The basicity of an active-site water molecule discriminates between tyrosinase and catechol oxidase activity

Matoba, Yasuyuki; Oda, Kosuke; Muraki, Yoshimi; Masuda, Taro

doi:10.1016/j.ijbiomac.2021.05.206

Cited by 14 publications

(6 citation statements)

References 42 publications

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“…The protein expression, purification, and redox-sensitive anaerobic preparation of Ty from S. glaucescens , as well as the computational protein model setup, were performed following the methodology we previously reported ( 26 ). SF-Abs spectra were collected using a SX.19 Applied Photophysics instrument placed in an anaerobic glovebox, and rR spectra were collected using 351-nm excitation (Innova Sabre Ar+ laser, 20 mW, 77 K).…”

Section: Methodsmentioning

confidence: 99%

“…However, this ternary intermediate remains elusive, despite previous efforts ( 21 ). In addition, given that under the typical range of physiological conditions for melanin biosynthesis (pH 4 to 7), the native substrate, L-tyrosine (pK a = 10), is predominantly in its fully protonated monophenolic form ( 3 ), the protonation state of the substrate during its binding to oxy-Ty and the identity of the putative phenolic proton acceptor site in the ternary intermediate remain subjects of debate ( 22 – 26 ). Consequently, the lack of critical experimental evidence on the nature of the catalytic ternary intermediate of Ty significantly limits our mechanistic understanding of its monooxygenation reactivity toward monophenols.…”

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confidence: 99%

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Elucidation of the tyrosinase/O₂/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis

Kipouros

Stańczak

Ginsbach

et al. 2022

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

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Melanins are highly conjugated biopolymer pigments that provide photoprotection in a wide array of organisms, from bacteria to humans. The rate-limiting step in melanin biosynthesis, which is the ortho -hydroxylation of the amino acid L-tyrosine to L-DOPA, is catalyzed by the ubiquitous enzyme tyrosinase (Ty). Ty contains a coupled binuclear copper active site that binds O 2 to form a μ:η 2 :η 2 -peroxide dicopper(II) intermediate (oxy-Ty), capable of performing the regioselective monooxygenation of para -substituted monophenols to catechols. The mechanism of this critical monooxygenation reaction remains poorly understood despite extensive efforts. In this study, we have employed a combination of spectroscopic, kinetic, and computational methods to trap and characterize the elusive catalytic ternary intermediate (Ty/O 2 /monophenol) under single-turnover conditions and obtain molecular-level mechanistic insights into its monooxygenation reactivity. Our experimental results, coupled with quantum-mechanics/molecular-mechanics calculations, reveal that the monophenol substrate docks in the active-site pocket of oxy-Ty fully protonated, without coordination to a copper or cleavage of the μ:η 2 :η 2 -peroxide O-O bond. Formation of this ternary intermediate involves the displacement of active-site water molecules by the substrate and replacement of their H bonds to the μ:η 2 :η 2 -peroxide by a single H bond from the substrate hydroxyl group. This H-bonding interaction in the ternary intermediate enables the unprecedented monooxygenation mechanism, where the μ-η 2 :η 2 -peroxide O-O bond is cleaved to accept the phenolic proton, followed by substrate phenolate coordination to a copper site concomitant with its aromatic ortho -hydroxylation by the nonprotonated μ-oxo. This study provides insights into O 2 activation and reactivity by coupled binuclear copper active sites with fundamental implications in biocatalysis.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Elucidation of the tyrosinase/O₂/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis

Kipouros

Stańczak

Ginsbach

et al. 2022

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

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“…Recently, it has been supposed that both monophenols and o -diphenols bind to the same copper ion since a reorganization of the phenolate around the two copper ions occurs in the diphenolase form. The presence of one molecule of water bridged with Cu(II) and the absence of bonds among dioxygen and Cu(II) is representative of the met form of the protein involved in the diphenolase activity [ 27 ]. The crystal structure resolved with an inhibitor as kojic acid (KA) showed that this molecule is positioned at the entrance of the catalytic site, at 7 Å away from the copper center.…”

Section: Resultsmentioning

confidence: 99%

Antamanide Analogs as Potential Inhibitors of Tyrosinase

Honisch¹,

Gazziero²,

Dallocchio³

et al. 2022

IJMS

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The tyrosinase enzyme, which catalyzes the hydroxylation of monophenols and the oxidation of o-diphenols, is typically involved in the synthesis of the dark product melanin starting from the amino acid tyrosine. Contributing to the browning of plant and fruit tissues and to the hyperpigmentation of the skin, leading to melasma or age spots, the research of possible tyrosinase inhibitors has attracted much interest in agri-food, cosmetic, and medicinal industries. In this study, we analyzed the capability of antamanide, a mushroom bioactive cyclic decapeptide, and some of its glycine derivatives, compared to that of pseudostellarin A, a known tyrosinase inhibitor, to hinder tyrosinase activity by using a spectrophotometric method. Additionally, computational docking studies were performed in order to elucidate the interactions occurring with the tyrosinase catalytic site. Our results show that antamanide did not exert any inhibitory activity. On the contrary, the three glycine derivatives AG9, AG6, and AOG9, which differ from each other by the position of a glycine that substitutes phenylalanine in the parent molecule, improving water solubility and flexibility, showed tyrosinase inhibition by spectrophotometric assays. Analytical data were confirmed by computational studies.

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“…From the chemical structure of the two compounds shown in Figure 2 a,b, a hydroxyl group can be seen in position 4. Docking studies in Figure 5 and Figure 6 show that the two compounds bind with high affinity for the hydroxyl in position 4, and also the distance of the oxygens of oxy-tyrosinase to the ortho position is adequate for the electrophilic aromatic substitution reaction to occur ( Table 3 ) [ 40 , 41 , 42 ]. In this way, these compounds would possibly behave as alternative substrates to L-dopa.…”

Section: Resultsmentioning

confidence: 99%

The Relationship between the IC50 Values and the Apparent Inhibition Constant in the Study of Inhibitors of Tyrosinase Diphenolase Activity Helps Confirm the Mechanism of Inhibition

et al. 2022

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Tyrosinase is the enzyme involved in melanization and is also responsible for the browning of fruits and vegetables. Control of its activity can be carried out using inhibitors, which is interesting in terms of quantitatively understanding the action of these regulators. In the study of the inhibition of the diphenolase activity of tyrosinase, it is intriguing to know the strength and type of inhibition. The strength is indicated by the value of the inhibition constant(s), and the type can be, in a first approximation: competitive, non-competitive, uncompetitive and mixed. In this work, it is proposed to calculate the degree of inhibition (iD), varying the concentration of inhibitor to a fixed concentration of substrate, L-dopa (D). The non-linear regression adjustment of iD with respect to the initial inhibitor concentration [I]0 allows for the calculation of the inhibitor concentration necessary to inhibit the activity by 50%, at a given substrate concentration (IC50), thus avoiding making interpolations between different values of iD. The analytical expression of the IC50, for the different types of inhibition, are related to the apparent inhibition constant (KIapp). Therefore, this parameter can be used: (a) To classify a series of inhibitors of an enzyme by their power. Determining these values at a fixed substrate concentration, the lower IC50, the more potent the inhibitor. (b) Checking an inhibitor for which the type and the inhibition constant have been determined (using the usual methods), must confirm the IC50 value according to the corresponding analytical expression. (c) The type and strength of an inhibitor can be analysed from the study of the variation in iD and IC50 with substrate concentration. The dependence of IC50 on the substrate concentration allows us to distinguish between non-competitive inhibition (iD does not depend on [D]0) and the rest. In the case of competitive inhibition, this dependence of iD on [D]0 leads to an ambiguity between competitive inhibition and type 1 mixed inhibition. This is solved by adjusting the data to the possible equations; in the case of a competitive inhibitor, the calculation of KI1app is carried out from the IC50 expression. The same occurs with uncompetitive inhibition and type 2 mixed inhibition. The representation of iD vs. n, with n=[D]0/KmD, allows us to distinguish between them. A hyperbolic iD vs. n representation that passes through the origin of coordinates is a characteristic of uncompetitive inhibition; the calculation of KI2app is immediate from the IC50 value. In the case of mixed inhibitors, the values of the apparent inhibition constant of meta-tyrosinase (Em) and oxy-tyrosinase (Eox), KI1app and the apparent inhibition constant of metatyrosinase/Dopa complexes (EmD) and oxytyrosinase/Dopa (EoxD), KI2app are obtained from the dependence of iD vs. n, and the results obtained must comply with the IC50 value.

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The basicity of an active-site water molecule discriminates between tyrosinase and catechol oxidase activity

Cited by 14 publications

References 42 publications

Elucidation of the tyrosinase/O₂/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis

Elucidation of the tyrosinase/O₂/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis

Antamanide Analogs as Potential Inhibitors of Tyrosinase

The Relationship between the IC50 Values and the Apparent Inhibition Constant in the Study of Inhibitors of Tyrosinase Diphenolase Activity Helps Confirm the Mechanism of Inhibition

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The basicity of an active-site water molecule discriminates between tyrosinase and catechol oxidase activity

Cited by 14 publications

References 42 publications

Elucidation of the tyrosinase/O2/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis

Elucidation of the tyrosinase/O2/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis

Antamanide Analogs as Potential Inhibitors of Tyrosinase

The Relationship between the IC50 Values and the Apparent Inhibition Constant in the Study of Inhibitors of Tyrosinase Diphenolase Activity Helps Confirm the Mechanism of Inhibition

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Elucidation of the tyrosinase/O₂/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis

Elucidation of the tyrosinase/O₂/monophenol ternary intermediate that dictates the monooxygenation mechanism in melanin biosynthesis