2-hydroxyestradiol and catechol

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.

Topics: Basidiomycota; Buffers; Butanols; Catechols; Chromatography, High Pressure Liquid; Estradiol; Glass; Monophenol Monooxygenase; Oxidation-Reduction; Quinones; Solvents; Spectrophotometry, Ultraviolet; Water

The hydroxylating activity of mushroom tyrosinase has been utilized for over a decade in the preparation of 2-hydroxyestradiol from estradiol, yet this same enzyme is known to function as an oxidant of o-dihydric compounds to the corresponding o-quinones. It was questioned why catechol estrogens do not react further, particularly since the tyrosinase activity (hydroxylating) is exceeded many fold by the diphenol oxidase activity of the enzyme. This report describes that the catechol estrogen will react in presence of enzyme but only if catechol is also present. Diphenol oxidase activity was measured either by the polarographic oxygen-utilization technique or by changes in the absorption spectrum at 206 and 256 nm. The enzyme activity was standardized with catechol (Km = 5.2 X 10(-4) M). The steroid did not react with the enzyme if catechol was absent. With catechol, the steroid reacted rapidly and completely (Km = 4.2 X 10(-4) M). The consumption of oxygen with catechol and 2-hydroxyestradiol was additive and stoichiometric, 1 g-atom oxygen/mol of either substrate. Kinetic analysis shows that catechol functions as an activator of the tyrosinase.

Topics: Basidiomycota; Catechol Oxidase; Catechols; Estradiol; Kinetics; Monophenol Monooxygenase; Oxidation-Reduction; Oxygen Consumption; Polarography; Spectrophotometry, Ultraviolet