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.

This work was supported by the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities, grant number 419/15 and by the Gurwin Fund for Scientific Research. We also acknowledge the Russell-Berrie Nanotechnology Institute (RBNI) at the Technion for supporting this research. This research benefited from use of the Technion Center of Structural Biology facility of the Lorry I. Lokey Center for Life Sciences and Engineering. We thank the staff of the European Synchrotron Radiation Facility (beamlines ID14-4 and ID 29) for provision of synchrotron radiation facilities and assistance. V.G. would like to thank the OxiDesign Spanish project (CTQ2013-48287-R) and D.L. acknowledges support from the Spanish Severo Ochoa Program (SEV-2011-00067).

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