Lorena G. Fenoll

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.

Enzyme inactivation analysis for industrial blanching applications: comparison of microwave, conventional, and combination heat treatments on mushroom polyphenoloxidase activity.

Browning reactions in fruits and vegetables are a serious problem for the food industry. In mushrooms, the principal enzyme responsible for the browning reaction is polyphenoloxidase (PPO). Microwaves have recently been introduced as an alternative for the industrial blanching of mushrooms. However, the direct application of microwave energy to entire mushrooms is limited by the important temperature gradients generated within the samples during heating, which can produce internal water vaporization and associated damage to the mushrooms texture. A microwave applicator has been developed, whereby irradiation conditions can be regulated and the heating process monitored. Whole edible mushrooms (Agaricus bisporus) were blanched by conventional, microwave, and combined heating methods to optimize the rate of PPO inactivation. A combined microwave and hot-water bath treatment has achieved complete PPO inactivation in a short time. Both the loss of antioxidant content and the increase of browning were minor in the samples treated with this combined method when compared to the control. This reduction in processing time also decreased mushroom weight loss and shrinkage.

Tyrosinase kinetics: discrimination between two models to explain the oxidation mechanism of monophenol and diphenol substrates.

The kinetic behaviour of tyrosinase is very complex because the enzymatic oxidation of monophenol and o-diphenol to o-quinones occurs simultaneously with the coupled non-enzymatic reactions of the latter. Both reaction types are included in the kinetic mechanism proposed for tyrosinase (Mechanism I [J. Biol. Chem. 267 (1992) 3801-3810]). We previously confirmed the validity of the rate equations by the oxidation of numerous monophenols and o-diphenols catalysed by tyrosinase from different fruits and vegetables. Other authors have proposed a simplified reaction mechanism for tyrosinase (Mechanism II [Theor. Biol. 203 (2000) 1-12]), although without deducing the rate equations. In this paper, we report new experimental work that provides the lag period value, the steady-state rate, o-diphenol concentration released to the reaction medium. The contrast between these experimental data and the respective numerical simulations of both mechanisms demonstrates the feasibility of Mechanism I. The need for the steps omitted from Mechanism II to interpret the experimental data for tyrosinase, based on the rate equations previously deduced for Mechanism I is explained.

Michaelis constants of mushroom tyrosinase with respect to oxygen in the presence of monophenols and diphenols

The complex reaction mechanism of tyrosinase involves three enzymatic forms, two overlapping catalytic cycles and a dead-end complex. Analytical expressions for the catalytic and Michaelis constants of tyrosinase towards phenols and oxygen were derived for both, monophenolase and diphenolase activities of the enzyme. Thus, the Michaelis constants of tyrosinase towards the oxygen (K(mO(2))) are related with the respective catalytic constants for monphenols (k(M)(cat)) and o-diphenols (k(D)(cat)), as well as with the rate constant, k(+8). We recently determined the experimental value of the rate constant for the binding of oxygen to deoxytyrosinase (k(+8)) by stopped-flow assays. In this paper, we calculate theoretical values of K(mO(2)) from the experimental values of catalytic constants and k(+8) towards several monophenols and o-diphenols. The reliability and the significance of the values of K(mO(2)) are discussed.

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.

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.

Stereospecificity of horseradish peroxidase.

Abstract We report here on the stereospecificity observed in the action of horseradish peroxidase (HRPC) on monophenol and diphenol substrates. Several enantiomers of monophenols and o-diphenols were assayed: L-tyrosinol, D-tyrosinol, L-tyrosine, DL-tyrosine, D-tyrosine, L-dopa, DL-dopa, D-dopa, L-α-methyldopa, DL-α-methyldopa, DL-adrenaline, D-adrenaline, L-isoproterenol, DL-isoproterenol and D-isoproterenol. The electronic density at the carbon atoms in the C-1 and C-2 positions of the benzene ring were determined by NMR assays (δ1 and δ2). This value is related to the nucleophilic power of the oxygen atom of the hydroxyl groups and to its oxidation-reduction capacity. The spatial orientation of the ring substituents resulted in lower K m values for L- than for D-isomers. The k cat values for substrates capable of saturating the enzyme were lower for D- than for L-isomers, although both have the same δ1 and δ2 NMR values for carbons C-1 and C-2, and therefore the same oxidation-reduction potential. In the case of substrates that cannot saturate the enzyme, the values of the binding constant for compound II (an intermediate in the catalytic cycle) followed the order: L-isomer>DL-isomer>D-isomer. Therefore, horseradish peroxidase showed stereospecificity in its affinity toward its substrates (K m) and in their transformation reaction rates (k cat).