Isoproterenol oxidation by tyrosinase: intermediates characterization and kinetic study

The present work deals with isoproterenol oxidation by mushroom tyrosinase and sodium metaperiodate. Intermediates produced at short reaction time were characterized by scanning repetitive spectrophotometry and the stoichiometry of the respective aminochrome appearance was established. The oxidation pathway from isoproterenol to aminochrome is parallel to the previously proposed for L-dopa oxidation by mushroom tyrosinase, whose steps are as follow: Isoproterenol----o-quinone-H+----o-quinone----leukoaminochrome---- aminochrome. The stoichiometry for the conversion of o-quinone-H+ into the aminochrome of isoproterenol followed the equation: 2 o-quinone-H+----isoproterenol + aminochrome. The kinetics of chemical reactions that take place from the o-quinone-H+ to aminochrome has been studied as a system of various chemical reactions coupled to an enzymatic reaction (EzCC: Enzymatic-Chemical-Chemical mechanism).     

Study of alpha-methyldopa oxidation by tyrosinase

The pathway for alpha-methyldopa oxidation to alpha-methyldopachrome, by mushroom tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometry determination have both been undertaken. The steps for alpha-methyldopa transformation into its aminochrome would be: alpha-methyldopa----o-alpha-methyldopaquinone-H+----o-alpha- methyldopaquinone----leuko-alpha-methyldopachrome----alpha- methyldopachrome. The stoichiometry for this conversion corresponded to the equation: 2 o-alpha-methyldopaquinone-H+----alpha-methyldopa + alpha-methyldopachrome. At very acid pH values, another route implying the addition of water to the quinonic ring, competes with the first one. Two chemical pathways can be proposed from alpha-methyldopaquinone-H+, the relative importance of which is determined by the pH. A theoretical and experimental kinetic approach was applied to this oxidative reaction. Rate constants and thermodynamic activation parameters of the chemical steps, have been evaluated. The results obtained confirmed that alpha-methyldopa oxidation by tyrosinase followed a scheme similar to that established for L-dopa and alpha-methylnoradrenaline.     

Chemical intermediates in dopamine oxidation by tyrosinase,and kinetic studies of the process

A minor pathway for dopamine oxidation to dopaminochrome, by tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometric determination have both been undertaken. After oxidizing dopamine with mushroom tyrosinase or sodium periodate in a pH range from 6.0 to 7.0, it was spectrophotometrically possible to detect o-dopaminoquinone-H⁺ as the first intermediate in this pathway. The steps for dopamine transformation to dopaminochrome are as follows: dopamine → o-dopaminequinone-H⁺ → o-dopaminequinone → leuko-dopaminochrome → dopaminochrome. No participation of oxygen was detected in the conversion of leukodopaminochrome to dopaminochrome. Scanning spectroscopy and graphical analysis of the obtained spectra also verified that dopaminequinone-H⁺ was transformed into aminochrome in a constant ratio. The stoichiometry equation for this conversion is 2 o-dopaminequinone-H⁺ → dopamine + dopaminochrome. The pathway for dopamine oxidation to dopaminochrome by tyrosinase has been studied as a system of various chemical reactions coupled to an enzymatic reaction. A theoretical and experimental kinetic approach is proposed for such a system; this type of mechanism has been named “Enzymatic-chemical-chemical” (E_ZCC). Rate constants for the implied chemical steps at different pH and temperature values have been evaluated from the measurement of the lag period arising from the accumulation of dopaminochrome that took place when dopamine was oxidized at acid pH. The thermodynamic activation parameters of the chemical steps, the deprotonation of dopaminequinone-H⁺ to dopaminequinone, and the internal cyclization of dopaminequinone to leukodopaminochrome have been calculated.     

Chemical intermediates in α-methylnoradrenaline oxidation by tyrosinase—I. Spectral properties and stoichiometry

• 1.1. A pathway for a-methylnoradrenaline oxidation to α-methylnoradrenochrome, by tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometry determination have both been performed.
• 2.2. It has been possible to detect spectrophotometrically o-quinone-H⁺ as the first intermediate in this pathway after oxidizing α-methylnoradrenaline with mushroom tyrosinase or sodium periodate in a pH range from 5 to 6.
• 3.3. The steps for α-methylnoradrenaline transformation into its aminochrome could be: α-methylnoradrenaline → o -α-methylnoradrenaline — H⁺ → o — α -methylnoradrenalinequinone → leuko — α — methylnora — drenochrome→α-methylnoradrenochrome.
• 4.4. No participation of oxygen was detected in the conversion of leuko-α-mehtylnoradrenochrome into α -methylnoradrenochrome.
• 5.5. Matrix analysis of the spectra obtained with a rapid scan spetrophotometer verified that o-quinone-H⁺ was transformed into aminochrome in a constant ratio.
• 6.6. The stoichiometry for this conversion followed the equation: 2 α-methylnoradrenalinequinone-H⁺ → α-methylnoradrenaline + α-methylnoradrenochrome.

     

The role of pH in the melanin biosynthesis pathway

Having oxidized 3,4-dihydroxyphenylalanine (dopa) with sodium periodate or mushroom tyrosinase in a pH range from 3.5 to 6.0, it has been possible to detect spectrophotometrically 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone with the amino group protonated (o-dopaquinone-H+), a postulated intermediate in the melanogenesis pathway. When the pH value was greater than 4, the final product obtained was 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome); however, for pH values lower than 4, two different products were identified by means of cyclic voltammetry: 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone and dopachrome. These products appeared when oxidation was achieved with the enzyme as well as with periodate. This suggests that two chemical pathways can arise from alpha-dopaquinone-H+, whose relative importance is determined by the pH. The steps of these pathways would be dopa leads to o-dopaquinone-H+ leads to o-dopaquinone leads to leukodopachrome leads to dopachrome, for the first one, and dopa leads to o-dopaquinone-H+ leads to 2,4,5-trihydroxyphenylalanine leads to 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone very slowly leads to intermediate compound leads to dopachrome, for the second one. The stoichiometry for the conversion of dopaquinone-H+ into dopachrome for pH values greater than 4 followed equation of 2 o-dopaquinone-H+ leads to dopa + dopachrome. No participation of oxygen was detected in the conversion of leukodopachrome (2,3-dihydro-5,6-dihydroxyindole-2-carboxylate) into dopachrome.     

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.     

Melanocyte-Directed enzyme prodrug therapy (MDEPT): development of second generation prodrugs for targeted treatment of malignant melanoma.

Evaluation of second generation prodrugs for MDEPT, by oximetry, has highlighted structural properties that are advantageous and disadvantageous for efficient oxidation using mushroom tyrosinase. In particular, a sterically undemanding prodrug bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28 was synthesised and found to be oxidised by mushroom tyrosinase at a superior rate to tyrosine methyl ester, the carboxylic acid of which is the natural substrate for tyrosinase. The more sterically demanding phenyl mustard prodrugs 9 and 10 were oxidised by mushroom tyrosinase at a similar rate to tyrosine methyl ester. In contrast, tyramine chain elongation via heteroatom insertion was detrimental and the rate of mushroom tyrosinase oxidation of phenyl mustard prodrugs 21 and 22 decreased by 10 nanomol/min.     

Water purification through bioconversion of phenol compounds by tyrosinase and chemical adsorption by chitosan beads

Enzymatic removal of various phenol compounds from artificial wastewater was undertaken by the combined use of mushroom tyrosinase (EC 1.14.18.1) and chitosan beads as function of pH value, temperature, tyrosinase dose, and hydrogen peroxide-to-substrate ratio. Chitosan film incubated in a p-crersol+tyrosinase mixture had the main peaks at 400-470 nm assigned to chemically adsorbed quinone derivatives, which increased over the immersion time. These results indicate that removal of phenol compounds is caused by their tyrosinase-catalyzed oxidation to the corresponding quinone derivatives and the subsequent chemical adsorption on the chitosan film. The optimum conditions for quinone adsorption were determined to be pH 7 and 45 degrees C for p-cresol. Some alkyl-substituted phenol compounds were removed by adsorption of quinone derivatives enzymatically generated on the chitosan beads, and the % removal for p-cresol, 4-ethylphenol, 4-n-propylphenol, 4-n-butylphenol, and p-chlorophenol went up to 93%. In addition, 4-tert-butylphenol underwent tyrosinase-catalyzed oxidation in the presence of hydrogen peroxide. This procedure was applicable to removal of chlorophenols and alkyl-substituted phenols.     

Comparative study of tyrosinases from different sources: relationship between halide inhibition and the enzyme active site

The inhibition of tyrosinases from frog epidermis (Rana esculenta ridibunda), mushroom (Agaricus bisporus) and Harding-Passey mouse melanoma by halides is compared. In all cases, the inhibition is pH dependent, increasing when the pH decreases. The order of inhibition is I- greater than Br- greater than Cl- much greater than F- for frog epidermis tyrosinase, F- greater than I- greater than Cl- greater than Br- for mushroom tyrosinase and F- greater than Cl- much greater than Br- greater than I- for the mouse melanoma enzyme. These results are discussed in terms of the active site accessibility to exogenous ligands. The activation energies of the enzyme-catalysed L-dopa oxidation were also calculated, being the values 6.86, 17.01 and 20.25 kcal/mol for frog epidermis, mushroom and Harding-Passey mouse melanoma, respectively. A relationship between these values and the evolutionary adaptation of these enzymes is proposed.     

Hen egg white lysozyme as an inhibitor of mushroom tyrosinase

We report a kinetics study on hen egg white lysozyme's (HEWL) inhibitory effect on mushroom tyrosinase catalysis of 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) or L-tyrosine. For the first time, we demonstrate HEWL as a robust inhibitor against mushroom tyrosinase in catalysis of both substrates. The kinetics pattern matches a mixed (mostly non-competitive) partial inhibition. Ki and ID50 value of HEWL are more than 20-fold lower than that of kojic acid, a well-known chemical inhibitor of mushroom tyrosinase. Ki, alpha value and beta value, are almost identical in both experiments (L-DOPA and L-tyrosine as substrates, respectively), which suggests this common inhibition mechanism affects both steps. The inhibitory effect increases as both proteins were mixed and pre-incubated for less than 1 h. HEWL-depletion only removed about half of the inhibitory effect. Here we propose a novel function of HEWL, which combines the reversible inhibition and the irreversible inactivation toward mushroom tyrosinase. Discovery of HEWL as an inhibitor to mushroom tyrosinase catalysis may be commercially valuable in the food, medical and cosmetic industries.     

Initial mushroom tyrosinase-catalysed oxidation product of 4-hydroxyanisole is 4-methoxy-ortho-benzoquinone

Evidence is presented that the first and major product of the oxidation of 4-hydroxyanisole (4HA) by tyrosinase is 4-methoxy ortho benzoquinone (4-MOB). 4-MOB was synthesized by oxidation of 4HA by potassium nitrodisulphonate and comparisons made between the synthetic quinone and an extract of a reaction mixture in which 4HA had been completely oxidized by mushroom tyrosinase. The chemical species were found to be identical in UV/visible absorption spectrum, 1H-NMR spectrum, and by thin-layer chromatography.     

Effect of Different Isomers of Dihydroxybenzoic Acids (DBA) on the Rate of DL-DOPA Oxidation by Mushroom Tyrosinase

Dihydroxybenzoic acids (DBA), such as 3,4-DRA, 3,5-DBA, and 2,4-DBA—at all concentrations tested—inhibited the rate of DL-DOPA oxidation to dopachrome (λmax = 475 nm) by mushroom tyrosinase. 2,3-DBA and 2,5-DBA at relatively low concentration had a synergistic effect on the reaction, whereas at relatively high concentrations they inhibited the rate of DL-DOPA oxidation. The synergistic effect of 0.6-13.3 mM 2,3-DRA on the rate of DL-DOPA oxidation to dopachrome (λmax = 475 nm) was found to be due to the ability of 2,3-DBA-o-quinone (formed by the oxidation of 2,3-DBA by mushroom tyrosinase or by sodium periodate) to oxidize DL-DOPA to dopachrome (via dopaquinone) non-enzymatically. A similar explanation is likely to be valid for the synergism exerted by 2,5-DBA on the rate of DL-DOPA oxidation by mushroom tyrosinase.     

N-Benzylbenzamides: a new class of potent tyrosinase inhibitors

A series of potent inhibitors of tyrosinase and their structure-activity relationships are described. N-Benzylbenzamide derivatives (1-21) with hydroxyl(s) were synthesized and tested for their tyrosinase inhibitory activity. With this series, compound 15 provided a potent tyrosinase inhibition: it effectively inhibited the oxidation of l-DOPA catalyzed by mushroom tyrosinase with an IC(50) of 2.2microM.     

The Stoichiometry of Tyrosinase-Catalyzed Oxidation of 4-Hydroxyanisole

Oxygen utilisation during tyrosinase-catalysed oxidation of 4-hydroxyanisole was investigated using an electron spin resonance technique which employs quantitative changes in the characteristics of the electron spin resonance spectrum of the spin label 3-carbamoyl-2,5-dihydro-2,2,5-5-tetramethyl-1-H-pyridoyl-1-yloxy (CTPO) to follow changes in the oxygen concentration. Reaction mixtures containing mushroom tyrosinase (15 μg ml^-1) and differing initial concentrations of 4-hydroxyanisole in aerated phosphate buffer at pH 6.8 were incubated at room temperature. The ratio of utilisation of oxygen was found to be in approximately 1:1 molar ratio with the initial 4-hydroxyanisole concentration in the reaction mixture between 50 and 200 μmol/1 4-hydroxyanisole. The results are consistent with the stoichiometry of oxygen utilisation being accounted for by the oxidation of 4-hydroxyanisole to anisyl quinone.     

Calibration of a Clark-Type oxygen electrode by tyrosinase-catalyzed oxidation of 4-tert-butylcatechol.

A procedure for calibrating a Clark-type oxygen electrode is described. This method is based on the oxidation of 4-tert-butylcatechol (TBC) by O2 catalyzed by tyrosinase, to yield 4-tert-butyl-o-benzoquinone (TBCQ). This reaction consumes known amounts of oxygen in accordance with the stoichiometry: 2TBC + O2----2TBCQ + 2H2O and can be used to determine the relation between the oxygen concentration and the oxygen electrode response. TBCQ is very stable in the reaction medium for more than 30 min and shows no significant breakdown, which makes the calibration possible. A kinetic study of the oxidation of 3,4-dihydroxyphenylalanine by tyrosinase using the oxygen electrode is shown to confirm the validity of the calibration method.     

Chronic ethanol feeding causes oxidative stress in rat liver mitochondria. Prevention by S-adenosyl methionine

Oxygen utilisation during tyrosinase-catalysed oxidation of 4-hydroxyanisole was investigated using an electron spin resonance technique which employs quantitative changes in the characteristics of the electron spin resonance spectrum of the spin label 3-carbamoyl-2,5-dihydro-2,2,5–5-tetramethyl-1-H-pyridoyl-1-yloxy (CTPO) to follow changes in the oxygen concentration. Reaction mixtures containing mushroom tyrosinase (15 μg ml^?1) and differing initial concentrations of 4-hydroxyanisole in aerated phosphate buffer at pH 6.8 were incubated at room temperature. The ratio of utilisation of oxygen was found to be in approximately 1:1 molar ratio with the initial 4-hydroxyanisole concentration in the reaction mixture between 50 and 200 μmol/1 4-hydroxyanisole. The results are consistent with the stoichiometry of oxygen utilisation being accounted for by the oxidation of 4-hydroxyanisole to anisyl quinone.     

Model sclerotization studies. 4. Generation of N-acetylmethionyl catechol adducts during tyrosinase-catalyzed oxidation of catechols in the presence of N-acetylmethionine

Incubation of catechol with mushroom tyrosinase in the presence of N-acetylmethionine resulted in the generation of an adduct. This product was identified to be N-acetylmethionyl catechol, on the basis of spectral characteristics and well-characterized chemical reaction of o-benzoquinone with N-acetylmethionine. Enzyme-catalyzed oxidation of catechol and the subsequent nonenzymatic addition of the resultant quinone to N-acetylmethionine accounted for the observed reaction. That the reaction is not confined to catechol alone, but is of general occurrence, can be demonstrated by the facile generation of similar adducts in incubation mixtures containing N-acetylmethionine, tyrosinase, and different N-acetylmethionines, such as 4-methylcatechol and N-acetyldopamine. Attempts to duplicate the reaction with insect cuticular phenoloxidases were not successful, as the excess N-acetylmethionine used in the reaction inhibited their activity. Nevertheless, occurrence of this nonenzymatic reaction between N-acetylmethionine and mushroom tyrosinase-generated quinones indicates that a similar reaction between enzymatically generated quinones in the cuticle with protein-bound methionine moiety is likely to occur during in vivo quinone tanning as well. Arch. Insect Biochem. Physiol. 38:44–52, 1998. © 1998 Wiley-Liss, Inc.     

Effect of different isomers of dihydroxybenzoic acids (DBA) on the rate of DL-dopa oxidation by mushroom tyrosinase.

Dihydroxybenzoic acids (DBA), such as 3,4-DBA, 3,5-DBA, and 2,4-DBA--at all concentrations tested--inhibited the rate of DL-DOPA oxidation to dopachrome (lambda max = 475 nm) by mushroom tyro0sinase. 2,3-DBA and 2,5-DBA at relatively low concentration had a synergistic effect on the reaction, whereas at relatively high concentrations they inhibited the rate of DL-DOPA oxidation. The synergistic effect of 0.6-13.3 mM 2,3-DBA on the rate of DL-DOPA oxidation to dopachrome (lambda max = 475 nm) was found to be due to the ability of 2,3-DBA-o-quinone (formed by the oxidation of 2,3-DBA by mushroom tyrosinase or by sodium periodate) to oxidize DL-DOPA to dopachrome (via dopaquinone) non-enzymatically. A similar explanation is likely to be valid for the synergism exerted by 2,5-DBA on the rate of DL-DOPA oxidation by mushroom tyrosinase.     

Structural insights into the hot spot amino acid residues of mushroom tyrosinase for the bindings of thujaplicins

Tyrosinase inhibitors are important agents for cosmetic products. We examined here the inhibitory effects of three isomers of thujaplicins (α, β and γ) on mushroom tyrosinase and analyzed their binding modes using a homology model from the crystal structure of Streptomyces castaneoglobisporus tyrosinase (PDB ID: 1wx2). All the thujaplicins were found to be competitive inhibitors and γ-thujaplicin has the most potent inhibitory activity (IC(50)=0.07μM). It is noted that there are good correlations between their observed IC(50) values and their binding free energies calculated by MM-GB/SA. The binding modes of thujaplicins were predicted to be similar to that of Tyr98 of caddie protein (ORF378), which was co-crystallized with S. castaneoglobisporus tyrosinase. Furthermore, free energy decomposition analysis indicated that the potent inhibitory activity of γ-thujaplicin is due to the interactions with His242, Val243 and Pro257 (hot spot amino acid residues) at the active site of tyrosinase. These results provide a novel structural insight into the hot spot of mushroom tyrosinase for the specific binding of γ-thujaplicin.     

Effect of thiohydroxyl compounds on tyrosinase: inactivation and reactivation study

An unusual thioether bridge (Cys-His) has been detected at the active site of mushroom tyrosinase, and the effects of thiohydroxyl compounds such as dithiothreitol (DTT) and beta-mercaptoethanol (beta-ME) on Cu2+ at the active site have been elucidated. Treatment with DTT and beta-ME on mushroom tyrosinase completely inactivated 3,4-dihydroxyphenylalanine oxidase activity in a dose-dependent manner. Sequential kinetic studies revealed that DTT and beta-ME caused different mixed-type inhibition mechanisms: the slope-parabolic competitive inhibition (Ki = 0.143 mM) by DTT and slope-hyperbolic noncompetitive inhibition (Ki = 0.0128 mM) by beta-ME, respectively. Kinetic Scatchard analysis consistently showed that mushroom tyrosinase had multiple binding sites for DTT and beta-ME with different affinities. Reactivation study of inactivated enzyme by addition of Cu2+ confirmed that DTT and beta-ME directly bound with Cu2+ at the active site. Our results may provide useful information regarding interactions of tyrosinase inhibitor for designing an effective whitening agent targeted to the tyrosinase active site.