Action mechanism of tyrosinase on meta- and para-hydroxylated monophenols

The relationship between the structure and activity of meta- and para-hydro xylated monophenols was studied during their tyrosinase-catalysed hydroxyla tion and the rate-limiting steps of the reaction mechanism were identified, The para-hydroxylated substrates permit us to study the effect of a substi tuent (R) in the carbon-1 position (C-l) of the benzene ring on the nucleop hilic attack step, while the meta group permits a similar study of the effe ct on the electrophilic attack step. Substrates with a -OCH3 group on C-1, as p-hydroxyanisol (4HA) and m-hydroxyanisol (3HA), or with a -CH2OH group, as p-hydroxybenzylalcohol (4HBA) and m-hydroxybenzylalcohol (3HBA), were u sed because the effect of the substituent (R) size was assumed to be simila r. However, the electron-donating effect of the -OCH3 group means that the carbon-4 position (C-4) is favoured for nucleophilic attack (para-hydroxyla ted substrates) or for electrophilic attack (meta-hydroxylated substrates), The electron-attracting effect of the -CH2OH group has the opposite effect , hindering nucleophilic (para) or electrophilic (meta) attack of C-4. The experimental data point to differences between the maximum steady-state rate (V-max(M)) of the different substrates, the value of this parameter d epends on the nucleophilic and electrophilic attack, However, differences a re greatest in the Michaelis constants (K-m(M)), with the meta-hydroxylated substrates having very large values. The catalytic efficiency k(cat)(M)/K- m(M) is much greater for the para-hydroxylated substrates although it varie s greatly between one substrate and the other, However, it varies much less in the meta-hydroxylated substrates since this parameter describes the pow er of the nucleophilic attack, which is weaker in the meta OH. The large in crease in the K-m(M) of the meta-hydroxylated substrates might suggest that the phenolic OH takes part in substrate binding. Since this is a weaker nu cleophil than the para-hydroxylated substrates, the binding constant decrea ses, leading to an increase in K-m(M). The catalytic efficiency of tyrosina se on a monophenol (para or meta) is directly related to the nucleophilic p ower of the oxygen of the phenolic OH, The oxidation step is not limiting s ince if this were the case, the para and meta substrates would have the sam e V-max(M). The small difference between the absolute values of V-max(M) su ggests that the rate constants of the nucleophilic and electrophilic attack s are on the same order of magnitude.