Reversible oxygenation of tyrosinase

doi:10.1016/S0006-291X(72)80223-7

Biochemical and Biophysical Research Communications

Volume 46, Issue 2, 31 January 1972, Pages 878-884

https://doi.org/10.1016/S0006-291X(72)80223-7 Get rights and content

Summary

When mushroom tyrosinase reacts aerobically with molar equivalents of H₂O₂, a hitherto unobserved absorption spectrum develops, apparently according to first order kinetics, and then decays. The spectrum has peaks, ɛ345 nm = 10 × 10³ M_Cu⁻¹ cm⁻¹ and ɛ600 nm = 7 × 10² M_Cu⁻¹ cm⁻¹. Both bands disappear upon deoxygenation, and reappear upon reoxygenation, in a cyclic manner. The reaction system can be interpreted in terms of four states of the enzyme in the reaction: T_r + H₂O₂ ⇌ T′ ⇌ T″ ⇌ T‴ + O₂, where T_r is the resting state, T′ a kinetically necessary intermediate, T″ an oxygenated state, and T‴ the cuprous enzyme.

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Tyrosinase-catalyzed hydroxylation of hydroquinone, a depigmenting agent, to hydroxyhydroquinone: A kinetic study
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Hydroquinone (HQ) is used as a depigmenting agent. In this work we demonstrate that tyrosinase hydroxylates HQ to 2-hydroxyhydroquinone (HHQ). Oxy-tyrosinase hydroxylates HQ to HHQ forming the complex met-tyrosinase-HHQ, which can evolve in two different ways, forming deoxy-tyrosinase and p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone or on the other way generating met-tyrosinase and HHQ. In the latter case, HHQ is rapidly oxidized by oxygen to generate 2-hydroxy-p-benzoquinone, and therefore, it cannot close the enzyme catalytic cycle for the lack of reductant (HHQ). However, in the presence of hydrogen peroxide, met-tyrosinase (inactive on hydroquinone) is transformed into oxy-tyrosinase, which is active on HQ. Similarly, in the presence of ascorbic acid, HQ is transformed into 2-hydroxy-p-benzoquinone by the action of tyrosinase; however, in this case, ascorbic acid reduces met-tyrosinase to deoxy-tyrosinase, which after binding to oxygen, originates oxy-tyrosinase. This enzymatic form is now capable of reacting with HQ to generate p-hydroxy-o-quinone, which rapidly isomerizes to 2-hydroxy-p-benzoquinone. The formation of HHQ during the action of tyrosinase on HQ is demonstrated by means of high performance liquid chromatography mass spectrometry (HPLC–MS) by using hydrogen peroxide and high ascorbic acid concentrations. We propose a kinetic mechanism for the tyrosinase oxidation of HQ which allows us the kinetic characterization of the process. A possible explanation of the cytotoxic effect of HQ is discussed.
Type-3 copper proteins: Recent advances on polyphenol oxidases
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Citation Excerpt :
Other mutants (as Phe197 to alanine, Met61 and Met184 to leucine) revealed that Phe197, Met61, and Met184, which are located at the entrance to the binding site, are also important for enhancing the diphenolase activity. First reports on tyrosinase reacting aerobically with molar equivalents of H2O2 were published by Jolley, Evans, and Mason (1972). For a review describing the electronic properties of the type-3 copper center, see Solomon et al. (1992).
Recent investigations in the study of plant, fungal, and bacterial type-3 copper proteins are reviewed. Focus is given to three enzymes: catechol oxidases (CO), tyrosinases, and aureusidin synthase. CO were mostly found in plants, however, in 2010 the first fungal CO was published. The first plant-originated tyrosinase was published in 2014, before tyrosinases were only reported in fungi, bacteria, and human. Aureusidin synthase from yellow snapdragon (Antirrhinum majus) was first published in 2000, as part of yellow flower coloration pathway. In the last years, many important results on type-3 copper enzymes originated from X-ray crystallographic investigations. In addition, studies on site-directed mutagenesis of amino acids around the active site were performed to identify the regions determining monophenolase and/or diphenolase activity. Although X-ray crystallographic structures of CO and tyrosinases are available, many questions like the response for the activation via proteases, sequence-based or structural-based differences between CO, as well as the physiological roles of many polyphenol oxidases still remain to be addressed.
Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus
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Citation Excerpt :
Both met- and oxy-forms show a diphenolase activity, whereas only the oxy-form shows a monophenolase activity [11,19]. The deoxy-form is a reduced and an instable form and binds oxygen to give the oxy-form [20–22]. Tyrosinase can accept a wide range of p-substituted monophenolic and diphenolic substrates.
Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5 mM dopachrome the oxygen consumption rate of TrT on 8 mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.
Comparison of substrate specificity of tyrosinases from Trichoderma reesei and Agaricus bisporus
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Understanding the substrate specificity of tyrosinases (EC 1.14.18.1) as well as their capability to oxidize peptide-bound tyrosine residues is important in a view of applicability of tyrosinases. In the present study, two fungal tyrosinases, an extracellular enzyme from the filamentous fungus Trichoderma reesei (TrT) and an intracellular enzyme from the edible mushroom Agaricus bisporus (AbT) were compared. Oxidation of various mono- and diphenolic compounds and tyrosine-containing tripeptides was examined and kinetic constants determined using spectrophotometric and oxygen consumption measurements. TrT and AbT were found to show notable differences in their substrate specificity. TrT generally showed 10-fold higher K_m values than AbT. The presence of a carboxylic and amine group in the substrate influenced the enzymes differently. While the substrates with a carboxyl group were observed not to be effectively oxidized by AbT, the amine group seemed to hider the oxidation in the TrT-catalyzed reactions. Moreover, the UV–visible absorption spectra on the oxidation of catechol and hydrocaffeic acid showed that the product patterns were different between the enzymes. The result is interesting as the primary products from tyrosinase-catalyzed reactions were assumed to be identical with both enzymes. Furthermore, a nucleophilic 3-methyl-2-benzothiazolinone hydrazone (MBTH) affected differently on the activity of the tyrosinases: the lag period related to the oxidation of monophenols was prolonged by MBTH with TrT, whereas with AbT the lag was shortened.
Opposite effects of peroxidase in the initial stages of tyrosinase-catalysed melanin biosynthesis
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The tyrosinase/oxygen enzymatic system catalyses the orthohydroxylation of l-tyrosine to l-dopa and the oxidation of this to dopaquinone, which evolves non-enzymatically towards to form melanins. The literature has demonstrated and revised the existence of peroxidase/hydrogen peroxide in the melanosomas of skin melanocytes, but points to controversy concerning the effects on melanogenesis. Some authors have recently proposed a new physiological function for tyrosinase, namely the direct scavenging of tyrosyl radicals, which are toxic oxidants of melanocytes. In this contribution, we describe and interpret four effects of peroxidase/hydrogen peroxide on melanogenesis. Two of these effects are its antagonism and synergy as regards the monophenolase and diphenolase activities, respectively, of tyrosinase/oxygen in the initial steps that trigger melanogenesis. Another effect concerns the increase in the oxidant character of the medium in the melanosome by increasing the synthesis of oxidising quinones (o-dopaquinone, p-topaquinone, dopachrome) and the consumption of antioxidant diphenols (l-dopa), which are intermediate biomolecules in melanogenesis. Lastly, we demonstrate that the tyrosyl radicals generated by light or by the peroxidase/hydrogen peroxide system are not directly trapped by the tyrosinase but by the antioxidant orthodiphenol, l-dopa, accumulated in the steady-state of melanogenesis. In conclusion, peroxidase/hydrogen peroxide may help regulate the development of melanogenesis and the oxidant environment within the melanosome. This enzyme deserves further study for its possible antitumoral and depigmentation capacities in skin cancer and hyperpigmentation.

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Reversible oxygenation of tyrosinase

Summary

J. Biol. Chem.

Biochem. Z.

Eur. J. Biochem.