Maria del Mar Garcia-Molina

Action of tyrosinase on ortho-substituted phenols: possible influence on browning and melanogenesis.

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).

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  mS ), 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.

Tyrosinase-catalyzed hydroxylation of hydroquinone, a depigmenting agent, to hydroxyhydroquinone: A kinetic study

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.

Hydroxylation of p-substituted phenols by tyrosinase: Further insight into the mechanism of tyrosinase activity

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(cat)(m) and the Michaelis constant, K(M)(m). Analysis of these data taking into account chemical shifts of the carbon atom supporting the hydroxyl group (δ) and σ(p)(+), 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(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 ρ of -1.75). These steps show the same dependency on 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 [Formula: see text] / [Formula: see text] against n (atom fractions of deuterium), where [Formula: see text] is the catalytic constant for a molar fraction of deuterium (n) and [Formula: see text] 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(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.

Discrimination between Alternative Substrates and Inhibitors of Tyrosinase

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.

Action of tyrosinase on hydroquinone in the presence of catalytic amounts of o-diphenol. A kinetic study

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.

Identification of p-hydroxybenzyl alcohol, tyrosol, phloretin and its derivate phloridzin as tyrosinase substrates.

In recent years, the hydroxyalkylphenols p-hydroxybenzyl alcohol and tyrosol, and the compound phloretin and its derivate phloridzin have been described as inhibitors of the enzyme tyrosinase. When the monophenolase and the diphenolase activities of tyrosinase on its physiological substrates l-dopa and/or l-tyrosine are measured in the presence of these compounds, the rate of action of the enzyme decreases. These findings led to the identification of these compounds as inhibitors. However, these molecules show an unusual behavior as inhibitors of the enzyme indeed, in this study, we demonstrate that they are not true inhibitors but alternative substrates of the enzyme.

Catalysis and inactivation of tyrosinase in its action on hydroxyhydroquinone

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 enzymes 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.

Kinetic characterization of substrate-analogous inhibitors of tyrosinase

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.

Linear compartmental systems. IV. A software, under MS-Windows, for obtaining the instantaneous species concentrations in enzyme systems

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.