A study of the monophenolase activity of tyrosinase by measuring the steady state rate with a group of p-substituted monophenols provides the following kinetic information: k(m)(cat) and the Michaelis constant, K-M(m). Analysis of these data taking into account chemical shifts of the carbon atom supporting the hydroxyl group (delta) and sigma(+)(p), enables a mechanism to be proposed for the transformation of monophenols into odiphenols, in which the first step is a nucleophilic attack on the copper atom on the form E-ox (attack of the oxygen of the hydroxyl group of C-1 on the copper atom) followed by an electrophilic attack (attack of the hydroperoxide group on the ortho position with respect to the hydroxyl group of the benzene ring, electrophilic aromatic substitution with a reaction constant rho of -1.75). These steps show the same dependency on the electronic effect of the substituent groups in C-4. Furthermore, a study of a solvent deuterium isotope effect on the oxidation of monophenols by tyrosinase points to an appreciable isotopic effect. In a proton inventory study with a series of p-substituted phenols, the representation of k(cat)(fn)/k(cat)(fo) against n (atom fractions of deuterium), where k(cat)(fn) is the catalytic constant for a molar fraction of deuterium (n) and k(cat)(fo) is the corresponding kinetic parameter in a water solution, was linear for all substrates. These results indicate that only one of the proton transfer processes from the hydroxyl groups involved the catalytic cycle is responsible for the isotope effects. We suggest that this step is the proton transfer from the hydroxyl group of C-1 to the peroxide of the oxytyrosinase form (E-ox). After the nucleophilic attack, the incorporation of the oxygen in the benzene ring occurs by means of an electrophilic aromatic substitution mechanism in which there is no isotopic effect. (C) 2012 Elsevier Inc. All rights reserved.