Oxymetric and spectrophotometric study of the ascorbate oxidase activity shown by frog epidermis tyrosinase

Many studies concerning the effect of ascorbic acid on the action of tyrosinase on several substrates have been carried out with contradictory results. The results shown in this work comprise a hypothetical reaction mechanism, which explains the ascorbate oxidase activity of frog epidermis tyrosinase. The reaction between frog epidermis tyrosinase and L-ascorbic acid was studied by oxymetric and spectrophotometric assays. The activity was linearly related to enzyme concentration, with a Michaelis constant for L-ascorbic acid of 0.160 +/- 0.009 mM and Vmax of 90 +/- 4 nM/s. Maximum activity was obtained at pH 7.5. The stoichiometry of the reaction was calculated by measuring the substrate (O2 and L-ascorbic acid) consumption as well as the initial rates of the consumption of oxygen and the disappearance of L-ascorbic acid. The stoichiometry was found to be 1:2 (O2:L-ascorbic acid). The action of the tyrosinase inhibitor tropolone was also studied. All the results present evidence concerning the ascorbate oxidase activity of frog epidermis tyrosinase and a possible reaction mechanism based on the different enzymatic forms of tyrosinase to explain such activity.