Oxidation of 2-hydroxyestradiol and its incorporation into melanin by mushroom tyrosinase.
The presence of catechol in a reaction mixture has been shown previously to promote oxidation of 2-hydroxyestradiol by mushroom tyrosinase. It was now found that the oxidized products of the catecholesterogen are incorporated into melanin under the influence of the enzyme. Whether the oxidation is restricted to tyrosinase or to enzymes with specific steroid oxidizing properties was examined by separating tyrosinase on agarose gel followed by hydroxylapatite. The effectiveness of separation was monitored electrophoretically. Two bands of enzyme activity of 127 kDa were found. One of these bands could be cleanly separated from the other. The fraction which contained the single band, as well as the one which contained both bands, had similar apparent Km values; i.e. 1.5 x 10(-4) and 2.1 x 10(-4) M. They both catalyzed oxidation of 2-hydroxyestradiol but only in the presence of catechol. All enzyme fractions showed the same pattern of activity towards the estrogen. HPLC analysis of reaction products of catechol indicated that not all of the substrate was consumed during the reaction. About 26% remained unreacted at an initial concentration of 100-400 microM of catechol. This remaining catechol, rather than its reaction products, appears to function as activator of the steroid reaction. The data are consistent with the presence on the enzyme of an allosteric activator site specific for catechol and an active site with a much lower structural specificity occupied by the catecholestrogen.
D001487:Basidiomycota / Q000201:enzymology*; D004156:Catechol Oxidase / Q000378:metabolism*; D002396:Catechols / Q000378:metabolism / Q000494:pharmacology; D002845:Chromatography; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000031:analogs & derivatives* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008543:Melanins / Q000378:metabolism*; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D002396:Catechols; D008543:Melanins; D004958:Estradiol; C001390:2-hydroxyestradiol; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase