is reported in the literature with contradicting inhibition mechanisms, while HQ is described as both a tyrosinase inhibitor and a substrate. By visualization of KA and HQ in the active site of TyrBm crystals, together with molecular modeling, binding constant analysis and kinetic experiments, we have elucidated their mechanisms of inhibition, which was ambiguous for both inhibitors. We confirm that while KA acts as a mixed inhibitor, HQ can act both as a TyrBm substrate and as an inhibitor.

Figure 1. Lineweaver–Burk plots for the inhibition of TyrBm by KA.

(a) L-tyrosine (0.03–1.4 mM) in the presence of KA concentrations (mM): (a ●) 0, (b×) 0.025, (c ▲) 0.05, (d ♦) 0.075, (e ■) 0.1 and (b) L-dopa (0.15–2.0 mM) in the presence of KA concentrations (mM): (a ●) 0, (b×) 0.025, (c ▲) 0.04, (d ♦) 0.05, (e ■) 0.075, (f -) 0.1. All measurements were performed in heptaplicates.

Figure 2. Lineweaver–Burk plots for the inhibition of TyrBm by HQ.

(a) L-tyrosine (0.03–1.4 mM) in the presence of HQ concentrations (mM): (a ●) 0, (b×) 0.025, (c ▲) 0.075, (d ♦) 0.5 and (b) L-dopa (0.15–2.0 mM) in the presence of HQ concentrations (mM): (a ■) 0, (b ●) 0.025, (c ▲) 0.05, (d×) 0.075, (e ♦) 0.1. All measurements were performed in heptaplicates.

Figure 3. Structures of KA bound to TyrBm.

(a) KA is observed inside the active site (purple) and at the entrance to the active site (green) (3NQ1). Copper ions are presented as brown spheres. (b) KA at the entrance to the active site (green) (3NQ1) is stabilized by second shell residues (light brown sticks). (c) Superposition with TyrBm structures contain KA (purple) and L-dopa (orange, 4P6S) oriented through hydrophobic interactions with His208. All the structures presented in this work were generated using PyMOL.

Figure 4. KA’s center of mass cluster analysis along the PELE simulation.

Clusters are presented as spheres and colors indicate the potential of mean field ∆G. Absolute standard binding free energies (with volume corrections) are shown for the active site and the surface bound minima, along with the ligand crystallographic complexes (cyan sticks). The two copper ions are presented as brown spheres.

Figure 5. Structures of HQ bound in the active site of TyrBm.

(a) Superposition with TyrBm structures contain HQ in orientation 2 (green) and L-tyrosine (blue, 4P6R), which forms a hydrogen bond with Arg209. Zinc ions are presented as grey spheres. (b) HQ, in orientation 1 (teal), forms a hydrogen bond with Asn205 His residues are in white.