Optimization of hydroxylation of tyrosine and tyrosine-containing peptides by mushroom tyrosinase

Free tyrosine and tyrosine residues in various peptides and proteins are converted into dopa and dopa residues by tyrosinase (monophenol,L-dopa:oxygen oxidoreductase, EC 1.14.18.1) in the presence of reductants. The efficiency of the tyrosine-to-dopa conversion was examined under varied conditions, such as the substrate-to-tyrosine ratio, concentrations of reductant and oxygen in the reaction solution, pH, temperature and reaction time. The highest dopa yields were achieved with the following optimal conditions for hydroxylation: 0.1 M phosphate buffer at pH 7, 25 mM ascorbic acid, 1 mM tyrosine, 50 micrograms/ml tyrosinase and 20 degrees C. Using these conditions, up to 70% of free tyrosine was converted into dopa, and tyrosine residues in several synthetic peptides were also hydroxylated to dopa residues at ratios as high as free tyrosine. The preparation of hydroxylated analogues of the decapeptide (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys), in particular, may contribute to a better understanding of adhesion in the dopa-containing mussel glue protein.     

Evidence against superoxide involvement in tyrosine hydroxylation by mushroom tyrosinase

The possible involvement of superoxide anions in the hydroxylation of tyrosine by mushroom tyrosinase was studied. Superoxide dismutase and scavengers of superoxide ions of smaller MW than superoxide dismutase, such as nitroblue tetrazolium and copper salicylate, had no direct effect on the monohydroxyphenolase activity of mushroom tyrosinase. The kinetics of tyrosine hydroxylation, but not of DOPA oxidation, by mushroom tyrosinase was atrected by the addition of a xanthine-xanthine oxidase system. In the presence of the xanthine-xanthine oxidase system, the lag period of tyrosine hydroxylation was shortened compared to the lag period in the absence of the xanthine-xanthine oxidase system. The xanthine- xanthine oxidase system alone (without mushroom tyrosinase) had no effect on tyrosine conversion to dopachrome. Superoxide dismutase, catalase and hydroxyl radical scavengers counteracted to some extent the shortening of the lag period of tyrosine hydroxylation by mushroom tyrosinase caused by the xanthin e-xanthine oxidase system. It is suggested that the shortening of the lag period is due mainly to hydroxyl radicals generated by the xanthine-xanthine oxidase system via interaction of O₂^?. and hydrogen paroxide (a Haber-Weiss type reaction). The data do not support the direct participation of superoxide anions in tyrosine hydroxylation by mushroom tyrosinase.     

A reexamination of the melanin formation assay of tyrosinase and an extension to estimate phaeomelanin formation

This paper presents some modifications of the melanin formation assay for tyrosinase from the point of view of both eu- and phaeomelanosynthesis. On the one hand, eumelanosynthesis can be estimated using neutral paper filters, such as the 3MM Whatman filters so far employed. The main advantages of this sort of paper are the very low blank values obtained in the absence of tyrosinase and its greater mechanical resistance in the successive washing steps. It is shown that the sensitivity of the assay can be enhanced by the addition of 1 mM Ni(II) to the incubation mixture or of NaOH to stop the enzymatic reaction and allow the incorporation of indolic intermediates into the polymer. Furthermore, the accuracy is also enhanced by the proposed modifications, since all reactions from dopaquinone are standardized, and the assay becomes only dependent on the tyrosinase activity. On the other hand, phaeomelanosynthesis cannot be estimated using neutral paper because of the slow rate of polymerization of the intermediates and the poor absorption of thiol-dopa conjugates to this kind of paper. It is shown that synthesis of this type of melanin can be estimated in the presence of glutathione by means of a cationic filter paper and by washing the excess of the radioactive substrate with distilled water instead of acidic media. Thus, the assay may be adapted to measure eu- or phaeomelanosynthetic activity by introducing slight modifications. This assay must be used with caution if detergent-solubilized tyrosinase is used, because detergents strongly inhibit melanin absorption to paper filters.     

Analogues of N-hydroxy-N'-phenylthiourea and N-hydroxy-N'-phenylurea as inhibitors of tyrosinase and melanin formation

A series of N-hydroxy-N'-phenylthiourea and N-hydroxy-N'-phenylurea analogues were prepared and evaluated as inhibitors of tyrosinase and melanin formation. The most active analogue 1 inhibited mushroom tyrosinase with an IC(50) of around 0.29 microM and also retained a substantial potency in cell culture by reducing pigment synthesis by 78%. Therefore, compound 1 could be considered as a promising candidate for preclinical drug development for skin hyperpigmentation application.     

D‐tyrosine negatively regulates melanin synthesis by competitively inhibiting tyrosinase activity

Although L‐tyrosine is well known for its melanogenic effect, the contribution of D‐tyrosine to melanin synthesis was previously unexplored. Here, we reveal that, unlike L‐tyrosine, D‐tyrosine dose‐dependently reduced the melanin contents of human MNT‐1 melanoma cells and primary human melanocytes. In addition, 500 μM of D‐tyrosine completely inhibited 10 μM L‐tyrosine‐induced melanogenesis, and both in vitro assays and L‐DOPA staining MNT‐1 cells showed that tyrosinase activity is reduced by D‐tyrosine treatment. Thus, D‐tyrosine appears to inhibit L‐tyrosine‐mediated melanogenesis by competitively inhibiting tyrosinase activity. Furthermore, we found that D‐tyrosine inhibited melanogenesis induced by α‐MSH treatment or UV irradiation, which are the most common environmental factors responsible for melanin synthesis. Finally, we confirmed that D‐tyrosine reduced melanin synthesis in the epidermal basal layer of a 3D human skin model. Taken together, these data suggest that D‐tyrosine negatively regulates melanin synthesis by inhibiting tyrosinase activity in melanocyte‐derived cells.     

The hydroxylation of phenylalanine and tyrosine: a comparison with salicylate and tryptophan.

The hydroxylation of phenylalanine by the Fenton reaction and gamma-radiolysis yields 2-hydroxy-, 3-hydroxy-, and 4-hydroxyphenylalanine (tyrosine), while the hydroxylation of tyrosine results in 2,3- and 3,4-dihydroxyphenylalanine (dopa). Yields are determined as a function of pH and the presence or absence of oxidants. During gamma-radiolysis and the Fenton reaction the same hydroxylated products are formed. The final product distribution depends on the rate of the oxidation of the hydroxyl radical adducts (hydroxycyclohexadiene radicals) relative to the competing dimerization reactions. The pH profiles for the hydroxylations of phenylalanine and tyrosine show a maximum at pH 5.5 and a minimum around pH 8. The lack of hydroxylated products around near pH 8 is due to the rapid oxidation of dopa to melanin. The relative abilities of iron chelates (HLFe(II) and HLFe(III) to promote hydroxyl radical formation from hydrogen peroxide are nitrilotriacetate (nta) greater than ethylenediaminediacetate (edda) much greater than hydroxyethylethylenediaminetriacetate greater than citrate greater than ethylenediaminetetraacetate greater than diethylenetriaminepentaacetate greater than adenosine 5'-triphosphate greater than pyrophosphate greater than adenosine 5'-diphosphate greater than adenosine 5'-monophosphate. The high activity of iron-nta and -edda chelates is explained by postulating the formation of a ternary Fe(III)-L-dopa complex in which dopa reduces Fe(III). The hydroxylations of phenylalanine and tyrosine are similar to that of salicylate (Z. Maskos, J. D. Rush, and W. H. Koppenol, 1990, Free Radical Biol. Med. 8, 153-162) and tryptophan (preceding paper) in that oxidants augment the formation of hydroxylated products by catalyzing the dismutation of hydroxyl radical adducts to the parent compound and a stable hydroxylated product. A comparison of salicylate and the amino acids tryptophan, phenylalanine, and tyrosine clearly shows that salicylate is the best indicator of hydroxyl radical production.     

Mammalian tyrosinase. Structural and functional interrelationship of isozymes

The isozymes of tyrosinase from normal and malignant melanocytes were studied; the data indicates that each consists of a basic tyrosinase polypeptide, and differs by post-translational modifications. T₃ represents the de novo form of the enzyme; it is converted to T₁ in vivo by the addition of sialic acids and neutral sugars, and in turn, to T₄ by complexing with melanosomal membrane constituents. The T₂ isomer is suggested to be an artefact of the electrophoretic procedure, and due to deamidation of T₃. It is shown that the apparent kinetics of enzyme activity are unaffected by any of these modifications.     

Studies on phenylalanine and tyrosine hydroxylation by rat brain tyrosine hydroxylase.

Tyrosine hydroxylase (EC1.14.16.2), presumably the rate-limiting enzyme in the biosynthesis of catecholamines, is known to catalyze the hydroxylation of both phenylalanine and tyrosine. Using both an isolated enzyme preparation and a synaptosomal preparation, where some architectural integrity of the tissue has been preserved, we have attempted to evaluate the manner in which these two substrates are hydroxylated by rat brain tyrosine hydroxylase. In the presence of tetrahydrobiopterin the isolated enzyme catalyzes the hydroxylation of phenylalanine to 3,4-dihydroxyphenylalanine with the release of free tyrosine as an obligatory intermediate. In contrast, the rat brain striatal synaptosomal preparation in the presence of endogenous cofactor converts phenylalanine to 3,4-dihydroxyphenylalanine without the release of free tyrosine.     

A study of the supposed hydroxylation of tyrosine catalysed by peroxidase.

The claim that peroxidase (rather than tyrosinase) is the enzyme responsible for the conversion of tyrosine into dopa (3,4-dihydroxyphenylalanine) in melanogenesis was investigated. The spectral changes that occurred during the action of horseradish peroxidase in the presence of H2O2 on dopa, tyrosine and mixtures of dopa with tyrosine or other phenolic compounds were studied. The effect of ascorbic acid or dihydroxyfumaric acid on some of these changes was also investigated. No evidence was found that tyrosine was hydroxylated by peroxidase in the presence of H2O2 and dopa as cofactor, although tyrosine or other phenolic compounds increased the rate of oxidation of dopa to dopachrome (indoline-5,6-quinone-2-carboxylic acid). Peroxidase was, however, effective in oxidizing tyrosine to dopa in the presence of dihydroxyfumaric acid and oxygen.     

Natural ortho-dihydroxyisoflavone derivatives from aged Korean fermented soybean paste as potent tyrosinase and melanin formation inhibitors

Natural o-dihydroxyisoflavone (ODI) derivatives with variable hydroxyl substituent at the aromatic ring of isoflavone and three known isoflavones were isolated from five-year-old Korean fermented soybean paste (Doenjang) and evaluated as potent inhibitors on tyrosinase activity and melanin formation in melan-a cells comparing with other known isoflavones, 7,8,4′-trihydroxyisoflavone (1) and 7,3′,4′-trihydroxyisoflavone (2) inhibited tyrosinase by 50% at a concentration of 11.21 ± 0.8 μM and 5.23 ± 0.6 μM (IC₅₀), respectively, whereas, 6,7,4′-trihydroxyisoflavone (3), daidzein (4), glycitein (5) and genistein (6) showed very low inhibition activity. Furthermore, those compounds significantly suppressed the cellular melanin formation by 50% at a concentration of 12.23 ± 0.7 μM (1), 7.83 ± 0.7 μM (2), and 57.83 ± 0.5(6) and show more activity than arbutin. But, compounds 3, 4, and 5 showed lower inhibition activity. This study shows that the position of hydroxyl substituent at the aromatic ring of isoflavone plays an important role in the intracellular regulation of melanin formation in cell-based assay system.     

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase.

Melanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.     

The hydroxylation of phenylalanine and tyrosine by tyrosine hydroxylase from cultured pheochromocytoma cells.

Pheochromocytoma tyrosine hydroxylase was reported to have unusual catalytic properties, which might be unique to the tumor enzyme (Dix, T. A., Kuhn, D. M., and Benkovic, S. J. (1987) Biochemistry 24, 3354-3361). Two such properties, namely the apparent inability to hydroxylate phenylalanine and an unprecedented reactivity with hydrogen peroxide were investigated further in the present study. Tyrosine hydroxylase was purified to apparent homogeneity from cultured pheochromocytoma PC12 cells. The purified tumor enzyme was entirely dependent on tetrahydrobiopterin (BH4) for the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine and hydrogen peroxide could not substitute for the natural cofactor. Indeed, in the presence of BH4, increasing concentrations of hydrogen peroxide completely inhibited enzyme activity. The PC12 hydroxylase exhibited typical kinetics of tyrosine hydroxylation exhibited typical kinetics of tyrosine hydroxylation, both as a function of tyrosine (S0.5 Tyr = 15 microM) and BH4 (apparent Km BH4 = 210 microM). In addition, the enzyme catalyzed the hydroxylation of substantial amounts of phenylalanine to tyrosine and 3,4-dihydroxyphenylalanine (apparent Km Phe = 100 microM). Phenylalanine did not inhibit the enzyme in the concentrations tested, whereas tyrosine showed typical substrate inhibition at concentrations greater than or equal to 50 microM. At higher substrate concentrations, the rate of phenylalanine hydroxylation was equal to or exceeded that of tyrosine. Essentially identical results were obtained with purified tyrosine hydroxylase from pheochromocytoma PC18 cells. The data suggest that the tumor enzyme has the same substrate specificity and sensitivity to hydrogen peroxide as tyrosine hydroxylase from other tissues.