Kinetic study of the oxidation of 3-hydroxyanisole catalysed by tyrosinase

Tyrosinase hydroxylates 3-hydroxyanisole in the 4-position. The reaction pr oduct accumulates in the reaction medium with a lag time (tau) which dimini shes with increasing concentrations of enzyme and lengthens with increasing concentrations of substrate, thus fulfilling all the predictions of the me chanism proposed by us for 4-hydroxyphenols. The kinetic constants obtained , k(cat)(M) = (46.87 +/- 2.06) s(-1) and K-m(M) = (5.40 +/- 0.60) mM, are d ifferent from those obtained with 4-hydroxyanisole, k(cat)(M) = (184.20 +/- 6.1) s(-1) and K-m(M) = (0.08 +/- 0.004) mM. The catalytic efficiency, k(c at)(M)/K-m(M) is, therefore, 265.3 times greater with 4-hydroxyanisole. The possible rate-determining steps for the reaction mechanism of tyrosinase o n 3- and 4-hydroxyanisole, based on the NMR spectra of both monophenols, ar e discussed. These possible rate-determining steps are the nucleophilic att ack of hydroxyl's oxygen on the copper and the electrophilic attack of the peroxide on the aromatic ring. Both steps may be of similar magnitude, i.e. take place in the same time scale. (C) 2000 Elsevier Science B.V. All righ ts reserved.