Mushroom tyrosinase has an ascorbate oxidase activity.

The reaction between mushroom tyrosinase and L-ascorbic acid was studied by oxymetric assays and evidence pointing to ascorbate oxidase activity of this enzyme has been obtained. The activity is clearly linear to enzyme concentration and the Michaelis constant for L-ascorbic acid has a value of 2.69 +/- 0.11 mM. Maximum activity is obtained at pH 7.5. A possible reaction mechanism, which is based on the different enzymatic forms of tyrosinase, is also presented.

Kinetics study of the oxidation of 4-tert-butylphenol by tyrosinase.

The reaction between 4-tert-butylphenol (BuPhOH) and mushroom tyrosinase was investigated by following 4-tert-butyl-ortho-benzoquinone, whose high stability permits the reaction to be used as a model for the study of the monophenolase activity of tyrosinase. The system evolves to a pseudo-steady state through an induction period (tau), the pseudo-steady-state rate (Vss) decreasing when the (BuPhOH) concentration increases. Increases in enzyme concentration result in a parabolic pattern with Vss, while tau is shortened. The addition of increasing catalytic amounts of 4-tert-butylcatechol at the start of the reaction reduces tau until it is totally abolished, an initial burst being observed at high 4-t-butylatechol concentrations. Initial bursts are also obtained at pH 4.5 or lower, indicating a lower affinity of the met-tyrosinase or oxidized form for the monophenol at low pH. These experimental results can be explained by the reaction mechanism of tyrosinase.

Tyrosinase: kinetic analysis of the transient phase and the steady state.

The transient phase of tyrosinase activity acting on monophenols has been investigated. Although an analytical solution for the lag period (tau) cannot be obtained, its dependence on reagent concentration and pH is studied. It is established that decreases as the quantity of enzyme increases, although it increases when monophenol or pH are increased. The computer simulation shows those rate constants whose variations affect the transient phase most significantly. In addition, the steady state of the pathway is studied using tyrosinases from several sources such as mushroom, frog epidermis and grape. The kinetic analysis, which is based on not imposing restrictions on the values of the rate constants involved in the mechanism, allows us to obtain analytical expressions for both monophenolase and diphenolase activities and explains the experimental results obtained with the different enzymes. The values determined for the kinetic parameter, R, point to the monophenol hydroxylation step as being the limiting step of the turnover, while the values obtained for n suggest the absence of fast equilibrium in the oxidation of diphenol by Emet.

Effect of L-ascorbic acid on the monophenolase activity of tyrosinase.

The effect of ascorbic acid on the monophenolase activity of tyrosinase, using tyrosine as substrate, has been studied. Over the ranges of ascorbic acid concentration used, no direct effect on the enzyme is found. However, a shortening of the characteristic induction period of the hydroxylation reaction is observed. The evolution of the reaction is dependent on the concentration of ascorbic acid. Low concentrations permit the system to reach the steady state when all ascorbic acid is consumed, whereas high concentrations do not. In the light of these results it is proposed that the influence of ascorbic acid on the reaction is due to its ability to reduce the enzymically generated o-quinones. A relationship between the ascorbic acid concentration, and the induction period generated by it, with the diphenolase activity of tyrosinase is established, which can be used as a basis for the determination of trace amounts of this reducing agent.

Oxygen Michaelis constants for tyrosinase.

The Michaelis constant of tyrosinase for oxygen in the presence of monophenols and o-diphenols, which generate a cyclizable o-quinone, has been studied. This constant depends on the nature of the monophenol and o-diphenol and is always lower in the presence of the former than of the latter. From the mechanism proposed for tyrosinase and from its kinetic analysis [Rodríguez-López, J. N., Tudela, J., Varón, R., García-Carmona, F. and García-Cánovas, F. (1992) J. Biol. Chem. 267, 3801-3810] a quantitative ratio has been established between the Michaelis constants for oxygen in the presence of monophenols and their o-diphenols. This ratio is used for the determination of the Michaelis constant for oxygen with monophenols when its value cannot be calculated experimentally.

Effect of ferrous ions on the monophenolase activity of tyrosinase.

The effect of ferrous ions on the monophenolase activity of tyrosinase has been studied. Although a shortening of the lag period which characterizes this hydroxylation reaction was observed, no direct effect on the enzyme was found. The reaction between ferrous ions and molecular oxygen in the presence of chelating agents, such as phosphate or EDTA, produces hydroxyl radicals. These radicals can hydroxylate tyrosine to generate L-3,4-dihydroxyphenylalanine (dopa). Catalase and scavengers of hydroxyl radicals inhibited both the shortening of the lag period and dopa formation. On the basis of these results, it is proposed that the influence of ferrous ions on tyrosinase is due to the formation of dopa in the chemical hydroxylation of tyrosine. Dopa transforms the Emet form of the enzyme (Cu2+Cu2+) into the Edeoxy form (Cu1+Cu1+) and, thus, shortens the lag period.