Pedro Antonio García-Ruiz

Analysis and interpretation of the action mechanism of mushroom tyrosinase on monophenols and diphenols generating highly unstable o-quinones.

Tyrosinase can act on monophenols because of the mixture of met- (E(m)) and oxy-tyrosinase (E(ox)) which exists in the native form of the enzyme. The latter form is active on monophenols, while the former is not. However, the kinetics are complicated because monophenols can bind to both enzyme forms. This situation becomes even more complex since the products of the enzymatic reaction, the o-quinones, are unstable and continue evolving to generate o-diphenols in the medium. In the case of substrates such as L-tyrosine, tyrosinase generates very unstable o-quinones, in which a process of cyclation and subsequent oxidation-reduction generates o-diphenol through non-enzymatic reactions. However, the release of o-diphenol through the action of the enzyme on the monophenol contributes to the concentration of o-diphenol in the first pseudo-steady-state [D(0)](ss). Hence, the system reaches an initial pseudo-steady state when t-->0 and undergoes a transition phase (lag period) until a final steady state is reached when the concentration of o-diphenol in the medium reaches the concentration of the final steady state [D(f)](ss). These results can be explained by taking into account the kinetic and structural mechanism of the enzyme. In this, tyrosinase hydroxylates the monophenols to o-diphenols, generating an intermediate, E(m)D, which may oxidise the o-diphenol or release it directly to the medium. We surmise that the intermediate generated during the action of E(ox) on monophenols, E(m)D, has axial and equatorial bonds between the o-diphenol and copper atoms of the active site. Since the orbitals are not coplanar, the concerted oxidation-reduction reaction cannot occur. Instead, a bond, probably that of C-4, is broken to achieve coplanarity, producing a more labile intermediate that will then release the o-diphenol to the medium or reunite it diaxially, involving oxidation to o-quinone. The non-enzymatic evolution of the o-quinone would generate the o-diphenol ([D(f)](ss)) necessary for the final steady state to be reached after the lag period.

Stopped-flow and steady-state study of the diphenolase activity of mushroom tyrosinase.

The reaction of mushroom (Agaricus bisporus) tyrosinase with dioxygen in the presence of several o-diphenolic substrates has been studied by steady-state and transient-phase kinetics in order to elucidate the rate-limiting step and to provide new insights into the mechanism of oxidation of these substrates. A kinetic analysis has allowed for the first time the determination of individual rate constants for several of the partial reactions that comprise the catalytic cycle. Mushroom tyrosinase rapidly reacts with dioxygen with a second-order rate constant k(+8) = 2.3 x 10(7) M(-)(1) s(-)(1), which is similar to that reported for hemocyanins [(1.3 x 10(6))-(5.7 x 10(7)) M(-)(1) s(-)(1)]. Deoxytyrosinase binds dioxygen reversibly at the binuclear Cu(I) site with a dissociation constant K(D)(O)()2 = 46.6 microM, which is similar to the value (K(D)(O)()2 = 90 microM) reported for the binding of dioxygen to Octopus vulgaris deoxyhemocyanin [Salvato et al. (1998) Biochemistry 37, 14065-14077]. Transient and steady-state kinetics showed that o-diphenols such as 4-tert-butylcatechol react significantly faster with mettyrosinase (k(+2) = 9.02 x 10(6) M(-)(1) s(-)(1)) than with oxytyrosinase (k(+6) = 5.4 x 10(5) M(-)(1) s(-)(1)). This difference is interpreted in terms of differential steric and polar effects that modulate the access of o-diphenols to the active site for these two forms of the enzyme. The values of k(cat) for several o-diphenols are also consistent with steric and polar factors controlling the mobility, orientation, and thence the reactivity of substrates at the active site of tyrosinase.

Tyrosinase action on monophenols: evidence for direct enzymatic release of o-diphenol

Using gas chromatography-mass spectrometry, the direct enzymatic release of o-diphenol (4-tert-butylcatechol) during the action of tyrosinase on a monophenol (4-tert-butylphenol) has been demonstrated for the first time in the literature. The findings confirm the previously proposed mechanism to explain the action of tyrosinase on monophenols (J.N. Rodríguez-López, J. Tudela, R. Varón, F. García-Carmona, F. García-Cánovas, J. Biol. Chem. 267 (1992)). Oxytyrosinase, the oxidized form of the enzyme with a peroxide group, is the only form capable of catalysing the transformation of monophenols into diphenols, giving rise to an enzyme-substrate complex in the process. The o-diphenol formed is then released from the enzyme-substrate complex or oxidized to the corresponding o-quinone. In order to detect the enzymatic release of o-diphenol, the non-enzymatic evolution of the o-quinone to generate o-diphenol by weak nucleophilic attack reactions and subsequent oxidation-reduction was blocked by the nucleophilic attack of an excess of cysteine. Furthermore, the addition of catalytic quantities of an auxiliary o-diphenol (e.g. catechol) considerably increases the accumulation of 4-tert-butylcatechol. The enzyme acting on 4-tert-butylphenol generates the enzyme-4-tert-butylcatechol complex and 4-tert-butylcatechol is then released (with k(-2)) generating mettyrosinase. The auxiliary o-diphenol added (catechol) and the 4-tert-butylcatechol generated by the enzyme then enter into competition. When [catechol] >> [4-tert-butylcatechol], the enzyme preferentially binds with the catechol to close the catalytic cycle, while 4-tert-butylcatechol is accumulated in the medium. In conclusion, we demonstrate that the enzyme produces 4-tert-butylcatechol from 4-tert-butylphenol, the concentration of which increases considerably in the presence of an auxiliary o-diphenol such as catechol.

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.

Action mechanism of tyrosinase on meta- and para-hydroxylated monophenols.

Abstract The relationship between the structure and activity of metaand parahydroxylated monophenols was studied during their tyrosinasecatalysed hydroxylation and the ratelimiting steps of the reaction mechanism were identified. The parahydroxylated substrates permit us to study the effect of a substituent (R) in the carbon-1 position (C-1) of the benzene ring on the nucleophilic attack step, while the meta group permits a similar study of the effect on the electrophilic attack step. Substrates with a OCH[3] group on C-1, as phydroxyanisol (4HA) and mhydroxyanisol (3HA), or with a CH[2]OH group, as phydroxybenzylalcohol (4HBA) and mhydroxybenzylalcohol (3HBA), were used because the effect of the substituent (R) size was assumed to be similar. However, the electrondonating effect of the OCH[3] group means that the carbon-4 position (C-4) is favoured for nucleophilic attack (parahydroxylated substrates) or for electrophilic attack (metahydroxylated substrates). The electronattracting effect of the CH[2]OH group has the opposite effect, hindering nucleophilic (para) or electrophilic (meta) attack of C-4. The experimental data point to differences between the maximum steadystate rate (V ) of the different substrates, the value of this parameter depends on the nucleophilic and electrophilic attack. However, differences are greatest in the Michaelis constants (K ), with the metahydroxylated substrates having very large values. The catalytic efficiency k /K is much greater for the parahydroxylated substrates although it varies greatly between one substrate and the other. However, it varies much less in the metahydroxylated substrates since this parameter describes the power of the nucleophilic attack, which is weaker in the meta OH. The large increase in the K of the metahydroxylated substrates might suggest that the phenolic OH takes part in substrate binding. Since this is a weaker nucleophil than the parahydroxylated substrates, the binding constant decreases, leading to an increase in K . The catalytic efficiency of tyrosinase on a monophenol (para or meta) is directly related to the nucleophilic power of the oxygen of the phenolic OH. The oxidation step is not limiting since if this were the case, the para and meta substrates would have the same V . The small difference between the absolute values of V suggests that the rate constants of the nucleophilic and electrophilic attacks are on the same order of magnitude.

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.

Production of o-diphenols by immobilized mushroom tyrosinase

The o-diphenols 4-tert-butyl-catechol, 4-methyl-catechol, 4-methoxy-catechol, 3,4-dihydroxyphenylpropionic acid and 3,4-dihydroxyphenylacetic acid were produced from the corresponding monophenols (4-tert-butyl-phenol, 4-methyl-phenol, 4-methoxy-phenol, p-hydroxyphenylpropionic acid and p-hydroxyphenylacetic acid) using immobilized mushroom tyrosinase from Agaricus bisporus. In all cases the yield was R(diphenol)> or =88-96%, which, according to the literature, is the highest yield so far, obtained using tyrosinase. The reaction was carried out in 0.5M borate buffer pH 9.0 which was used to minimize the diphenolase activity of tyrosinase by complexing the o-diphenols generated. Hydroxylamine and ascorbic acid were also present in the reaction medium, the former being used to reduce mettyrosinase to deoxytyrosinase, closing the catalytic cycle, and the latter to reduce the o-quinone produced to o-diphenol. Inactivation of the tyrosinase by ascorbic acid was also minimized due to the formation of an ascorbic acid-borate complex. Concentrations of the o-diphenolic compounds obtained at several reaction times were determined by gas chromatography-mass spectrometry (GC-MS) and UV-vis spectroscopy. The experimental results are discussed.

Deuterium isotope effect on the oxidation of monophenols and o-diphenols by tyrosinase

A solvent deuterium isotope effect on the catalytic affinity (km) and catalytic constant (kcat) of tyrosinase in its action on different monophenols and o-diphenols was observed. The catalytic constant decreased in all substrates as the molar fraction of deuterated water in the medium increased, while the catalytic affinity only decreased for the o-diphenols with an R group in C-1 [-H, -CH3 and -CH(CH3)2]. In a proton inventory study of the oxidation of o-diphenols, the representation of kcat fn/kcat f0 against n (atom fractions of deuterium), where kcat fn is the catalytic constant for a molar fraction of deuterium (n) and kcat f0 is the corresponding kinetic parameter in a water solution, was linear for all substrates, indicating that only one of the four protons transferred from the hydroxy groups of the two molecules of substrate, which are oxidized in one turnover, is responsible for the isotope effects, the proton transferred from the hydroxy group of C-4 to the peroxide of the oxytyrosinase form (Eox). However, in the representation of Km fn/Km f0 against n, where Km fn represents the catalytic affinity for a molar fraction of deuterium (n) and Km f0 is the corresponding kinetic parameter in a water solution, a linear decrease was observed as n increased in the case of o-diphenols with the R group [-H, -CH3 and -CH(CH3)2], and a parabolic increase with other R groups, indicating that more than one proton is responsible for the isotope effects on substrate binding. In the case of monophenols with six protons transferred in the catalytic cycle, the isotope effect occurs in the same way as for o-diphenols. In the present paper, the fractionation factors of different monophenols and o-diphenols are described and possible mechanistic implications are discussed.

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).