Kinetic evaluation of catalase and peroxygenase activities of tyrosinase

Tyrosinase is a copper monooxygenase containing a coupled dinuclear copper active site (type-3 copper), which catalyzes oxygenation of phenols (phenolase activity) as well as dehydrogenation of catechols (catecholase activity) using O-2 as the oxidant. In this study, catalase activity (conversion of H2O2 to (1/2)O-2 and H2O) and peroxygenase activity (H2O2-dependent oxygenation of substrates) of mushroom tyrosinase have been examined kinetically by using amperometric O-2 and H2O2 sensors. The catalase activity has been examined by monitoring the initial rate of O-2 production from H2O2 in the presence of a catalytic amount of tyrosinase in 0.1 M phosphate buffer (pH 7.0) at 25 degreesC under initially anaerobic conditions. It has been found that the catalase activity of mushroom tyrosinase is three-order of magnitude greater than that of mollusk hemocyanin. The higher catalase activity of tyrosinase could be attributed to easier accessibility of H2O2 to the dinuclear copper site of tyrosinase. Mushroom tyrosinase has also been demonstrated for the first time to catalyze oxygenation reaction of phenols with H2O2 (peroxygenase activity). The reaction has been investigated kinetically by monitoring the H2O2 consumption rate in 0.5 M borate buffer (pH 7.0) under aerobic conditions. Similarity of the substituent effects of a series of p-substituted phenols in the peroxygenase reaction with H2O2 to those in the phenolase reaction with O-2 as well as the absence of kinetic deuterium isotope effect with a perdeuterated substrate (p-Cl-C6D4OH VS p-Cl-C6H4OH) clearly demonstrated that the oxygenation mechanisms of phenols in both systems are the same, that is, the electrophilic aromatic substitution reaction by a (mu-eta(2):eta(2)-peroxo)-dicopper(II) intermediate of oxy-tyrosinase.