Jose Luis Muñoz-Muñoz

Suicide inactivation of the diphenolase and monophenolase activities of tyrosinase

The suicide inactivation mechanism of tyrosinase acting on its phenolic substrates has been studied. Kinetic analysis of the proposed mechanism during the transition phase provides explicit analytical expressions for the concentrations of o‐quinone versus time. The electronic, steric, and hydrophobic effects of the phenolic substrates influence the enzymatic reaction, increasing the catalytic speed by three orders of magnitude and the inactivation by one order of magnitude. To explain this suicide inactivation, we propose a mechanism in which the enzymatic form oxy‐tyrosinase is responsible for the inactivation. In this mechanism, the rate constant of the reaction would be directly related with the strength of the nucleophilic attack of the C‐1 hydroxyl group, which depends on the chemical shift of the carbon C‐1 (δ1) obtained by 13C‐NMR. The suicide inactivation would occur if the C‐2 hydroxyl group transferred the proton to the protonated peroxide, which would again act as a general base. In this case, the coplanarity between the copper atom, the oxygen of the C‐1 and the ring would only permit the oxidation/reduction of one copper atom, giving rise to copper (0), hydrogen peroxide, and an o‐quinone, which would be released, thus inactivating the enzyme. One possible application of this property could be the use of these suicide substrates as skin depigmenting agents.

Phenolic substrates and suicide inactivation of tyrosinase: kinetics and mechanism.

The suicide inactivation mechanism of tyrosinase acting on its substrates has been studied. The kinetic analysis of the proposed mechanism during the transition phase provides explicit analytical expressions for the concentrations of o-quinone against time. The electronic, steric and hydrophobic effects of the substrates influence the enzymatic reaction, increasing the catalytic speed by three orders of magnitude and the inactivation by one order of magnitude. To explain the suicide inactivation, we propose a mechanism in which the enzymatic form E(ox) (oxy-tyrosinase) is responsible for such inactivation. A key step might be the transfer of the C-1 hydroxyl group proton to the peroxide, which would act as a general base. Another essential step might be the axial attack of the o-diphenol on the copper atom. The rate constant of this reaction would be directly related to the strength of the nucleophilic attack of the C-1 hydroxyl group, which depends on the chemical shift of the carbon C-1 (delta(1)) obtained by (13)C-NMR. Protonation of the peroxide would bring the copper atoms together and encourage the diaxial nucleophilic attack of the C-2 hydroxyl group, facilitating the co-planarity with the ring of the copper atoms and the concerted oxidation/reduction reaction, and giving rise to an o-quinone. The suicide inactivation would occur if the C-2 hydroxyl group transferred the proton to the protonated peroxide, which would again act as a general base. In this case, the co-planarity between the copper atom, the oxygen of the C-1 and the ring would only permit the oxidation/reduction reaction on one copper atom, giving rise to copper(0), hydrogen peroxide and an o-quinone, which would be released, thus inactivating the enzyme.

Generation of hydrogen peroxide in the melanin biosynthesis pathway

The generation of H(2)O(2) in the melanin biosynthesis pathway is of great importance because of its great cytotoxic capacity. However, there is controversy concerning the way in which H(2)O(2) is generated in this pathway. In this work we demonstrate that it is generated in a series of chemical reactions coupled to the enzymatic formation of o-quinones by tyrosinase acting on monophenols and o-diphenols and during the auto-oxidation of the o-diphenols and other intermediates in the pathway. The use of the enzymes such as catalase, superoxide dismutase and peroxidase helps reveal the H(2)O(2) generated. Based on the results obtained, we propose a scheme of enzymatic and non-enzymatic reactions that lead to the biosynthesis of melanins, which explains the formation of H(2)O(2).

Quantification of the Antioxidant Capacity of Different Molecules and Their Kinetic Antioxidant Efficiencies

A kinetic method has been developed to determine the antioxidant capacity of a variety of molecules. In this method, named the enzymatic kinetic method, the free radical of ABTS is generated continuously in the reaction medium by a peroxidase/ABTS/H(2)O(2) system. The presence of an antioxidant in the solution provokes a lag period in the accumulation of the free radical in the medium, and by studying this lag period it is possible to calculate the antioxidant capacity of the molecule in question. This antioxidant capacity, named the primary antioxidant capacity, will be quantified by n, the number of electrons donated per molecule of antioxidant, the effective concentration, EC50, and the antioxidant or antiradical power (ARP) (ARP = 1/EC50 = 2n). If the products arising from the reaction between the antioxidant and the free radical evolve by consuming more radical, a secondary antioxidant capacity is generated. To calculate this, a nonenzymatic test is proposed.

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.

Kinetic Characterization of the Enzymatic and Chemical Oxidation of the Catechins in Green Tea

The oxidation of green tea catechins by polyphenol oxidase/O2 and peroxidase/H2O2 gives rise to o-quinones and semiquinones, respectively, which inestability, until now, have hindered the kinetic characterization of enzymatic oxidation of the catechins. To overcome this problem, ascorbic acid (AH2) was used as a coupled reagent, either measuring the disappearance of AH2 or using a chronometric method in which the time necessary for a fixed quantity of AH2 to be consumed was measured. In this way, it was possible to determine the kinetic constants characterizing the action of polyphenol oxidase and peroxidase toward these substrates. From the results obtained, (-) epicatechin was seen to be the best substrate for both enzymes with the OH group of the C ring in the cis position with respect to the B ring. The next best was (+) catechin with the OH group of the C ring in the trans position with respect to the B ring. Epigallocatechin, which should be in first place because of the presence of three vecinal hydroxyls in its structure (B ring), is not because of the steric hindrance resulting from the hydroxyl in the cis position in the C ring. The epicatechin gallate and epigallocatechin gallate are very poor substrates due to the presence of sterified gallic acid in the OH group of the C ring. In addition, the production of H2O2 in the auto-oxidation of the catechins by O2 was seen to be very low for (-) epicatechin and (+) catechin. However, its production from the o-quinones generated by oxidation with periodate was greater, underlining the importance of the evolution of the o-quinones in this process. When the [substrate] 0/[IO4 (-)] 0 ratio = 1 or >>1, H2O2 formation increases in cases of (-) epicatechin and (+) catechin and practically is not affected in cases involving epicatechin gallate, epigallocatechin, or epigallocatechin gallate. Moreover, the antioxidant power is greater for the gallates of green tea, probably because of the greater number of hydroxyl groups in its structure capable of sequestering and neutralizing free radicals. Therefore, we kinetically characterized the action of polyphenol oxidase and peroxidase on green tea catechins. Furthermore, the formation of H2O2 during the auto-oxidation of these compounds and during the evolution of their o-quinones is studied.

Stereospecific inactivation of tyrosinase by L- and D-ascorbic acid.

A kinetic study of the inactivation of tyrosinase by L- and D-ascorbic acid isomers has been carried out. In aerobic conditions, a suicide inactivation mechanism operates, which was attributed to the enzymatic form oxytyrosinase. This suicide inactivation is stereospecific as regards the affinity of the enzyme for the substrate but not as regards the speed of the process, which is the same for both isomers, reflecting the influence of the chemical shift of the carbon C-2 (delta(2)) and C-3 (delta(3)) as seen by (13)C-NMR. The inactivation of deoxytyrosinase and mettyrosinase observed in anaerobic conditions, is irreversible and faster than the suicide inactivation process, underlining the fact that the presence of oxygen protects the enzyme against inactivation.

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.

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.