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The development of effective tyrosinase inhibitors has become increasingly important in the cosmetic, medicinal, and agricultural industries for application as antibrowning and depigmenting agents. The kinetic mechanisms of action of tyrosinase on monophenols and o-diphenols are complex, particularly in the case of monophenols because of the lag period that occurs at the beginning of the reaction. When enzyme inhibitors are studied, the problem becomes more complicated because the lag period increases, which has led to erroneous identification of the type of inhibition that many compounds exert on the monophenolase activity and the inaccurate determination of their inhibition constants. When the degrees of inhibition of an inhibitor which is analogous to tyrosinase substrates are the same for both monophenolase and diphenolase activities, this means that the inhibitor binds to the same enzymatic species and so the inhibition constants should be similar for both activities. In this study, we demonstrate this typical behavior of substrate-analogous inhibitors and propose a methodology for determining the type of inhibition and the inhibition constants for the monophenolase and diphenolase activities of the enzyme. Benzoic acid and cinnamic acid were used as inhibitors and the monophenol/o-diphenol pairs l-tyrosine/l-dopa and α-methyl-L-tyrosine/α-methyl-L-dopa as substrates.

Many phenolic compounds have been described in the scientific literature as inhibitors of tyrosinase. In this work a test is proposed that allows us to distinguish whether a molecule is an enzyme inhibitor or substrate. The test has several stages. First, the degree of inhibition of the studied molecule is determined on the monophenolase activity (i(M)) and on the diphenolase activity (i(D)). If i(M) = i(D), it is an inhibitor. If i(M) ≠ i(D), the molecule could be substrate or inhibitor. Several additional stages are proposed to solve this ambiguity. The study described herein was carried out using the following molecules: benzoic acid, cinnamic acid, guaiacol, isoeugenol, carvacrol, 4-tert-butylphenol, eugenol, and arbutin.

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.

The formation of hydroxyhydroquinone during the action of tyrosinase on hydroquinone is demonstrated for the first time by means of high performance liquid chromatography mass spectrometry. A kinetic mechanism is proposed to explain this action in the presence of catalytic amounts of 4-tert-butylcatechol. Based on a kinetic analysis of this mechanism, an experimental design is proposed that permits the system to be characterized kinetically.

In this paper we extend to enzyme systems the results previously obtained in paper I of this series for linear compartmental systems. We obtain the time course equations for both the enzyme and ligand species involved in the reaction mechanisms, which fit a general enzyme system model when the connections between the different enzyme species are of first or pseudofirst order. The kinetic equations obtained here for a given species, enzyme or ligand have the advantage over all previous equations described in the literature, in that they are in the most simplified form possible, since they only contain the kinetic parameters and initial concentrations of the enzymatic reaction which really have some influence on the time progress curves of the species under study. These kinetic equations are denominated optimized equation to distinguish them from the others, which shall call non-optimized equations. We discuss those cases when both types of equation coincide and we show how, when they do not coincide, the non-optimized equations can be simplified to the optimized ones. Therefore, we show that the optimized equations could be used in all cases to avoid the need of subsequent simplifications to eliminate the parameters that play no role in the corresponding time equations. To illustrate the use of this procedure we will apply it to two simple examples of enzymatic reactions.

Software application is implemented in this work to take full advantage of the characteristics of current operating systems and to provide the optimized symbolic kinetic equations for both enzyme and ligand species involved in enzyme reactions. This software, called SKEE-w2013, is implemented using C# language and runs under all operating systems from Windows XP up to Windows 8. It is applicable to any enzyme reaction mechanism that fits the general reaction scheme proposed previously by our group. It can be downloaded, free of charge, from http://oretano.iele-ab.uclm.es/~BioChem-mg/software.php . Besides the optimized equations, the software can provide non-optimized equations, so that the user can compare the advantage of using optimized equations rather than the non-optimized ones, whenever they do not coincide. Moreover, the software circumvents the limitations of other existing previous software tools implemented with what are nowadays obsolete programming languages and that, moreover, are limited to non-optimized kinetic equations.

Hydroxyhydroquinone (HHQ) was characterized kinetically as a tyrosinase substrate. A kinetic mechanism is proposed, in which HHQ is considered as a monophenol or as an o-diphenol, depending on the part of the molecule that interacts with the enzyme. The kinetic parameters obtained from an analysis of the measurements of the initial steady state rate of 2-hydroxy p-benzoquinone formation were kcatapp=229.0±7.7 s(-1) and KMapp,HHQ=0.40±0.05 mM. Furthermore, the action of tyrosinase on HHQ led to the enzyme's inactivation through a suicide inactivation mechanism. This suicide inactivation process was characterized kinetically by λmaxapp (the apparent maximum inactivation constant) and r, the number of turnovers made by 1 mol of enzyme before being inactivated. The values of λmaxapp and r were (8.2±0.1)×10(-3) s(-1) and 35,740±2,548, respectively.

A solvent deuterium isotope effect on the inactivation suicide of tyrosinase in its action on o-diphenols (catechol, 4-methylcatechol, and 4-tert-butylcatechol) was observed. This isotope effect, observed during kinetic studies in the transition phase, was higher than that described previously in the steady state, indicating that there is an additional slow step in the suicide inactivation mechanism, which we believe to be responsible for the inactivation. In a proton inventory study of oxidation of o-diphenols, the representation of versus n (atom fractions of deuterium), where is the maximum apparent inactivation constant for a molar fraction of deuterium (n) and is the corresponding kinetic parameter in a water solution, was linear for all substrates. This suggests that only one of the protons transferred from the two hydroxyl groups of the substrate, which are oxidized in one turnover, is responsible for the isotope effects. We propose that this proton could be the proton transferred from the hydroxyl group of C-2 to the hydroperoxide of the oxytyrosinase form (Eox) and that it probably causes enzyme inactivation through the reduction of the Cu2+A to Cu0 and its subsequent release from the active site. © 2013 IUBMB Life, 65(9):793–799, 2013

A study of the diphenolase, o-aminophenol oxidase, and the aromatic o-diamine oxidase activities of tyrosinase carried out by measuring the catalysis and suicide inactivation kinetics provides the following information: catalytic constant, k cat S , Michaelis constant, K M S , the maximum apparent inactivation constant, λ max S , and the partition ratio “r” between the suicide inactivation pathway and catalytic pathway or the number of turnovers made by one mol of enzyme before its inactivation. Analysis of these data, taking into account chemical shifts of the carbon atom supporting the hydroxyl or amino group, (δ) and σ p + , enables a mechanism to be proposed for the transformation of o-diphenols, o-aminophenols and o-phenylendiamines into their products (o-quinones, o-quinoneimine and o-diimine), and, at the same time, for the suicide inactivation. The reaction constants in the representations of log k cat X / k cat H vs. σ p + according to Hammett's equation for the three types of substrate (o-diphenols, o-aminophenols and o-phenylendiamines) confirm that the catalysis mechanism is similar (simultaneous oxidation/reduction process on the two copper atoms). The dependence of log λ max X / λ max H vs. σ p + for the three substrate types reflects a lower reaction constant (in absolute value), which might indicate a similar reaction mechanism for the different molecules, but different from the previous mechanism, in that the oxidation/reduction only involves one copper atom. We also discuss the proposed mechanism and compare it with those described by other authors. Knowledge and quantification of the catalysis/inactivation process of tyrosinase might be of interest for optimizing applications such as wastewater treatment.

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.

The development of effective tyrosinase inhibitors has become increasingly important in the cosmetic, medicinal, and agricultural industries for application as antibrowning and depigmenting agents. The kinetic mechanisms of action of tyrosinase on monophenols and o-diphenols are complex, particularly in the case of monophenols because of the lag period that occurs at the beginning of the reaction. When enzyme inhibitors are studied, the problem becomes more complicated because the lag period increases, which has led to erroneous identification of the type of inhibition that many compounds exert on the monophenolase activity and the inaccurate determination of their inhibition constants. When the degrees of inhibition of an inhibitor which is analogous to tyrosinase substrates are the same for both monophenolase and diphenolase activities, this means that the inhibitor binds to the same enzymatic species and so the inhibition constants should be similar for both activities. In this study, we demonstrate this typical behavior of substrate-analogous inhibitors and propose a methodology for determining the type of inhibition and the inhibition constants for the monophenolase and diphenolase activities of the enzyme. Benzoic acid and cinnamic acid were used as inhibitors and the monophenol/o-diphenol pairs l-tyrosine/l-dopa and α-methyl-L-tyrosine/α-methyl-L-dopa as substrates.

Hydroxyhydroquinone (HHQ) was characterized kinetically as a tyrosinase substrate. A kinetic mechanism is proposed, in which HHQ is considered as a monophenol or as an o-diphenol, depending on the part of the molecule that interacts with the enzyme. The kinetic parameters obtained from an analysis of the measurements of the initial steady state rate of 2-hydroxy p-benzoquinone formation were kcatapp=229.0±7.7 s(-1) and KMapp,HHQ=0.40±0.05 mM. Furthermore, the action of tyrosinase on HHQ led to the enzyme's inactivation through a suicide inactivation mechanism. This suicide inactivation process was characterized kinetically by λmaxapp (the apparent maximum inactivation constant) and r, the number of turnovers made by 1 mol of enzyme before being inactivated. The values of λmaxapp and r were (8.2±0.1)×10(-3) s(-1) and 35,740±2,548, respectively.

Under anaerobic conditions, the o-diphenol 4-tert-butylcatechol (TBC) irreversibly inactivates met and deoxytyrosinase enzymatic forms of tyrosinase. However, the monophenol 4-tert-butylphenol (TBF) protects the enzyme from this inactivation. Under aerobic conditions, the enzyme suffers suicide inactivation when it acts on TBC. We suggest that TBF does not directly cause the suicide inactivation of the enzyme in the hydroxylase activity, but that the o-diphenol, which is necessary for the system to reach the steady state, is responsible for the process. Therefore, monophenols do not induce the suicide inactivation of tyrosinase in its hydroxylase activity, and there is a great difference between the monophenols that give rise to unstable o-quinones such as L-tyrosine, which rapidly accumulate L-dopa in the medium and those like TBF, after oxidation, give rise to a very stable o-quinone.

The action of tyrosinase on ortho-substituted monophenols (thymol, carvacrol, guaiacol, butylated hydroxyanisole, eugenol, and isoeugenol) was studied. These monophenols inhibit melanogenesis because they act as alternative substrates to L-tyrosine and L-Dopa in the monophenolase and diphenolase activities, respectively, despite the steric hindrance on the part of the substituent in ortho position with respect to the hydroxyl group. We kinetically characterize the action of tyrosinase on these substrates and assess its possible effect on browning and melanognesis. In general, these compounds are poor substrates of the enzyme, with high Michaelis constant values, K(m), and low catalytic constant values, k(cat), so that the catalytic efficiency k(cat)/K(m) is low: thymol, 161 ± 4 M(-1) s(-1); carvacrol, 95 ± 7 M(-1) s(-1); guaiacol, 1160 ± 101 M(-1) s(-1).

Captopril and mesna are molecules with a free thiol group, used as active ingredients due to their hypotensor and mucolytic properties, respectively. These compounds cross the hematoencephalic barrier and, due to the reactivity of their thiol group, can form adducts with the o-quinones formed during the oxidation of mono- and o-diphenols. Polyphenol oxidase from plants and fungi can be used as a tool for generating o-quinones in their action on o-diphenols and facilitate the formation of adducts in the presence of captopril or mesna. The spectrophotometric characterization of these adducts is useful from several points of view. Here, using the end-point method, which involves the exhaustion of oxygen in the medium, we determined the molar absorptivity of the adducts of different o-diphenols with captopril and mesna. Besides the analytical interest of this approach, we also use it to make a kinetic characterization of polyphenol oxidase as it acts on o-diphenolic substrates that produce unstable o-quinones.

Highlights: Black-Right-Pointing-Pointer The action the copper complexes and tyrosinase on phenols is equivalent. Black-Right-Pointing-Pointer Isotope effect showed that nucleophilic attack to copper atom may be the slower step. Black-Right-Pointing-Pointer The value of {rho} (Hammett constant) supports an electrophilic aromatic substitution. Black-Right-Pointing-Pointer Data obtained in steady state pH 7 conditions support the mechanism of Scheme 1SM. -- Abstract: A study of the monophenolase activity of tyrosinase by measuring the steady state rate with a group of p-substituted monophenols provides the following kinetic information: k{sub cat}{sup m} and the Michaelis constant, K{sub M}{sup m}. Analysis of these data taking into account chemical shifts of the carbon atom supporting the hydroxyl group ({delta}) and {sigma}{sub p}{sup +}, enables a mechanism to be proposed for the transformation of monophenols into o-diphenols, in which the first step is a nucleophilic attack on the copper atom on the form E{sub ox} (attack of the oxygen of the hydroxyl group of C-1 on the copper atom) followed by an electrophilic attack (attack of the hydroperoxide group on the ortho position with respect to the hydroxyl group of the benzene ring, electrophilic aromatic substitution with a reaction constant {rho} of -1.75). These steps show the same dependency onmore » the electronic effect of the substituent groups in C-4. Furthermore, a study of a solvent deuterium isotope effect on the oxidation of monophenols by tyrosinase points to an appreciable isotopic effect. In a proton inventory study with a series of p-substituted phenols, the representation of k{sub cat}{sup f{sub n}}/k{sub cat}{sup f{sub 0}} against n (atom fractions of deuterium), where k{sub cat}{sup f{sub n}} is the catalytic constant for a molar fraction of deuterium (n) and k{sub cat}{sup f{sub 0}} is the corresponding kinetic parameter in a water solution, was linear for all substrates. These results indicate that only one of the proton transfer processes from the hydroxyl groups involved the catalytic cycle is responsible for the isotope effects. We suggest that this step is the proton transfer from the hydroxyl group of C-1 to the peroxide of the oxytyrosinase form (E{sub ox}). After the nucleophilic attack, the incorporation of the oxygen in the benzene ring occurs by means of an electrophilic aromatic substitution mechanism in which there is no isotopic effect.« less