Papers by JOSE LUIS MUÑOZ
Molecules
Tyrosinase is the enzyme involved in melanization and is also responsible for the browning of fru... more Tyrosinase is the enzyme involved in melanization and is also responsible for the browning of fruits and vegetables. Control of its activity can be carried out using inhibitors, which is interesting in terms of quantitatively understanding the action of these regulators. In the study of the inhibition of the diphenolase activity of tyrosinase, it is intriguing to know the strength and type of inhibition. The strength is indicated by the value of the inhibition constant(s), and the type can be, in a first approximation: competitive, non-competitive, uncompetitive and mixed. In this work, it is proposed to calculate the degree of inhibition (iD), varying the concentration of inhibitor to a fixed concentration of substrate, L-dopa (D). The non-linear regression adjustment of iD with respect to the initial inhibitor concentration [I]0 allows for the calculation of the inhibitor concentration necessary to inhibit the activity by 50%, at a given substrate concentration (IC50), thus avoidi...
International Journal of Biological Macromolecules, 2020
The pathways of melanization and sclerotization of the cuticle in insects are carried out by the ... more The pathways of melanization and sclerotization of the cuticle in insects are carried out by the action of laccases on dopamine and related compounds. In this work, the laccase action of Trametes versicolor (TvL) on catecholamines and related compounds has been kinetically characterized. Among them, dopamine, L-dopa, Lepinephrine, L-norepinephrine, L-isoprenaline L-α-methyldopa and L-dopa methylester. A chronometric method has been used for this characterization, with the help of a small amount of ascorbic acid. The use of TvL has allowed docking studies of these molecules to be carried out at the active site of this enzyme. The hydrogen bridge interaction between the hydroxyl oxygen at C-4 with His-458, and with the acid group of Asp-206, would make it possible to transfer the electron to the copper centre of the enzyme. The presence of an isopropyl group bound to nitrogen (isoprenaline) makes it especially difficult to catalyse. The formation of the ester (L-dopa methyl ester) practically does not affect catalysis. The addition of a methyl group (α-methyl dopa) increases the rate but decreases the affinity for catalysis. L-epinephrine and Lnorepinephrine have a similar affinity to isoprenaline, but faster catalysis, probably due to the greater nucleophilic power of their phenolic hydroxyl.
International Journal of Biological Macromolecules, 2019
We studied the laccase-catalysed oxygenation of methoxyphenolic food ingredients, such as 2-metho... more We studied the laccase-catalysed oxygenation of methoxyphenolic food ingredients, such as 2-methoxyphenol (guaiacol) and 2,6-dimethoxyphenol (syringol), isomers such as 3- and 4-methoxyphenol, and 2,3-, 3,4- and 3,5-dimethoxyphenol. These methoxyphenolic substrates generate unstable free radicals, which leads to the erroneous determination of steady state rates. The addition of small quantities of ascorbic acid as coupling reagent generates a lag period because it reduces free radicals to methoxyphenols. Measurement of the length of the lag period provides the reliable determination of true steady state rates. We describe the application of this chronometric method to the kinetic characterization of the oxidation of the above methoxyphenolic substrates by Trametes versicolor laccase.
Acta Biochimica Polonica, 2011
Tyrosinase shows kinetic cooperativity in its action on o-diphenols, but not when it acts on mono... more Tyrosinase shows kinetic cooperativity in its action on o-diphenols, but not when it acts on monophenols, confirming that the slow step is the hydroxylation of monophenols to o-diphenols. This model can be generalised to a wide range of substrates; for example, type S(A) substrates, which give rise to a stable product as the o-quinone evolves by means of a first or pseudo first order reaction (α-methyl dopa, dopa methyl ester, dopamine, 3,4-dihydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid, α-methyl-tyrosine, tyrosine methyl ester, tyramine, 4-hydroxyphenylpropionic acid and 4-hydroxyphenylacetic acid), type S(B) substrates, which include those whose o-quinone evolves with no clear stoichiometry (catechol, 4-methylcatechol, phenol and p-cresol) and, lastly, type S(C) substrates, which give rise to stable o-quinones (4-tert-butylcatechol/4-tert-butylphenol).
Journal of Molecular Catalysis B: Enzymatic, 2016
Abstract The pH has a great physiological effect on the catalysis of tyrosinase because protons c... more Abstract The pH has a great physiological effect on the catalysis of tyrosinase because protons can act as competitive-type inhibitors. In this work, a reaction mechanism is proposed whereby only the protonation of the free forms Em (met-tyrosinase) and Eox (oxy-tyrosinase) has a significant kinetic effect. The pKa value calculated for tyrosinase is 4.63 ± 0.04. This pKa value could correspond to a residue that acts as a gatekeeper of the active site, controlling the access of the substrate depending on its protonation state. Regarding the location and the nature of the responsible residue, we propose that glutamic acid E322 could be a key residue in the pH effect on the enzyme activity. Moreover, the substrate nature has a large effect on the kinetic behavior as a function of pH, depending on the pKa values of the phenolic hydroxyl groups and, to a lesser extent, on the charge of the R group. The sigmoid behavior of the initial rate vs. pH due to the pKa of the enzyme in the pH range studied could change to a bell-shaped behavior when the pKa values of the hydroxyl groups are low enough, and the R group is negatively charged. These aspects are interesting for controlling food browning and understanding the relationship melanosome pH plays in human skin pigmentation.
Reaction Kinetics, Mechanisms and Catalysis, 2014
ABSTRACT The formation of hydroxyhydroquinone during the action of tyrosinase on hydroquinone is ... more ABSTRACT 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.
Bioorganic & Medicinal Chemistry, 2014
Hydroquinone (HQ) is used as a depigmenting agent. In this work we demonstrate that tyrosinase hy... more 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.
Journal of Molecular Catalysis B: Enzymatic, 2012
ABSTRACT The suicide inactivation kinetics of tyrosinase acting on 3-isopropyl-6-methylcatechol, ... more ABSTRACT The suicide inactivation kinetics of tyrosinase acting on 3-isopropyl-6-methylcatechol, 3-tert-butyl-6-methylcatechol and 3,6-difluorocatechol was studied. All three substrates act as suicide substrates despite the fact that their 3 and 6 positions are occupied, confirming the mechanism proposed in Biochem. J. (2008) 416, 431-440. Although the most active substrate for the suicide inactivation was 3,6-difluorocatechol, its efficiency was much lower than that of the catechol used as reference. Its r value, the number of turnovers made by one mol of enzyme before its inactivation, is the highest described in the bibliography, highlighting the great difference between the catalytic and inactivation constants. A study of the effect of the pH on the enzymatic activity of tyrosinase showed that both 3-isopropyl-6-methylcatechol and 3-tert-butyl-6-methylcatechol behave as typical substrates of tyrosinase, while 3,6-difluorocatechol behaves differently. The remarkable behavior of 3,6-difluorocatechol when reacts with tyrosinase may be due to the fact that its two hydroxyl groups have very low pK values as a result of the strong electron-withdrawing effect of the fluorine atoms in the ortho positions, so that, at pH 7.0, the substrate would be mainly negatively charged. The apparent Michaelis constant shows a minimum value at pH 6.0, but increases at both high and low pH. However, the values of the catalytic constant and maximum apparent inactivation constant do not vary with the pH, so that the r remains practically constant. Under anaerobic conditions, 3,6-difluorocatechol acts as an irreversible inhibitor of the deoxy- and met-tyrosinase forms. (C) 2011 Elsevier B.V. All rights reserved.
Journal of Mathematical Chemistry, 2010
Tyrosinase has two types of enzymatic activities: the hydroxylation of monophenols to o-diphenols... more Tyrosinase has two types of enzymatic activities: the hydroxylation of monophenols to o-diphenols (monophenolase activity) and oxidation of o-diphenols to o-quinones (diphenolase activity). The action on o-diphenols involves two substrates: oxygen and o-diphenol, while the mechanism proposed is a Uni Uni Bi Bi ping-pong. In this contribution, we demonstrate experimentally that there is a kinetically preferred pathway, which translates into the appearance of curves of initial velocity vs. initial diphenol concentration shows inhibition by an excess of substrate, while sigmoid curves are obtained when the initial velocity vs. initial oxygen concentration are graphed. However, the action mechanism of the enzyme on monophenols, which is more complex because it involves three substrates (monophenol, oxygen and o-diphenol), does behave differently from the hyperbolic behaviour as regards the initial velocity vs. initial monophenol concentration, results that can be explained if the limiting step in the action of tyrosinase is the hydroxylation of monophenol to o-diphenol.
Journal of Enzyme Inhibition and Medicinal Chemistry, 2013
Under anaerobic conditions, the o-diphenol 4-tert-butylcatechol (TBC) irreversibly inactivates me... more 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.
Journal of Enzyme Inhibition and Medicinal Chemistry, 2011
Tetrahydrobiopterin (BH 4), methyl-tetrahydropterin (MBH 4) and dimethyl-tetrahydropterin (DMBH 4... more Tetrahydrobiopterin (BH 4), methyl-tetrahydropterin (MBH 4) and dimethyl-tetrahydropterin (DMBH 4) are oxidized by tyrosinase in a process during which the suicide inactivation of tyrosinase may occur. From the kinetic study of this process, l E S ox R (max) (apparent maximum constant for the suicide inactivation), K m S R (Michaelis constant for the substrate) and r (number of turnovers that the enzyme makes before the inactivation) can be obtained. From the results obtained, it can be deduced that the velocity of the inactivation governed by (l E
Journal of Agricultural and Food Chemistry, 2011
The coenzyme tetrahydrofolic acid is the most rapid suicide substrate of tyrosinase that has been... more The coenzyme tetrahydrofolic acid is the most rapid suicide substrate of tyrosinase that has been characterized to date. A kinetic study of the suicide inactivation process provides the kinetic constants that characterize it: λ max , the maximum apparent inactivation constant; r, the partition ratio or the number of turnovers made by one enzyme molecule before inactivation; and k cat and K m , the catalytic and Michaelis constants, respectively. From these values, it is possible to establish the ratio λ max /K m , which represents the potency of the inactivation process. Besides acting as a suicide substrate of tyrosinase, tetrahydrofolic acid reduces o-quinones generated by the enzyme in its action on substrates, such as L-tyrosine and L-DOPA (o-dopaquinone), thus inhibiting enzymatic browning.
Journal of Agricultural and Food Chemistry, 2012
The action of tyrosinase on ortho-substituted monophenols (thymol, carvacrol, guaiacol, butylated... more 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
IUBMB Life, 2014
Hydroxyhydroquinone (HHQ) was characterized kinetically as a tyrosinase substrate. A kinetic mech... more 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 k app cat 5 229.0 6 7.7 s 21 and K app;HHQ M 5 0.40 6 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 k app max (the apparent maximum inactivation constant) and r, the number of turnovers made by 1 mol of enzyme before being inactivated. The values of k app max and r were (8.2 6 0.1) 3
IUBMB Life, 2010
The suicide inactivation mechanism of tyrosinase acting on its phenolic substrates has been studi... more 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 (delta(1)) obtained by (13)C-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.
IUBMB Life, 2009
Ellagic acid has been described as an inhibitor of tyrosinase or polyphenol oxidase and, therefor... more 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 S m), 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.
IUBMB Life, 2013
A solvent deuterium isotope effect on the inactivation suicide of tyrosinase in its action on o-d... more 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 k D;fn max =k D;f0 max versus n (atom fractions of deuterium), where k D;fn max is the maximum apparent inactivation constant for a molar fraction of deuterium (n) and k D;f0 max 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 thisproton could be the proton transferred from the hydroxyl group of C-2 to the hydroperoxide of the oxytyrosinase form (E ox) and that it probably causes enzyme inactivation through the reduction of the Cu 21 A to Cu 0 and its subsequent release from the active site.
Biochemical Journal, 2008
The suicide inactivation mechanism of tyrosinase acting on its substrates has been studied. The k... more 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 Eox (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...
Biochemical and Biophysical Research Communications, 2012
A study of the monophenolase activity of tyrosinase by measuring the steady state rate with a gro... more A study of the monophenolase activity of tyrosinase by measuring the steady state rate with a group of psubstituted monophenols provides the following kinetic information: k m cat and the Michaelis constant, K m M. Analysis of these data taking into account chemical shifts of the carbon atom supporting the hydroxyl group (d) and r þ p , enables a mechanism to be proposed for the transformation of monophenols into odiphenols, 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 q of À1.75). 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 k fn cat /k f 0 cat against n (atom fractions of deuterium), where k fn cat is the catalytic constant for a molar fraction of deuterium (n) and k f 0 cat 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.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2011
The kinetics of tyrosinase acting on o-aminophenols and aromatic amines as substrates was studied... more The kinetics of tyrosinase acting on o-aminophenols and aromatic amines as substrates was studied. The catalytic constants of aromatic monoamines and o-diamines were both low, these results are consistent with our previous mechanism in which the slow step is the transfer of a proton by a hydroxyl to the peroxide in oxy-tyrosinase (Fenoll et al., Biochem. J. 380 (2004) 643-650). In the case of o-aminophenols, the hydroxyl group indirectly cooperates in the transfer of the proton and consequently the catalytic constants in the action of tyrosinase on these compounds are higher. In the case of aromatic monoamines, the Michaelis constants are of the same order of magnitude than for monophenols, which suggests that the monophenols bind better (higher binding constant) to the enzyme to facilitate the π-π interactions between the aromatic ring and a possible histidine of the active site. In the case of aromatic o-diamines, both the catalytic and Michaelis constants are low, the values of the catalytic constants being lower than those of the corresponding odiphenols. The values of the Michaelis constants of the aromatic o-diamines are slightly lower than those of their corresponding o-diphenols, confirming that the aromatic o-diamines bind less well (lower binding constant) to the enzyme.
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Papers by JOSE LUIS MUÑOZ