Catalysis and inhibition of tyrosinase in the presence of cinnamic acid and some of its derivatives.

The kinetic action of tyrosinase on l-tyrosine and l-Dopa as substrates in the presence of cinnamic acid and some of its derivatives has been characterized. Cinnamic acid, 2-hydroxycinnamic, 2,3 and 4-methoxycinnamic acids were seen to be inhibitors of tyrosinase being determined the type of inhibition and inhibition constants of all of them. However, 3-hydroxycinnamic, 4-hydroxycinnamic and 3,4-dihydroxycinnamic acids were seen to be substrates of tyrosinase at the same time. The kinetic constants of the catalysis of these substrates were determined and found to be perfectly correlated with the chemical shifts of the carbon with the phenolic hydroxyl group revealed by NMR. Docking studies of 2-hydroxycinnamic and 3-hydroxycinnamic acids showed that tyrosinase is able to hydroxylate 3-hydroxycinnamic acid but is unable to hydroxylate 2-hydroxycinnamic acid.

Chitin hydrolysis assisted by cell wall degrading enzymes immobilized of Thichoderma asperellum on totally cinnamoylated D-sorbitol beads.

In this study, cell wall degrading enzymes produced by Thrichoderma asperellum (TCWDE) were immobilized on totally cinnamoylated D-sorbitol (TCNSO) beads and used for chitin hydrolysis. In order to optimize immobilization efficiency, the reaction time was varied from 2 to 12 h and reactions were conducted in the presence or absence of Na2SO4. Immobilized enzymes were analysed concerning to thermal and operational stability. Immobilization in presence of Na2SO4 was 54% more efficient than immobilization in absence of salt. After optimization, 32% of the total enzyme offered was immobilized, with 100% of bounding efficiency, measured as the relation between protein and enzyme immobilized. Free and TCNSO-TCWDE presented very similar kinetics with maximum hydrolysis reached at 90 min of reaction. Thermal stability of both free and TCNSO-TCWDE was similar, with losses in activity after 55 °C. Moreover, free and TCNSO-TCWDE retained 100% activity after 3h incubation at 55 °C. TCNSO-TCWDE were used in a bath-wise reactor during 14 cycles, producing 1825 µg of N-acetylglucosamine (NAG) maintaining 83% of initial activity.

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.

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.

Interpretation of the reactivity of peroxidase compound II with phenols and anilines using the Marcus equation.

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.

Mushroom tyrosinase: catalase activity, inhibition, and suicide inactivation.

Mushroom tyrosinase exhibits catalase activity with hydrogen peroxide (H(2)O(2)) as substrate. In the absence of a one-electron donor substrate, H(2)O(2) is able to act as both oxidizing and reducing substrate. The kinetic parameters V(max) and K(m) that characterize the reaction were determined from the initial rates of oxygen gas production (V(0)(O)()2) under anaerobic conditions. The reaction can start from either of the two enzyme species present under anaerobic conditions: met-tyrosinase (E(m)) and deoxy-tyrosinase (E(d)). Thus, a molecule of H(2)O(2) can reduce E(m) to E(d) via the formation of oxy-tyrosinase (E(ox)) (E(m) + H(2) <==> O(2) right harpoon over left harpoon E(ox)), E(ox) releases oxygen into the medium and is transformed into E(d), which upon binding another molecule of H(2)O(2) is oxidized to E(m). The effect of pH and the action of inhibitors have also been studied. Catalase activity is favored by increased pH, with an optimum at pH = 6.4. Inhibitors that are analogues of o-diphenol, binding to the active site coppers diaxially, do not inhibit catalase activity but do reduce diphenolase activity. However, chloride, which binds in the equatorial orientation to the protonated enzyme (E(m)H), inhibits both catalase and diphenolase activities. Suicide inactivation of the enzyme by H(2)O(2) has been demonstrated. A kinetic mechanism that is supported by the experimental results is presented and discussed.

Opposite effects of peroxidase in the initial stages of tyrosinase-catalysed melanin biosynthesis.

The tyrosinase/oxygen enzymatic system catalyses the orthohydroxylation of L-tyrosine to L-dopa and the oxidation of this to dopaquinone, which evolves non-enzymatically towards to form melanins. The literature has demonstrated and revised the existence of peroxidase/hydrogen peroxide in the melanosomas of skin melanocytes, but points to controversy concerning the effects on melanogenesis. Some authors have recently proposed a new physiological function for tyrosinase, namely the direct scavenging of tyrosyl radicals, which are toxic oxidants of melanocytes. In this contribution, we describe and interpret four effects of peroxidase/hydrogen peroxide on melanogenesis. Two of these effects are its antagonism and synergy as regards the monophenolase and diphenolase activities, respectively, of tyrosinase/oxygen in the initial steps that trigger melanogenesis. Another effect concerns the increase in the oxidant character of the medium in the melanosome by increasing the synthesis of oxidising quinones (o-dopaquinone, p-topaquinone, dopachrome) and the consumption of antioxidant diphenols (L-dopa), which are intermediate biomolecules in melanogenesis. Lastly, we demonstrate that the tyrosyl radicals generated by light or by the peroxidase/hydrogen peroxide system are not directly trapped by the tyrosinase but by the antioxidant orthodiphenol, L-dopa, accumulated in the steady-state of melanogenesis. In conclusion, peroxidase/hydrogen peroxide may help regulate the development of melanogenesis and the oxidant environment within the melanosome. This enzyme deserves further study for its possible antitumoral and depigmentation capacities in skin cancer and hyperpigmentation.

Stereospecificity of horseradish peroxidase.

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-alpha-methyldopa, DL-alpha-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 (delta1 and delta2). 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 Km values for L- than for D-isomers. The kcat values for substrates capable of saturating the enzyme were lower for D- than for L-isomers, although both have the same delta1 and delta2 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).

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.