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.

Indirect inactivation of tyrosinase in its action on 4-tert-butylphenol.

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.

Kinetic characterisation of o-aminophenols and aromatic o-diamines as suicide substrates of tyrosinase.

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-diamineso-aminophenols>aromatic o-diamines.

Hydrogen peroxide helps in the identification of monophenols as possible substrates of tyrosinase.

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.

Catalytic oxidation of o-aminophenols and aromatic amines by mushroom tyrosinase.

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.

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.

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).

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.

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 (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.

A kinetic study of a ternary cycle between adenine nucleotides.

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.

Contribution of the intra- and intermolecular routes in autocatalytic zymogen activation: application to pepsinogen activation.

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.

Action of ellagic acid on the melanin biosynthesis pathway.

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.

Kinetics of intra- and intermolecular zymogen activation with formation of an enzyme-zymogen complex.

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.

Kinetic analysis of the transient phase and steady state of open multicyclic enzyme cascades.

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.

Kinetic characterisation of the reaction mechanism of mushroom tyrosinase on tyramine/dopamine and L-tyrosine methyl esther/L-dopa methyl esther.

Tyrosinase or polyphenol oxidase is the key enzyme in melanin biosynthesis and for the enzymatic browning of fruits and vegetables. Our research group previously proposed a kinetic reaction mechanism for tyrosinase acting on some phenolic substrates, whose reliability was demonstrated for tyrosinases from several fruits and vegetables. A kinetic analysis and an experimental design for testing the reliability of the kinetic reaction mechanism of tyrosinase are reported. The applicability of the mechanism to the oxidation of tyramine/dopamine and L-tyrosine methyl esther/L-dopa methyl esther has been checked. Some structure/activity topics are discussed. A complete kinetic characterisation of the oxidation of these phenolic substrates has been made. This will be useful for further studies about the control of depigmenting agents, antimelanome drugs and antibrowning reagents acting on tyrosinase.

Kinetic study of the effects of calcium ions on cationic artichoke (Cynara scolymus L.) peroxidase: calcium binding, steady-state kinetics and reactions with hydrogen peroxide.

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.

Enzymatic synthesis of 3'-hydroxyacetaminophen catalyzed by tyrosinase.

3'-Hydroxyacetaminophen, a catechol metabolite of N-acetyl-p-aminophenol (acetaminophen) and N-acetyl-m-aminophenol (a structural analogue of acetaminophen and considered as a possible alternative because it is not hepatotoxic), is enzymatically synthesized for the first time using mushroom tyrosinase. Although reported to be weakly hepatotoxic in vivo, this catechol derivative of acetaminophen is not commercially available. This compound was obtained from its monophenolic precursor, acetaminophen, using the enzyme tyrosinase in the presence of an excess of ascorbic acid, thus reducing back the o-quinone product of catalytic activity to the catechol acetaminophen derivative. A mathematical model of the system is proposed, which is also applicable to the tyrosinase-mediated synthesis of any o-diphenolic compound from its corresponding monophenol. This synthesis procedure is continuous, easy to perform and control, and adaptable to a bioreactor with the immobilized enzyme for industrial purposes in a nonpolluting way.

Kinetic study of the oxidation of quercetin by mushroom tyrosinase.

The kinetic behavior of mushroom tyrosinase in the presence of the flavonol quercetin was studied. This flavonol was oxidized by mushroom tyrosinase and the reaction was followed by recording spectral changes over time. The spectra obtained during the reaction showed two isosbectic points, indicating a stable o-quinone. When quercetin was oxidized by tyrosinase in the presence of cysteine and 3-methyl-2-benzothiazolone hydrazone (Besthorn's hydrazone, MBTH) isosbestic points were also observed indicating a definite stoichiometry. From the data analysis of the initial rate in the presence of MBTH, the kinetic parameters: = (16.2 +/- 0.6) microM/min, = (0.12 +/- 0.01) mM, (/) = (V(max)/K(S)(')()) = (13.5 +/- 1.4) x 10(-)(2) min(-)(1), = (6.2 +/- 0.6) s(-)(1) were determined. We propose that quercetin acts simultaneously as a substrate and a rapid reversible inhibitor of mushroom tyrosinase, depending on how it binds to the copper atom of the enzyme active site. Thus, if the binding occurs through the hydroxylic groups at the C3' and C4' positions, quercetin acts as a substrate, while if it occurs through the hydroxylic group at the C3 position of the pyrone ring, quercetin acts as an inhibitor.

A mathematical model for colour loss in paprikas containing differing proportions of seed.

In this paper a general kinetic model for predicting the colour of paprikas with differing proportions of seed as a function of the storage conditions is developed. We apply this model to study the effect of time and storage conditions on colour loss in paprikas made from the same pepper variety, Ocal, but with differing proportions of seed. The agreement between the experimental colour data for this paprika and the results predicted by the mathematical model is very good. Both the model and the experimental data show that the rate of colour loss decreases with increasing proportion of seed, and after a determined inversion time the samples with the lowest initial colour values actually show the highest values. Likewise, the experimental data show that for a given proportion of seed the colour loss decreases as the storage relative humidity increases. © 2000 Society of Chemical Industry.

Kinetic cooperativity of tyrosinase. A general mechanism.

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).