INHIBITION OF TYROSINASE BY KOJIC ACID AND HYDROQUINONE - BIOCHEMICAL AND STRUCTURAL INVESTIGATIONS

Tyrosinases are type-3 copper proteins ubiquitously distributed in all domains of life. They are mainly responsible for melanin formation in skin pigmentation and in fruit and vegetable browning. They catalyze the hydroxylation of phenols to form ortho-diphenols and the subsequent oxidation of o-diphenols to o-quinones that spontaneously polymerize to form melanin. Tyrosinase inhibitors are important for treating skin disorders, cosmetic whitening agents and preventing browning in agricultural produce. The most widely used and effective agents are hydroquinone (HQ) and kojic acid (KA) however, the literature contains contradicting mechanistic studies on these inhibitors.

In this research we work with tyrosinase from Bacillus megaterium (TyrBm) that was isolated and characterized in our lab. The activity of TyrBm on tyrosine (monophenol) and L-dopa (diphenol) was measured in the presence of KA and HQ. The results of this study showed a mixed inhibition mechanism on monophenolase activity in the presence of KA. The K_m and k_cat values obtained for the non-inhibited reaction were 0.037 mM and 5.52 s^-1, whereas with 0.1 mM KA the values were 1.132 mM and 0.95 s^-1, respectively. On diphenolase activity, a mixed inhibition mechanism was determined as well. The K_m and k_cat values changed from 0.18 mM and 20.32 s^-1 to 2.33 mM and 8.29 s^-1, respectively. The k_d value of the KA-TyrBm interaction was 0.3 nM as measured using Micro Scale Thermophoresis. A crystal structure with bound KA (2.5Å) revealed similar binding of the inhibitor to tyrosine. We also obtained an X-ray structure of TyrBm with bound HQ that supports the competitive inhibition mechanism. Informative inhibition mechanism and structural data can assist us in designing unique inhibitors such as peptides or peptidomimetics.