Tyrosinase Reaction Research Articles

Evaluation of the effect of red guava (<em>Psidium guajava</em>) fruit extract on tyrosinase (EC 1.14.18.1) activity by spectrophotometry

Tyrosinase is one of the most important enzymes in melanin biosynthesis. Inhibition of tyrosinase activity will cause a decrease in melanin production. Tyrosinase inhibitory activity by ascorbic acid has been studied before. Based on reported experiments, ascorbic acid can inhibit tyrosinase activity in enzymatic reaction competitively. As an effort to find out a skin whitening agent which is effective, safe and have minimum adverse effect, the inhibitory activities of Psidium guajava extract was studied on tyrosinase activity. This is one of the fruits that contains high amount of ascorbic acid. To determine the efficacy of tyrosinase inhibition, L-tyrosine was used as the substrate, P. guajava ex-tract as inhibitor and ascorbic acid as positive control that was measured by spectrophotometry. The optimization of method was also performed. The inhibitory kinetics was determined by measuring the absorbance of dopachrome as the end product of the tyrosinase reaction. Michaelis-Menten constant (Km) and maximum velocity (Vmax) of the ty-rosinase were determined by Lineweaver-Burk’s plots. The Km and Vmax without the fruit extract were 0.315mM and 0.0265?mol/min. The Km value with the fruit extract of 1%, 2% & 3% w/v were 0.4824, 0.698 & 0.543mM, while the Vmax value were 0.0269, 0.0283 & 0.0255 ?mol/min, respectively. Lineweaver-Burk’s plots in presence of P. guajava fruit extract showed that the extract inhibited tyrosinase competitively. From the plots, the IC50 of the fruit extract was determined as 0.26mM; the control in 0.26mM concentration inhibited 56.523%. Finally, P. guajava fruit extract showed higher effect than ascorbic acid on Tyrosinase activity.DOI: http://dx.doi.org/10.3329/icpj.v1i5.10280International Current Pharmaceutical Journal 2012, 1(5): 92-97

The First Catalytic Tyrosinase Model System Based on a Mononuclear Copper(I) Complex: Kinetics and Mechanism

Ready…︁ steady…︁ go! The copper(I) complex 1 not only catalyzes the oxygenation of di-tert-butylphenol (DTBP-H) to di-tert-butylquinone (DTBQ) in a tyrosinase-like fashion, but also allows the reactive cycle to be studied in a stepwise and controlled manner. This feature opens new insights into the individual stages of the tyrosinase reaction, phenol hydroxylation, and release of the product as quinone. The implications for the enzymatic reaction are discussed.

Reexamination of the mechanisms of oxidative transformation of the insect cuticular sclerotizing precursor, 1,2-dehydro-N-acetyldopamine

1,2-dehydro-N-acetyldopamine (dehydro NADA) is an important catecholamine derivative formed during the sclerotization of insect cuticle. Earlier we have reported that tyrosinase-catalyzed oxidation of dehydro NADA produces a reactive quinone methide imine amide that forms adducts and cross-links through its side chain, thereby accounting for sclerotization reactions. Recently, laccase has also been identified as a key enzyme associated with sclerotization. Hence, we re-examined oxidation of dehydro NADA by tyrosinase and laccase using high performance liquid chromatography – tandem mass spectrometry. Tyrosinase-catalyzed oxidation of dehydro NADA not only generated dimers as reported earlier, but also generated significant amounts of oligomers. The course of laccase-catalyzed oxidation of dehydro NADA significantly differed from the tyrosinase reaction kinetically and mechanistically. Laccase failed to produce any detectable quinone or quinone methide as the primary two-electron oxidation product. Since laccases are known to generate primarily semiquinones as the initial products, lack of accumulation of two-electron oxidation products indicated that laccase reaction is primarily occurring via free radical coupling mechanism. Consistent with this proposal, laccase-catalyzed oxidation of dehydro NADA, resulted in the production of largely dimeric products and failed to produce any significant amount of oligomeric materials. These studies call for radical coupling as yet another major mechanism for sclerotization of insect cuticle.

Dopamine- and tyramine-based derivatives of triazenes: Activation by tyrosinase and implications for prodrug design

A range of triazene derivatives were synthesized and investigated as prodrug candidates for melanocyte-directed enzyme prodrug therapy (MDEPT). The prodrugs contained a tyramine or dopamine promoiety required for tyrosinase activation and this was joined via a urea functional group to the cytotoxic triazene. The stability of each of the prodrugs in phosphate buffer, human plasma and in mushroom tyrosinase is discussed. The identification of the main peak formed after the tyrosinase reaction was attempted by LC–MS and the conversion of prodrug to the quinone was confirmed.

Ellagic acid: Characterization as substrate of polyphenol oxidase

Ellagic acid has been described as an inhibitor of tyrosinase or polyphenol oxidase and, therefore, of melanogenesis. In this work, we demonstrate that ellagic acid is not an inhibitor, but a substrate of mushroom polyphenol oxidase, an enzyme which oxidizes ellagic acid, generating its o-quinone. Because o-quinones are very unstable, we used an oxymetric method to characterize the kinetics of this substrate, based on measurements of the oxygen consumed in the tyrosinase reaction. The catalytic constant is very low at both pH values used in this work (4.5 and 7.0), which means that the Michaelis constant for the oxygen is low. The affinity of the enzyme for the substrate is high (low K(m) (S)), showing the double possibility of binding the substrate. Moreover, a new enzymatic method is applied for determining the antioxidant activity. Ellagic acid shows high antioxidant activity (EC50 = 0.05; number of electrons consumed by molecule of antioxidant = 10), probably because of the greater number of hydroxyl groups in its structure capable of sequestering and neutralizing free radicals.

Inhibitory Mechanism of Melanin Synthesis by Glutathione

Glutathione dose dependently inhibited melanin synthesis in the reaction of tyrosinase and L-DOPA. The inhibition of melanin synthesis was recovered by increasing the concentration of L-DOPA, but not recovered by increasing tyrosinase. Glutathione inhibited the binding between tyrosinase and L-DOPA. Although the synthesized melanin was aggregated within 1 h, the aggregation was inhibited by the addition of glutathione. These results indicate that glutathione inhibits the synthesis and agglutination of melanin by interrupting the function of L-DOPA.

Modulating effects of a novel skin-lightening agent, α-lipoic acid derivative, on melanin production by the formation of DOPA conjugate products

Sodium zinc dihydrolipoylhistidinate (DHLHZn) is a compound of Zn 2+/dihydrolipoic acid derivate complex, which was developed for cosmetic/medical use. To characterize DHLHZn as a novel skin-lightening agent, inhibitory actions of DHLHZn on tyrosinase (including its reaction pathway) have been elucidated in this study. In a B16 melanoma cell system, DHLHZn was active in suppressing the synthesis of melanins as well as α-arbutin, well known as a depigmenting drug. Furthermore, in a tyrosinase assay, DHLHZn showed stronger inhibitory effect on DOPAchrome formation than other tyrosinase inhibitors such as kojic acid. Our previous report demonstrated that the sulfhydryl groups of lipoyl motif react with DOPAquinone to form lipoyl DOPA conjugates. We therefore postulated that conjugated products between DHLHZn and DOPAquinone might be formed. Upon reaction of DHLHZn with l-DOPA following tyrosinase-catalyzed oxidation, the formation of DHLH DOPA conjugated products was confirmed by HPLC-tandem mass spectrometry using reserpine as the internal standard. In addition, the inhibitory kinetics analyzed by a Lineweaver–Burk plot exhibited the reversibility of DHLHZn as a competitive inhibitor with a K I value of 0.35 μM. These results indicate that this covalent reaction might contribute to alternating DOPAquinone, which is a tyrosinase reaction product, and result in the competitive inhibitory effect of DHLHZn on DOPAchrome formation. DHLHZn may thus serve as a potentially effective skin-lightening agent, an effectiveness that is based on the compound’s covalent scavenging of DOPAquinone resulting in depigmentation.

Enzymatic synthesis of arbutin undecylenic acid ester and its inhibitory effect on mushroom tyrosinase

A novel tyrosinase inhibitor, an arbutin derivative having undecylenic acid at the 6-position of its glucose moiety, was enzymatically synthesized. Its inhibitory activity was studied in vitro by using catechol and phenol as substrates. The IC(50) value of the arbutin ester on tyrosinase using catechol (4 x 10(-4) M) was 1% of that when arbutin (4 x 10(-2) M) was used. Using phenol, IC(50) of the arbutin ester (3 x 10(-4) M) as substrate was 10% of that of arbutin (3 x 10(-3) M). These results suggest that the arbutin ester inhibits the latter part of the tyrosinase reaction, which consists of hydroxylation and oxidation.

Significant Enhancement of Monooxygenase Activity of Oxygen Carrier Protein Hemocyanin by Urea

Oxygenation of a series of p-substituted phenols to the corresponding catechols (phenolase activity) by the (mu-eta2:eta2-peroxo)dicopper(II) species of Octopus hemocyanin has been directly examined for the first time by using a UV-vis spectroscopic method in a 0.5 M borate buffer solution containing 8 M urea under anaerobic conditions. Preliminary kinetic studies have indicated that the reaction involves an electrophilic aromatic substitution mechanism as in the case of phenolase reaction of tyrosinase. The oxygenation of phenols by hemocyanin also proceeded catalytically when the reaction was carried out under aerobic conditions.

Tyrosinase scavenges tyrosyl radical

Melanosomes scavenged tyrosyl radical that was generated by ultraviolet irradiation of tyrosine. Purified mushroom tyrosinase also removed tyrosyl radical in a dose-dependent manner. To elucidate the underlying mechanism, we analyzed the reaction of mushroom tyrosinase with tyrosyl radical generated by horseradish peroxidase and hydrogen peroxide. Resting tyrosinase, which contained a small amount of oxytyrosinase, did not oxidize tyrosine to DOPAchrome until horseradish peroxidase exhausted H 2O 2 and thereafter the enzyme recovered its full activity. During the inhibition period most tyrosine was converted to dityrosine, suggesting that only a small amount of tyrosyl radical was enough to interact with a fraction of tyrosinase which was in the active oxy-form. When horseradish peroxidase and H 2O 2 were added to oxytyrosinase, which was prepared by allowing it to turn over beforehand, DOPAchrome production was abolished with an accelerated consumption of H 2O 2. Dityrosine formation was totally suppressed and tyrosine concentration stayed constant during the inhibition period with a concomitant production of O 2. The results are accounted for by a mechanism in which tyrosyl radical is reduced to tyrosine by oxytyrosinase and the resulting met-form reacts with H 2O 2 to return to the oxy-form.

Kinetic evaluation of phenolase activity of tyrosinase using simplified catalytic reaction system.

A very simple tyrosinase reaction system has been developed using borate anion as a trapping agent of catechols and hydroxylamine as an external reductant to evaluate the phenolase activity without the interference of catecholase activity. Reactivities of variously para-substituted phenols in this system were compared directly to those of the phenols in the model reactions, demonstrating that the enzymatic oxygenation reaction of phenols proceeds via the same mechanism as the model reaction, that is, electrophilic aromatic substitution mechanism.

Catecholate Adducts of Binuclear Copper Complexes Modelling the Type 3 Copper Active Site — Spectroscopic Characterization and Relevance to the Tyrosinase Reaction

AbstractThe p‐nitrochatecholate (NCat) adducts 2 and 4 of the two copper(II) complexes [Cu2(L66)](ClO4)4 (1; L66 = α, α′‐bis{bis[2‐(1′‐methyl‐2′‐benzimidazolyl)ethyl]amino}‐m‐xylene) and [Cu(L6)](ClO4)2 (3; L6 = N, N‐bis[2‐(1′‐methyl‐2′‐benzimidazolyl)ethyl]amine), respectively, have been prepared and investigated by infrared, Raman and electronic absorption spectroscopies. The combined spectroscopic information suggests that the binding mode of p‐nitrocatecholate in 2 and 4 is similar for both adducts although for catecholate complexes of mononuclear Cu complexes like 4 an η2 coordination applies and for binuclear copper sites like 2 an η1:η1 bridging modes has been postulated. In addition a double catecholate adduct of 1, [Cu2(L66)(NCat)2] (5), has been prepared which again shows spectral features similar to 1 and 3. Based on these results it is suggested in all cases catecholate is binding in an η2 mode to one copper atom, eventually exhibiting an additional η1 bridging coordination to a second copper atom. This arrangement also plays an important role in inhibitor complexes of type 3 copper proteins like tyrosinase and catechol oxidase. The relevance of the η1:η2 binding mode of catecholate to the mechanism of the tyrosinase reaction is discussed.

Dioxygen Activation by Copper Complexes. Mechanistic Insights into Copper Monooxygenases and Copper Oxidases

Abstract Reactions of copper(I) complexes with molecular oxygen have been examined using a series of N-alkyl-bis[2-(2-pyridyl)ethyl]amine tridentate ligands (R′Py2R) and N,N-dialkyl-2-(2-pyridyl)ethylamine didentate ligands (R′Py1R1,R2) at low temperature. The tridentate ligands predominantly provide (μ-η2 : η2-peroxo)dicopper(II) complexes (side-on type peroxo complex), while the didentate ligands enhance O–O bond homolysis of the peroxo species to produce bis(μ-oxo)dicopper(III) complexes. With the (μ-η2 : η2-peroxo)dicopper(II) complexes supported by the tridentate ligand, efficient oxygenation of phenolates to the corresponding catechols has been accomplished to provide a good model reaction of tyrosinase. The bis(μ-oxo)dicopper(III) complexes, on the other hand, undergo aliphatic ligand hydroxylation as well as oxygen atom transfer to sulfides to give the corresponding sulfoxides. In the reaction of bis(μ-oxo)dicopper(III) complex with 10-methyl-9,10-dihydroacridine (AcrH2) and 1,4-cyclohexadiene (CHD), a new active oxygen intermediate such as a (μ-oxo)(μ-oxyl radical)dicopper(III) or a tetranuclear copper-oxygen complex has been suggested to be involved as the real active oxygen species for the C–H bond activation of the external substrates. A mixed valence bis(μ3-oxo) trinuclear copper(II,II,III) complex has also been assessed using the didentate ligand with the smallest N-alkyl substituent (methyl). Mechanistic details of the above reactions as well as ligand effects on the copper(I)-dioxygen reactivity are discussed systematically.

A New CuI Complex that Mimics the Cresolase Reaction of Tyrosinase and the Crystal Structure of its Oxygenated CuII Complex

A new dinuclear CuII complex with a unique structural arrangement and containing a doubly bridged macrocyclic ligand has been prepared from two different routes, one of which involves the reaction of oxygen and a CuI precursor, thus mimicking the cresolase activity of tyrosinase

Composite electrochemical biosensors: a comparison of three different electrode matrices for the construction of amperometric tyrosinase biosensors

A comparison of the behaviour of three different rigid composite matrices for the construction of amperometric tyrosinase biosensors, which are widely used for the detection of phenolic compounds, is reported. The composite electrode matrices were, graphite–Teflon; reticulated vitreous carbon (RVC)–epoxy resin; and graphite–ethylene/propylene/diene (EPD) terpolymer. After optimization of the experimental conditions, different aspects regarding the stability of the three composite tyrosinase electrode designs were considered and compared. A better reproducibility of the amperometric responses was found with the graphite–EPD electrodes, whereas a longer useful lifetime was observed for the graphite–Teflon electrodes. The kinetic parameters of the tyrosinase reaction were calculated for eight different phenolic compounds, as well as their corresponding calibration plots. The general trend in sensitivity was graphite–EPD>graphite–Teflon≫RVC–epoxy resin. A correlation between sensitivity and the catalytic efficiency of the enzyme reaction for each phenolic substrate was found. Furthermore, differences in the sensitivity order for the phenolic compounds were observed among the three biocomposite electrodes, which suggests that the nature of the electrode matrix influences the interactions in the tyrosinase catalytic cycle.

Functional Expression and Characterization of Aedes aegypti Dopachrome Conversion Enzyme

A full-length mosquito dopachrome conversion enzyme (DCE) and its truncated form lacking the last 54 carboxyl-terminal amino acid residues are expressed using a baculovirus/insect cell expression system. The full-length recombinant DCE displayed multiple bands during native PAGE with substrate staining, but only one active band was detected when the truncated recombinant DCE was analyzed under identical analysis conditions. Our data suggest that the last 50 some carboxyl-terminal residues are involved in the polymerization of the DCE molecules and that the proposed DCE isozymes likely reflect the presence of multimers of the same DCE molecules. The significance of the recombinant DCE in accelerating the melanization pathway is demonstrated by a rapid production of melanin in a dopa and tyrosinase reaction mixture in the presence of recombinant DCE. The DCE sequence data obtained in our previous study, together with results of functional expression and biochemical characterization achieved in this study, provide a necessary reference for the study of other insect DCEs.

Formation of nitric oxide from nitroxyl anion: role of quinones and ferricytochrome c.

1. Our previous finding that copper ions oxidize nitroxyl anion released from Angeli's salt to nitric oxide prompted us to examine if copper-containing enzymes shared this property. 2. The copper-containing enzyme, tyrosinase, which catalyses the hydroxylation of monophenols to diphenols and the subsequent oxidation of these to the respective unstable quinone, failed to generate nitric oxide from Angeli's salt by itself, but did so in the presence of tyrosine. 3. L-DOPA, the initial product of the reaction of tyrosinase with tyrosine, was not the active species, since it failed to generate nitric oxide from Angeli's salt. Nevertheless, L-DOPA and two other substrates, namely, catechol and tyramine did produce nitric oxide from Angeli's salt in the presence of tyrosinase, suggesting involvement of the respective unstable quinones. In support, we found that 1,4-benzoquinone produced a powerful nitric oxide signal from Angeli's salt. 4. Coenzyme Q(o), an analogue of ubiquinone, failed to generate nitric oxide from Angeli's salt by itself, but produced a powerful signal in the presence of its mitochondrial complex III cofactor, ferricytochrome c. 5. Experiments conducted on rat aortic rings with the mitochondrial complex III inhibitor, myxothiazol, to determine if this pathway was responsible for the vascular conversion of nitroxyl to nitric oxide were equivocal: relaxation to Angeli's salt was inhibited but so too was that to unrelated relaxants. 6. Thus, certain quinones oxidize nitroxyl to nitric oxide. Further work is required to determine if endogenous quinones contribute to the relaxant actions of nitroxyl donors such as Angeli's salt.

Hydrogen Peroxide Sensor with Bienzyme Membrane Based on Antagonism of Peroxidase Reaction to Tyrosinase Reaction Using Common Substrate

The oxidations of catechol to 1,2-benzoquinone by oxygen and hydrogen peroxide are catalyzed by tyrosinase and peroxidase, respectively. The tyrosinase reaction consumes oxygen but peroxidase reaction does not. Then, when hydrogen peroxide is added to the peroxidase containing solution in which tyrosinase reaction of catechol had taken place the consumption rate of oxygen in tyrosinase reaction should be decreased by the commencing peroxidase reaction because catechol works as a common substrate of both enzymatic reactions. In this work, the hydrogen peroxide sensor was fabricated by combining a bienzyme membrane with an oxygen electrode. This sensor response increased proportional to the added H2O2 (linear concentration range is from 5×10–6 M to 2×10–4 M with a detection limit of 2×10–6 M). The use of the oxygen electrode with gas permeable membrane should increase the selectivity of the present H2O2 sensor because nonvolatile electroactive species in the sample solution are not detected.

Hydrogen Peroxide Sensor with Bienzyme Membrane Based on Antagonism of Peroxidase Reaction to Tyrosinase Reaction Using Common Substrate

Hydrogen Peroxide Sensor with Bienzyme Membrane Based on Antagonism of Peroxidase Reaction to Tyrosinase Reaction Using Common Substrate

Tyrosinase inactivation in organic solvents

The inactivation of the catecholase activity of mushroom tyrosinase was investigated under nonaqueous conditions. The enzyme was immobilized on glass beads, and assays were conducted in chloroform, toluene, amyl acetate, isopropyl ether, and butanol. The reaction components were pre-equilibrated for 2 weeks with a saturated salt solution at a water activity of 0.90. The initial reaction velocity varied between 1.3 x 10(3) mol product/((mol enzyme)(min)) in toluene and 8.7 x 10(3) mol product/((mol enzyme)(min)) in amyl acetate. The turnover number varied between 8.1 x 10(3) mol product/mol enzyme in toluene and 7.2 x 10(4) mol product/mol enzyme in amyl acetate. In each solvent, the tyrosinase reaction inactivation parameters were represented by a probabilistic model. Changes in the probability of inactivation were followed throughout the course of the reaction using a second model which relates the reaction velocity to the amount of product formed. These models reveal that the inactivation rate of tyrosinase decreases as the reaction progresses, and that the inactivation kinetics are independent of the quinone concentration in toluene, chloroform, butanol, and amyl acetate. Significant effects of quinone concentration were, however, observed in isopropyl ether. The likelihood of inactivation of the enzyme was found to be greatest toward the beginning of the reaction. In the latter phase of the reaction, inactivation probability was less and tended to remain constant until the completion of the reaction.