Action of tyrosinase on alpha and beta-arbutin: A kinetic study

The known derivatives from hydroquinone, a and beta-arbutin, are used as depigmenting agents. In this work, we demonstrate that the oxy form of tyrosinase (oxytyrosinase) hydroxylates a and beta-arbutin in ortho position of the phenolic hydroxyl group, giving rise to a complex formed by met-tyrosinase with the hydroxylated a or beta-arbutin. This complex could evolve in two ways: by oxidizing the originated o-diphenol to o-quinone and deoxy-tyrosinase, or by delivering the o-diphenol and met-tyrosinase to the medium, which would produce the self-activation of the system. Note that the quinones generated in both cases are unstable, so the catalysis cannot be studied quantitatively. However, if 3-methyl-2-benzothiazolinone hydrazone hydrochloride hydrate is used, the o-quinone is attacked, so that it becomes an adduct, which can be oxidized by another molecule of o-quinone, generating o-diphenol in the medium. In this way, the system reaches the steady state and originates a chromophore, which, in turn, has a high absorptivity in the visible spectrum. This reaction allowed us to characterize a and beta-arbutin kinetically as substrates of tyrosinase for the first time, obtaining a Michaelis constant values of 6.5 +/- 0.58 mM and 3 +/- 0.19 mM, respectively. The data agree with those from docking studies that showed that the enzyme has a higher affinity for beta-arbutin. Moreover, the catalytic constants obtained by the kinetic studies (catalytic constant = 4.43 +/- 0.33 s(-1) and 3.7 +/- 0.29 s(-1) for a and beta-arbutin respectively) agree with our forecast based on 13 C NMR considerations. This kinetic characterization of alpha and beta-arbutin as substrates of tyrosinase should be taken into account to explain possible adverse effects of these compounds.