Activity of mushroom tyrosinase on catechol and on a catechol estrogen in an organic solvent.

A suspension of tyrosinase-coated glass beads in butanol effectively oxidizes catechol substrate. The enzyme is not soluble in the organic solvent and activity can be stopped by removal of the solid state enzyme after low-speed centrifugation or decantation. The product was assayed by HPLC and by its reactivity towards Besthorn's reagent, which gave a reaction typical for o-quinones. Addition of water to the extent of 0.5 to 4% raised the rate of substrate utilization but the accumulation of quinone first increased and then began to decrease. It is suggested that the product in dry butanol is prevented from reacting further by lack of water, which is necessary to promote secondary reactions causing free radical formation and leading ultimately to polymerization to melanin. Successive washes of the solid state enzyme with butanol increased enzyme activity, indicating presence of a butanol extractable inhibitor in the tyrosinase preparation. The enzyme on glass beads in butanol suspension was stabilized by the presence of substrate. 2-Hydroxyestradiol acted as an inhibitor of the tyrosinase-catalyzed oxidation of catechol. The data obtained can be interpreted to mean that the oxidation of the estrogen in the presence of tyrosinase, as previously reported, may be dependent upon the enzyme-catalyzed oxidation of catechol. The oxidation product of catechol, the o-quinone, is likely to function as oxidant towards 2-hydroxyestradiol.