Discrimination between Alternative Substrates and Inhibitors of Tyrosinase

Ortiz-Ruiz, Carmen Vanessa; García-Molina, Maria del Mar; Tudela, José; Pérez-Ruíz, Tomás; Garcı́a-Cánovas, Francisco

doi:10.1021/jf5051816

Cited by 32 publications

(28 citation statements)

References 38 publications

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“…Regarding the diphenolase activity, the effect of these compounds is depicted in S3 (α-arbutin) and S3 Inset (β-arbutin) Fig, which point to an apparent competitive inhibition and values of 4 ± 0.29 mM and 0.9 ± 0.05 mM, respectively. These data agree with the behaviour of an alternative substrate of tyrosinase [61,62]. …”

Section: Resultssupporting

confidence: 86%

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

et al. 2017

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The known derivatives from hydroquinone, α and β-arbutin, are used as depigmenting agents. In this work, we demonstrate that the oxy form of tyrosinase (oxytyrosinase) hydroxylates α and β-arbutin in ortho position of the phenolic hydroxyl group, giving rise to a complex formed by met-tyrosinase with the hydroxylated α or β-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 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.

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Section: Resultssupporting

confidence: 86%

“…Taking into account that the assay without arbutin does not have a lag period due to the addition of L-dopa (in catalytic amounts ([D] / [M] = 0.042 [1,61]), when α-arbutin is added, the activity rate of the enzyme on L-tyrosine varies (Fig 2A Inset). When the degree of inhibition ( i ) was calculated, a hyperbole was obtained (Fig 2A).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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“…and Supporting Information Fig. 1SI suggests that this compound is another alternative substrate of tyrosinase , rather than inhibitor or inactivator. It was therefore decided to test the possible activity of tyrosinase on BR.…”

Section: Resultsmentioning

confidence: 96%

4-n-butylresorcinol, a depigmenting agent used in cosmetics, reacts with tyrosinase

et al. 2016

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4-n-Butylresorcinol (BR) is considered the most potent inhibitor of tyrosinase, which is why it is used in cosmetics as a depigmenting agent. However, this work demonstrates that BR is a substrate of this enzyme. The E m (met-tyrosinase) form is not active on BR, but E ox (oxy-tyrosinase) can act on this molecule, hydroxylating it to o-diphenol. In turn, this is oxidized to an oquinone, which isomerizes to a red p-quinone. Thus, for tyrosinase to act on this compound, a mechanism to generate E ox in the medium is required, which can be achieved by means of hydrogen peroxide or ascorbic acid. A kinetic analysis of the proposed mechanism allows its kinetic characterization: catalytic constant k BR cat (8.49 6 0.20 s 21 ) and Michaelis-constant K BR M (60.26 6 8.76 lM). These findings are compared with those for other monophenolic substrates of tyrosinase. Studies of BR docking to the E m form of the enzyme show that the hydroxyl group in C-1 position is oriented toward the copper atom A (CuA), as in it is L-tyrosine. As regards E ox , BR is oriented with the carbon in C-6 position ready to be hydroxylated. The reaction of BR originates o-quinones, which isomerize to p-quinones, which in turn, could react with thiol compounds, a finding that could have important implications for pharmacology and the cosmetic industry. , initial p-quinone formation rate; V p2Q max , maximum action rate of tyrosinase on 4-n-butylresorcinol

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“…Furthermore, we suggest varying the conditions by adding reducing agents (to avoid polymerization or enzyme inactivation) or hydrogen peroxide. The latter is useful not only in omitting the lag phase of the hydroxylation reaction but also in detecting hydroxylase activity toward compounds that were previously reported to be tyrosinase inhibitors but in fact are hydroxylized and oxidized by tyrosinase (41) [e.g., arbutin and isoeugenol (42), phloridzin and phloretin (43)]. …”

Section: Reactivity Of Aus1 and Insights Into The Reaction Mechanism mentioning

confidence: 99%

Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

Molitor

Mauracher

Rompel

2016

Proc. Natl. Acad. Sci. U.S.A.

121

109

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Tyrosinases and catechol oxidases belong to the family of polyphenol oxidases (PPOs). Tyrosinases catalyze the o-hydroxylation and oxidation of phenolic compounds, whereas catechol oxidases were so far defined to lack the hydroxylation activity and catalyze solely the oxidation of o-diphenolic compounds. Aurone synthase from Coreopsis grandiflora (AUS1) is a specialized plant PPO involved in the anabolic pathway of aurones. We present, to our knowledge, the first crystal structures of a latent plant PPO, its mature active and inactive form, caused by a sulfation of a copper binding histidine. Analysis of the latent proenzyme's interface between the shielding C-terminal domain and the main core provides insights into its activation mechanisms. As AUS1 did not accept common tyrosinase substrates (tyrosine and tyramine), the enzyme is classified as a catechol oxidase. However, AUS1 showed hydroxylase activity toward its natural substrate (isoliquiritigenin), revealing that the hydroxylase activity is not correlated with the acceptance of common tyrosinase substrates. Therefore, we propose that the hydroxylase reaction is a general functionality of PPOs. Molecular dynamics simulations of docked substrate-enzyme complexes were performed, and a key residue was identified that influences the plant PPO's acceptance or rejection of tyramine. Based on the evidenced hydroxylase activity and the interactions of specific residues with the substrates during the molecular dynamics simulations, a novel catalytic reaction mechanism for plant PPOs is proposed. The presented results strongly suggest that the physiological role of plant catechol oxidases were previously underestimated, as they might hydroxylate their-so far unknown-natural substrates in vivo.crystal structure | tyrosinase | catechol oxidase | type III copper protein | mechanism

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Discrimination between Alternative Substrates and Inhibitors of Tyrosinase

Cited by 32 publications

References 38 publications

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

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

4-n-butylresorcinol, a depigmenting agent used in cosmetics, reacts with tyrosinase

Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

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