RESUMEN
BACKGROUND: Tyrosinase is an enzyme involved in the first steps of the melanogenesis process. It catalyzes the hydroxylation of monophenols to o-diphenols and the oxidation of the latter to o-quinones. Ellagic acid (EA) is a phenolic compound which has been described as a tyrosinase inhibitor and is used in the cosmetic industry as a whitening agent. However, it has hydroxyl groups in ortho position and could act as a substrate rather than inhibitor. This aspect should be taken into consideration when using this compound as a cosmetic ingredient due to the reactive character of o-quinones. OBJECTIVE: To determine whether ellagic acid is a substrate or an inhibitor of tyrosinase, to characterize it kinetically and interpret its role in the melanogenesis process. METHODS: UV-vis spectrophotometry was used to follow the action of tyrosinase on typical substrates and ellagic acid. A chronometric method was chosen for the kinetic characterization of ellagic acid. RESULTS: Ellagic acid is not an inhibitor per se but an alternative substrate of tyrosinase. It is oxidized by the enzyme to an unstable o-quinone. Its kinetic characterization provided low Michaelis and catalytic constants (KM(EA)=138±13µM and kcat(EA)=0.47±0.02s(-1)). Furthermore, ellagic acid, which is a powerful antioxidant, may chemically reduce the o-quinones (o-dopaquinone) and semiquinones, in this way inhibiting the melanogenesis. CONCLUSION: Ellagic acid is oxidized by tyrosinase, producing reactive o-quinones. As an antioxidant it can inhibit the melanogenesis process. This first aspect should be taken into consideration in its application as a cosmetic ingredient due to the toxicity of o-quinones and its ability to modify the redox status of the cell.
Asunto(s)
Ácido Elágico/metabolismo , Ácido Elágico/farmacología , Melaninas/biosíntesis , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/metabolismo , Preparaciones para Aclaramiento de la Piel/metabolismo , Preparaciones para Aclaramiento de la Piel/farmacología , Antioxidantes/metabolismo , Antioxidantes/farmacología , Benzoquinonas/metabolismo , Vías Biosintéticas/efectos de los fármacos , Dihidroxifenilalanina/análogos & derivados , Dihidroxifenilalanina/metabolismo , Pruebas de Enzimas , Humanos , Cinética , Fenoles/metabolismo , Quinonas/metabolismo , Piel/efectos de los fármacos , Piel/enzimología , Espectrofotometría Ultravioleta/métodos , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Inhibidores Enzimáticos/química , Proteínas Fúngicas/química , Levodopa/química , Monofenol Monooxigenasa/química , Tirosina/química , Cinamatos/química , Evaluación Preclínica de Medicamentos , Proteínas Fúngicas/antagonistas & inhibidores , Cinética , Monofenol Monooxigenasa/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Catecoles/química , Inhibidores Enzimáticos/química , Proteínas Fúngicas/química , Oxígeno/química , Fenoles/química , Agaricales/química , Agaricales/enzimología , Pruebas de Enzimas , Proteínas Fúngicas/aislamiento & purificación , Cinética , Levodopa/química , Oxidación-Reducción , Soluciones , Especificidad por Sustrato , Tirosina/químicaRESUMEN
Tyrosinase exists in three forms in the catalytic cycle depending on the oxidation state of the copper: met- (Em), oxy- (E(ox)), and deoxy- (Ed). When O-quinones, products of the enzymatic reaction, evolve chemically to generate an O-diphenol in the reaction medium, the enzyme acts on a monophenol with O-diphenol as reductant, converting Em to Ed. The binding of Ed to molecular oxygen gives E(ox), which is active on monophenols, but when the O-quinone product does not generate O-diphenol through chemical evolution, the monophenol does not act as an enzyme substrate. The fact that E(ox) can be formed from Em with hydrogen peroxide can be used to help identify whether a monophenol is a substrate of tyrosinase. The results obtained in this study confirm that compounds previously described as inhibitors of the enzyme are true substrates of it.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Peróxido de Hidrógeno/metabolismo , Monofenol Monooxigenasa/metabolismo , Fenoles/metabolismo , Agaricales/enzimología , Hidroxilación , Monofenol Monooxigenasa/antagonistas & inhibidores , Oxidación-Reducción , Unión Proteica , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Monofenol Monooxigenasa/química , Oxidorreductasas/química , Fenoles/química , Agaricales/enzimología , Hidroxilación , CinéticaRESUMEN
We study the suicide inactivation of tyrosinase acting on o-aminophenols and aromatic o-diamines and compare the results with those obtained for the corresponding o-diphenols. The catalytic constants follow the order aromatic o-diamines
Asunto(s)
Aminofenoles/química , Diaminas/química , Proteínas Fúngicas/química , Monofenol Monooxigenasa/química , Fenoles/química , Ácido Ascórbico/química , Proteínas Fúngicas/antagonistas & inhibidores , Cinética , Monofenol Monooxigenasa/antagonistas & inhibidores , Oxidación-Reducción , Oxígeno/químicaRESUMEN
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).
Asunto(s)
Monofenol Monooxigenasa/química , Monofenol Monooxigenasa/metabolismo , Ácido 3,4-Dihidroxifenilacético/química , Ácido 3,4-Dihidroxifenilacético/metabolismo , Ácidos Cafeicos/química , Ácidos Cafeicos/metabolismo , Catecoles/química , Catecoles/metabolismo , Cresoles/química , Cresoles/metabolismo , Desoxiepinefrina/análogos & derivados , Desoxiepinefrina/química , Desoxiepinefrina/metabolismo , Dopamina/química , Dopamina/metabolismo , Modelos Químicos , Fenoles/química , Fenoles/metabolismo , Fenilacetatos/química , Fenilacetatos/metabolismo , Fenilpropionatos/química , Fenilpropionatos/metabolismo , Quinonas/química , Quinonas/metabolismo , Especificidad por SustratoRESUMEN
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 o-diphenols. 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.
Asunto(s)
Agaricales/enzimología , Aminoácidos Aromáticos/metabolismo , Aminofenoles/metabolismo , Monofenol Monooxigenasa/metabolismo , Aminas/química , Aminas/metabolismo , Aminoácidos Aromáticos/química , Aminofenoles/química , Catálisis , Cinética , Modelos Biológicos , Modelos Químicos , Monofenol Monooxigenasa/química , Oxidación-Reducción , Fenoles/metabolismo , Análisis Espectral , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Monofenol Monooxigenasa/antagonistas & inhibidores , Animales , Humanos , Cinética , Monofenol Monooxigenasa/química , Oxidorreductasas/antagonistas & inhibidores , Fenoles/metabolismo , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Antioxidantes/análisis , Antioxidantes/farmacología , Análisis de los Alimentos , Antioxidantes/química , Depuradores de Radicales Libres/análisis , Radicales Libres/química , Concentración de Iones de Hidrógeno , Cinética , EspectrofotometríaRESUMEN
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).
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Melaninas/biosíntesis , Catalasa/metabolismo , Monofenol Monooxigenasa/metabolismo , Oxidación-Reducción , Peroxidasas/metabolismo , Fenoles/metabolismo , Espectrofotometría/métodos , Superóxido Dismutasa/metabolismoRESUMEN
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.
Asunto(s)
Ácido Ascórbico/química , Monofenol Monooxigenasa/química , Cinética , Espectroscopía de Resonancia Magnética , Monofenol Monooxigenasa/antagonistas & inhibidores , Oxígeno/química , Estereoisomerismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
In the present paper, a kinetic analysis of a general model for proenzyme activation, where the activating enzyme and also the activated one are reversibly inhibited in two steps by two different inhibitors, has been performed. The cases in which both inhibitors are the same, or in which the inhibition is irreversible (only one or the two inhibition routes) are treated as particular cases of the general model. In addition, the kinetic behaviour of many other proenzyme activation systems involving inhibition, particular cases of the reaction scheme under study, can be obtained. The total number of particular cases for the general model under study is 370, so this approach offers to the scientific community working in limited proteolysis regulation for the first time a method based on general solutions which only needs to be specified to their concrete problem of zymogen activation. Finally, new adimensional parameters are introduced, allowing the knowledgement, in the case that any of the inhibition routes is irreversible, the relative weight of both activation and irreversible inhibition routes.
Asunto(s)
Inhibidores Enzimáticos/metabolismo , Precursores Enzimáticos/metabolismo , Animales , Activación Enzimática , Precursores Enzimáticos/antagonistas & inhibidores , Matemática , Modelos Químicos , Péptido Hidrolasas/metabolismo , Factores de TiempoRESUMEN
In the present paper, a kinetic study is made of the behavior of a moiety-conserved ternary cycle between the adenine nucleotides. The system contains the enzymes S-acetyl coenzyme A synthetase, adenylate kinase and pyruvate kinase, and converts ATP into AMP, then into ADP and finally back to ATP. L-Lactate dehydrogenase is added to the system to enable continuous monitoring of the progress of the reaction. The cycle cannot work when the only recycling substrate in the reaction medium is AMP. A mathematical model is proposed whose kinetic behavior has been analyzed both numerically by integration of the nonlinear differential equations describing the kinetics of the reactions involved, and analytically under steady-state conditions, with good agreement with the experimental results being obtained. The data obtained showed that there is a threshold value of the S-acetyl coenzyme A synthetase/adenylate kinase ratio, above which the cycle stops because all the recycling substrate has been accumulated as AMP, never reaching the steady state. In addition, the concept of adenylate energy charge has been applied to the system, obtaining the enabled values of the rate constants for a fixed adenylate energy charge value and vice versa.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Cromatografía Líquida de Alta Presión , Simulación por Computador , Cinética , Modelos TeóricosRESUMEN
Taking as the starting point a recently suggested reaction scheme for zymogen activation involving intra- and intermolecular routes and the enzyme-zymogen complex, we carry out a complete analysis of the relative contribution of both routes in the process. This analysis suggests the definition of new dimensionless parameters allowing the elaboration, from the values of the rate constants and initial conditions, of the time course of the contribution of the two routes. The procedure mentioned above related to a concrete reaction scheme is extrapolated to any other model of autocatalytic zymogen activation involving intra- and intermolecular routes. Finally, we discuss the contribution of both of the activating routes in pepsinogen activation into pepsin using the values of the kinetic parameters given in the literature.
Asunto(s)
Algoritmos , Precursores Enzimáticos/metabolismo , Pepsinógeno A/metabolismo , Catálisis , Activación Enzimática , Precursores Enzimáticos/química , Cinética , Modelos Químicos , Pepsinógeno A/químicaRESUMEN
A kinetic study was performed of a model for an autocatalytic zymogen activation process involving both intra- and intermolecular routes, to which a chromogenic reaction in which the active enzyme acts upon one of its substrates was coupled to continuously monitor the reaction. Kinetic equations describing the evolution of species involved in the system with time were obtained. These equations are valid for any zymogen autocatalytic activation process under the same initial conditions. Experimental design and kinetic data analysis procedures to evaluate the kinetic parameters, based on the derived kinetic equations, are suggested. In addition, a dimensionless distribution coefficient was defined, which shows mathematically whether the intra- or the intermolecular route prevails once the kinetic parameters involved in the system are known. The validity of the results obtained was checked using simulated curves for the species involved. As an example of application of the method, the system is experimentally illustrated by the continuous monitoring of pepsinogen transformation to pepsin.
Asunto(s)
Precursores Enzimáticos/metabolismo , Pepsinógeno A/metabolismo , Activación Enzimática , CinéticaRESUMEN
This paper presents a kinetic analysis of the whole reaction course, i.e. of both the transient phase and the steady state, of open multicyclic enzyme cascade systems. Equations for fractional modifications are obtained which are valid for the whole reaction course. The steady state expressions for the fractional modifications were derived from the latter equations since they are not restricted to the condition of rapid equilibrium. Finally, the validity of our results is discussed and tested by numerical integration. Apart from the intrinsic value of knowing the kinetic behaviour of any of the species involved in any open multicyclic enzyme cascade, the kinetic analysis presented here can be the basis of future contributions concerning open multicyclic enzyme cascades which require the knowledge of their time course equations (e.g. evaluation of the time needed to reach the steady state, suggestion of kinetic data analysis, etc.), analogous to those already carried out for open bicyclic cascades.
Asunto(s)
Enzimas/metabolismo , Simulación por Computador , Cinética , Modelos Teóricos , Reproducibilidad de los ResultadosRESUMEN
The catalytic cycle of heme peroxidases involves three processes: the formation of compound I, its conversion to compound II and regeneration of the native enzyme. Each of the processes consists of a reversible binding stage followed by an irreversible transformation stage. Our group has proposed a continuous, sensitive and reliable chronometric method for measuring the steady-state rate of peroxidase activity. Furthermore, we have derived an analytical expression for the steady-state rate and simplified it, taking into consideration the experimental values of the rate constants of some stages previously determined by other authors in stopped-flow assays. We determined the value of the constant for the transformation of a series of phenols and anilines by compound II, and found that it involves a deprotonation step and an electron transfer step. Study of the solvent deuterium isotope effect on the oxidation of phenol revealed the non-rate-limiting character of the deprotonation step in a proton inventory study. Usage of the Marcus equation showed that the electronic transfer step is rate-limiting in both cases, while phenols and anilines were oxidised at different rates for the same potentials. This can be attributed to the shorter electron-tunnelling distance for electron transfer to the iron ion in the phenols than in the anilines.
Asunto(s)
Compuestos de Anilina/metabolismo , Modelos Químicos , Peroxidasa/química , Peroxidasa/metabolismo , Fenoles/química , Fenoles/metabolismo , CinéticaRESUMEN
A mathematical description was made of an autocatalytic zymogen activation mechanism involving both intra- and intermolecular routes. The reversible formation of an active intermediary enzyme-zymogen complex was included in the intermolecular activation route, thus allowing a Michaelis-Menten constant to be defined for the activation of the zymogen towards the active enzyme. Time-concentration equations describing the evolution of the species involved in the system were obtained. In addition, we have derived the corresponding kinetic equations for particular cases of the general model studied. Experimental design and kinetic data analysis procedures to evaluate the kinetic parameters, based on the derived kinetic equations, are suggested. The validity of the results obtained were checked by using simulated progress curves of the species involved. The model is generally good enough to be applied to the experimental kinetic study of the activation of different zymogens of physiological interest. The system is illustrated by following the transformation kinetics of pepsinogen into pepsin.
Asunto(s)
Precursores Enzimáticos/metabolismo , Pepsinógeno A/metabolismo , Animales , Cinética , Reproducibilidad de los Resultados , PorcinosRESUMEN
The apparent catalytic constant (k(cat)) of artichoke (Cynara scolymus L.) peroxidase (AKPC) with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) increased 130-fold in the presence of calcium ions (Ca2+) but the affinity (K(m)) of the enzyme for ABTS was 500 times lower than for Ca2+-free AKPC. AKPC is known to exhibit an equilibrium between 6-aquo hexa-coordinate and penta-coordinate forms of the haem iron that is modulated by Ca2+ and affects compound I formation. Measurements of the Ca2+ dissociation constant (K(D)) were complicated by the water-association/dissociation equilibrium yielding a global value more than 1000 times too high. The value for the Ca2+ binding step alone has now been determined to be K(D) approximately 10 nM. AKPC-Ca2+ was more resistant to inactivation by hydrogen peroxide (H(2)O(2)) and exhibited increased catalase activity. An analysis of the complex H(2)O(2) concentration dependent kinetics of Ca2+-free AKPC is presented.