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

<div><p>The known derivatives from hydroquinone, α and β-arbutin, are used as depigmenting agents. In this work, we demonstrate that the <i>oxy</i> form of tyrosinase (oxytyrosinase) hydroxylates α and β-arbutin in <i>ortho</i> position of the phenolic hydroxyl group, giving rise to a complex formed by <i>met</i>-tyrosinase with the hydroxylated α or β-arbutin. This complex could evolve in two ways: by oxidizing the originated <i>o</i>-diphenol to <i>o</i>-quinone and <i>deoxy</i>-tyrosinase, or by delivering the <i>o</i>-diphenol and <i>met</i>-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 <i>o</i>-quinone is attacked, so that it becomes an adduct, which can be oxidized by another molecule of <i>o</i>-quinone, generating <i>o</i>-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 α and β-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 β-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 α and β-arbutin respectively) agree with our forecast based on 13 C NMR considerations. This kinetic characterization of α and β-arbutin as substrates of tyrosinase should be taken into account to explain possible adverse effects of these compounds.</p></div

Action of tyrosinase on TBC in the presence of D-Arb.

<p>The increase of absorbance was followed at 410 nm by means of a total oxygen consumption test, carried out in the presence of TBC and different concentrations of D-Arb (mM): a) 0, b) 0.1, c) 0.2 and d) 0.4. The rest of the experimental conditions were [<i>E</i>]₀ = 50 nM and [TBC]₀ = 1 mM. <b>Inset.</b> Spectrophotometric recordings of the action of tyrosinase on TBC and D-Arb. The experimental conditions were [<i>E</i>]₀ = 20 nM, [TBC]₀ = 0.5 mM and [D-Arb]₀ = 0.4 mM. The spectrophotometric recordings were made every 60 seconds.</p