Action Mechanism of Tyrosinase on meta- and para-Hydroxylated Monophenols

Lorena G. Fenoll, José Neptuno Rodríguez-López, Ramón Varón, Pedro Antonio García-Ruiz, Francisco García-Cánovas,  and José Tudela

Abstract

The relationship between the structure and activity of metaand parahydroxylated monophenols was studied during their tyrosinasecatalysed hydroxylation and the ratelimiting steps of the reaction mechanism were identified. The parahydroxylated substrates permit us to study the effect of a substituent (R) in the carbon-1 position (C-1) of the benzene ring on the nucleophilic attack step, while the meta group permits a similar study of the effect on the electrophilic attack step. Substrates with a OCH[3] group on C-1, as phydroxyanisol (4HA) and mhydroxyanisol (3HA), or with a CH[2]OH group, as phydroxybenzylalcohol (4HBA) and mhydroxybenzylalcohol (3HBA), were used because the effect of the substituent (R) size was assumed to be similar. However, the electrondonating effect of the OCH[3] group means that the carbon-4 position (C-4) is favoured for nucleophilic attack (parahydroxylated substrates) or for electrophilic attack (metahydroxylated substrates). The electronattracting effect of the CH[2]OH group has the opposite effect, hindering nucleophilic (para) or electrophilic (meta) attack of C-4. The experimental data point to differences between the maximum steadystate rate (V ) of the different substrates, the value of this parameter depends on the nucleophilic and electrophilic attack. However, differences are greatest in the Michaelis constants (K ), with the metahydroxylated substrates having very large values. The catalytic efficiency k /K is much greater for the parahydroxylated substrates although it varies greatly between one substrate and the other. However, it varies much less in the metahydroxylated substrates since this parameter describes the power of the nucleophilic attack, which is weaker in the meta OH. The large increase in the K of the metahydroxylated substrates might suggest that the phenolic OH takes part in substrate binding. Since this is a weaker nucleophil than the parahydroxylated substrates, the binding constant decreases, leading to an increase in K . The catalytic efficiency of tyrosinase on a monophenol (para or meta) is directly related to the nucleophilic power of the oxygen of the phenolic OH. The oxidation step is not limiting since if this were the case, the para and meta substrates would have the same V . The small difference between the absolute values of V suggests that the rate constants of the nucleophilic and electrophilic attacks are on the same order of magnitude.

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Biological Chemistry keeps you up-to-date with the latest advances in the molecular life sciences. The journal publishes Research Articles, Short Communications, Reviews and Minireviews. Areas include: general biochemistry/pathobiochemistry, structural biology, molecular and cellular biology, genetics and epigenetics, virology, molecular medicine, plant molecular biology/biochemistry and novel experimental methodologies.

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