Phloretin as both a substrate and inhibitor of tyrosinase: Inhibitory activity and mechanism

Tyrosinase is the rate-limiting enzyme for controlling the production of melanin in the human body, and overproduction of melanin can lead to a variety of skin disorders. In this paper, the inhibitory kinetics of phloretin on tyrosinase and their binding mechanism were determined using spectroscopy, molecular docking, antioxidant assays and chromatography. The spectroscopic results indicate that phloretin reversibly inhibits tyrosinase in a mix-type manner through a multiphase kinetic process with the IC₅₀ of 169.36 μmol/L. It is shown that phloretin has a strong ability to quench the intrinsic fluorescence of tyrosinase mainly through a static quenching procedure, suggesting that a stable phloretin-tyrosinase complex is generated. Molecular docking results suggest that the dominant conformation of phloretin binds to the gate of the active site of tyrosinase. Moreover, the antioxidant assays demonstrate that phloretin has powerful antioxidant capacity and has the ability to reduce o-dopaquinone to L-dopa just like ascorbic acid. Interestingly, the results of spectroscopy and chromatography indicate that phloretin is a substrate of tyrosinase but also an inhibitor. The possible inhibitory mechanism is proposed, which will be helpful to design and search for tyrosinase inhibitors.