Tyrosinase inhibition kinetics of anisic acid

Anisic acid (p-methoxybenzoic acid) was characterized as a tyrosinase inhibitor from ani-seed, a common food spice. It inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by tyrosinase with an IC50 of 0.60 mM. The inhibition of tyrosinase by anisic acid is a reversible reaction with residual enzyme activity. This phenolic acid was found to be a classical noncompetitive inhibitor and the inhibition constant K(I) was obtained as 0.603 mM. Anisic acid also inhibited the hydroxylation of L-tyrosine catalyzed by tyrosinase. The lag phase caused by the monophenolase activity was lengthened and the steady-state activity of the enzyme was decreased by anisic acid.     

Methyl p-coumarate, a melanin formation inhibitor in B16 mouse melanoma cells

p-Coumaric acid (4-hydroxycinnamic acid) and methyl p-coumarate (methyl 4-hydroxycinnamate) inhibit the oxidation of L-tyrosine catalyzed by mushroom tyrosinase. However, both were oxidized as monophenol substrate analogues at an extremely slow rate. This oxidation was significantly accelerated as soon as catalytic amounts (0.01 mM) of L-3,4-dihydroxyphenylalanine (L-DOPA) became available as a co-factor. Methyl p-coumarate significantly suppressed the melanin formation in B16 mouse melanoma cells, whereas p-coumaric acid did not show this activity.     

A study on the in vitro interaction between tyrosinase and glutathione S-transferase

The actions of glutathione S-transferase and tyrosinase on the in vitro production of glutathionyl-3,4-dihydroxyphenylalanine and the dopachrome level in the presence of GSH and L-3,4-dihydroxyphenylalanine were studied. No clear evidence of complementarity between tyrosinase and glutathione S-transferase was observed; on the contrary, in the presence of glutathione S-transferase the glutathionyl-3,4-dihydroxyphenylalanine yield was lower than with tyrosinase only, as measured by HPLC. It is concluded that the spontaneous conjugation of GSH with dopaquinone should probably be high enough to scavenge the toxic quinone and to produce precursors for phaeomelanogenesis.     

Association of the Acetylcholine-phosphatidyl Inositol Effect with a “Receptor” Proteolipid from Cerebral Cortex

IN spite of continuing research on the treatment of Parkinson's disease^1–3, no drug with clear advantages over L-dopa (the L-isomer of 3,4-dihydroxyphenylalanine) has yet been found. The problems of supply of L-dopa and reduction of its side effects⁴ are therefore still of interest. L-Dopa can be obtained from L-tyrosine by a hydroxylation reaction catalysed by the enzyme tyrosinase (EC 1.10.3.1). Such a reaction using immobilized tyrosinase could form the basis of an industrial method because L-tyrosine is cheap. Alternatively, in view of the fact that L-tyrosine is present in human serum, immobilized tyrosinase suitably implanted in the blood stream might be used to synthesize L-dopa in situ. We have been studying tyrosinase immobilized by covalent attachment to a cellulosic support. In the absence of a readily available mammalian tyrosinase or tyrosine hydroxylase which would be more suitable for clinical purposes we have used a polyphenol oxidase with tyrosinase activity, obtainable from mushrooms.     

Liposome-entrapped tyrosinase: a tool to investigate the regulation of the Raper-Mason pathway

The effect of the entrapment of mushroom tyrosinase (EC 1.14.18.1) within liposomes on the enzyme activity and Km vs. L-3,4-dihydroxyphenylalanine is reported in the present work; the effect of cholesterol insertion within liposome membranes on the enzyme activity has also been studied. The oxidation rates of various monophenols and diphenols by free and liposome-integrated mushroom tyrosinase were measured and the oxidation latencies vs. different substrates investigated. The different substrates are apparently oxidized according to the properties of the substituents as electron donors or acceptors; the Km values vs. L-3,4-dihydroxyphenylalanine calculated on measuring O2 consumption are higher than those calculated on measuring the dopachrome production rates. It is interesting that natural substrates of tyrosinase are oxidized according to a negative catalysis by the liposome-entrapped enzyme; this point is discussed in relation to the well known cytotoxicity of some intermediates of the Raper-Mason pathway.     

Effects of hydroxystilbene derivatives on tyrosinase activity

Synthesis of melanin starts from the conversion of L-tyrosine to 3,4-dihydroxyphenylalanine (L-dopa) and then the oxidation of L-dopa yields dopaquinone by tyrosinase. Therefore, tyrosinase inhibitors have been established as important constituents of depigmentation agents. Recently, polyhydroxystilbene compounds, which are trans-resveratrol (3,4('),5-trihydroxy-trans-stilbene) analogs, have been demonstrated as potent tyrosinase inhibitors. However, their detailed inhibitory mechanisms are not clearly understood. In the present study, a variety of synthesized hydroxystilbene compounds were tested for their inhibitory effects against murine tyrosinase activity. The inhibitory potencies of the hydroxy-trans-stilbene compounds were remarkably elevated by increasing number of phenolic hydroxy substituents. Methylated hydroxy-trans-stilbene lost the inhibitory activity. Furthermore, hydrogenated hydroxystilbene or hydroxy-cis-stilbene exerted little or no inhibitory effect compared with hydroxy-trans-stilbene on tyrosinase activity. The structure-activity relationships demonstrated in the present study suggest that the phenolic hydroxy groups and trans-olefin structure of the parent stilbene skeleton contribute to the inhibitory potency of hydroxystilbene for tyrosinase activity.     

Inhibition of tyrosinase by indole compounds and reaction products protection by albumin

Tyrosinase activity decreases as the reaction proceeds and is inhibited by L-3,4-dihydroxyphenylalanine oxidation products. Indole and tryptophan inhibit tyrosinase reaction and bovine albumin protects against end-products(s) inhibiton or inactivation. Since the same tyrosinase reaction products are indole compounds and some authors reported the binding of indole derivatives with albumin, it is here suggested that indole intermediates of melanin synthesis inhibit or inactivate tyrosinase.     

Inhibition of tyrosinase by indole compounds and reaction products. Protection by albumin.

Tyrosinase activity decreases as the reaction proceeds and is inhibited by L-3,4-dihydroxyphenylalanine oxidation products. Indole and tryptophan inhibit tyrosinase reaction and bovine albumin protects against end-product(s) inhibition or inactivation. Since the same tyrosinase reaction products are indole compounds and some authors reported the binding of indole derivatives with albumin, it is here suggested that indole intermediates of melanin synthesis inhibit or inactivate tyrosinase.     

An actinomycete producing L-3,4-dihydroxyphenylalanine from L-tyrosine

An actinomycete was isolated during a soil screening programme to obtain L-3,4-dihydroxyphenylalanine producers. A mutant of this organism was isolated by chemical mutagenesis and it accumulated 1 g/litre L-dihydroxyphenylalanine when grown on L-tyrosine. Resting cells converted 30% of tyrosine in the reaction mixture. The use of resting cells for dihydroxyphenylalanine production is advantageous as it eliminates interfering substances which accumulate during fermentation.     

Immunoelectron microscopic localization of tyrosinase in the mouse melanocyte

In the melanocyte, tyrosinase is known as the dey enzyme for melanin formation. Purified tyrosinase protein was prepared that was capable of oxidizing tyrosine. The localization of tyrosinase antigen in the melanocyte was studied using antiserum against tyrosinase. DOPA (L-3,4-dihydroxyphenylalanine)-reaction product and tyrosinase antigen were found on the same organelles i.e., premelanosomes, melanosomes, GERL, and Golgi vesicles. This result seems to suggest that it is cytochemically appropriate to use DOPA as the substrate of tyrosinase. It appeared that tyrosinase antigen was present as granule-like structures inside GERL cisterna and associated with its membrane.     

Novel Biological Process for L-DOPA Production from L-Tyrosine by P-Hydroxyphenylacetate 3-Hydroxylase

A novel biological method was developed for the production of L-3,4-dihydroxyphenylalanine (L-DOPA) from L-tyrosine by p-hydroxyphenylacetate 3-hydroxylase of Escherichia coli strain W (ATCC 11105). About 48 mM (or 1% w/v) L-DOPA was obtained by a fed batch operation in 50 h when a recombinant strain constitutively producing the enzyme was used.     

Phenylalanine as substrate for tyrosine hydroxylase in bovine adrenal chromaffin cells.

Incubation of bovine chromaffin cells with L-[14C]phenylalanine resulted in label accumulation in catecholamines at about 30% of the rate seen with L-tyrosine as precursor. Studies with purified tyrosine hydroxylase (EC 1.14.16.2) showed that the enzyme catalysed the hydroxylation of L-phenylalanine first to L-p-tyrosine and then to 3,4-dihydroxyphenylalanine (DOPA). No evidence for a significant involvement of an L-m-tyrosine intermediate in DOPA formation was found.     

2-hydroxy-4-isopropylbenzaldehyde, a potent partial tyrosinase inhibitor

Chamaecin (2-hydroxy-4-isopropylbenzaldehyde) was synthesized and tested for its tyrosinase inhibitory activity. It partially inhibits the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase with an IC(50) of 2.3 microM. The inhibition kinetics analyzed by Dixon plots found that chamaecin is a mixed type inhibitor. This inhibition may come in part from its ability to form a Schiff base with a primary amino group in the enzyme.     

Synthesis of tyrosine and 3,4-dihydroxyphenylalanine by Citrobacter freundii bacteria from lactic acid

A possibility of using lactic acid as a precursor for the synthesis of L-tyrosine and L-3,4-dihydroxyphenylalanine (DOPA) by Citrobacter freundii 62 and 63 was established. The synthesis of tyrosine from lactic acid occurs at a phenol concentration of less than 0.6%. The conditions were found which enable C. freundii 62 and 63 to synthesize from lactic acid tyrosine and DOPA with the yield of 35-38 g/l and 32-33 g/l, respectively.     

The influence of hydroquinone on tyrosinase kinetics

In vitro studies, using combined spectrophotometry and oximetry together with hplc/ms examination of the products of tyrosinase action demonstrate that hydroquinone is not a primary substrate for the enzyme but is vicariously oxidised by a redox exchange mechanism in the presence of either catechol, L-3,4-dihydroxyphenylalanine or 4-ethylphenol. Secondary addition products formed in the presence of hydroquinone are shown to stimulate, rather than inhibit, the kinetics of substrate oxidation.     

Proanthocyanidins Extracted from Rhododendron pulchrum Leaves as Source of Tyrosinase Inhibitors: Structure,Activity, and Mechanism

The objective of this study was to assess the structure, anti-tyrosinase activity, and mechanism of proanthocyanidins extracted from Rhododendron pulchrum leaves. Results obtained from mass spectra of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) revealed that proanthocyanidins were complex mixtures of procyanidins, prodelphinidins, propelargonidins, and their derivatives, among which procyanidins were the main components. The anti-tyrosinase analysis results indicated that the mixtures were reversible and mixed competitive inhibitors of tyrosinase. Interactions between proanthocyanidins with substrate (L-tyrosine and 3,4-dihydroxyphenylalanine) and with copper ions were the important molecular mechanisms for explaining their efficient inhibition. This research would provide scientific evidence for the use of R. pulchrum leaf proanthocyanidins as new novel tyrosinase inhibitors.     

Effects of salicylic acid on mushroom tyrosinase and B16 melanoma cells

Salicylic acid slightly inhibited the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase noncompetitively without being oxidized. In contrast, 4-hydroxybenzoic acid did not inhibit this enzymatic oxidation if a longer reaction time was observed, although it suppressed the initial rate of the oxidation to a certain extent. Neither acid showed noticeable effects on cultured murine B16-F10 melanoma cells except weak cytotoxicity.     

Contribution of L-3,4-dihydroxyphenylalanine metabolism to the inhibition of gluconeogenesis in rabbit kidney-cortex tubules

The circulating L-3,4-dihydroxyphenylalanine, the drug of choice in the therapy of Parkinson's disease (PD), is efficiently extracted by kidney and converted to dopamine, known to control several renal functions. As: (i) in addition to liver, kidney is an important source of glucose in mammals and (ii) the action of this drug on renal gluconeogenesis has not yet been studied, the aim of the present investigation was to estimate the influence of L-3,4-dihydroxyphenylalanine metabolism on glucose formation in isolated kidney-cortex tubules incubated with various gluconeogenic substrates. The data indicate that a rapid intracellular degradation of L-3,4-dihydroxyphenylalanine and tyramine (at 100 and 200 microM concentrations) is accompanied by 25-40% decrease in glucose production from pyruvate, alanine + glycerol + octanoate and dihydroxyacetone due to augmented generation of hydrogen peroxide via monoamine oxidase B, resulting in a decline of glutathione redox state by 40%. Moreover, following inhibition of monoamine oxidase B by deprenyl or substitution of pyruvate by aspartate + glycerol + octanoate both L-3,4-dihydroxyphenylalanine and tyramine affect neither the rate of gluconeogenesis nor glutathione redox state. In view of: (i) L-3,4-dihydroxyphenylalanine- and tyramine-induced changes in intracellular levels of gluconeogenic intermediates, and (ii) a significant decline of phosphoenolpyruvate carboxykinase activity by 500 microM oxidized glutathione, it is likely that L-3,4-dihydroxyphenylalanine- and tyramine-evoked disturbances in the glutathione redox state might diminish flux through phosphoenolpyruvate carboxykinase and in consequence decrease glucose formation in renal tubules, suggesting a new potential side-action of L-3,4-dihydroxyphenylalanine treatment.     

5,6-Dihydroxyindole oxidation by mammalian, mushroom and amphibian tyrosinase preparations

The oxidation of 5,6-dihydroxyindole by tyrosinases from mushroom, Harding-Passey melanoma, bovine eye and Bufo bufo embryo has been investigated. The apparent Km values for this substrate were measured and found to be of the same order of magnitude as those for L-tyrosine and L-3,4-dihydroxyphenylalanine, as reported in the literature (5 x 10(-4) M). The 5,6-dihydroxyindole oxidases of mushroom and T4 melanoma isozyme are sensitive to phenylthiourea, while, on the other hand, those from crude preparations of bovine and B. bufo tyrosinases are not sensitive to the inhibitor in an evident manner. The action of some indole derivatives on the 5,6-dihydroxyindole oxidase of mushroom has also been investigated.     

Ultrastructural and chemical distinction of melanins formed by Verticillium dahliae from (+)-scytalone, 1,8-dihydroxynaphthalene, catechol, and L-3,4-dihydroxyphenylalanine

Microsclerotia of three melanin-deficient mutants of Verticillium dahliae formed malanin from (+)-scytalone, 1,8-dihydroxynaphthalene, catechol, and L-3,4-dihydroxyphenylalanine. The melanins formed from (+)-scytalone or 1,8-dihydroxynaphthalene resembled wild-type melanin chemically and ultrastructurally, whereas the melanins formed from catechol and L-3,4-dihydroxyphenlalanine were different. This suggests that scytalone and 1,8-dihydroxynaphthalene but no catechol or L-3,4-dihydroxyphenylalanine are natural intermediates of melanin biosynthesis in V. dahliae.