Jacobsohn M K, Byler D M, Jacobsohn G M
Department of Biological Chemistry, Hahnemann University, Philadelphia, PA 19102.
Biochim Biophys Acta. 1991 Jan 23;1073(1):1-10. doi: 10.1016/0304-4165(91)90175-g.
We have reported previously that 2-hydroxyestradiol can be oxidized in the presence of catechol by mushroom tyrosinase, with a stoichiometric requirement of molecular oxygen (Jacobsohn, G.M. and Jacobsohn, M.K. (1984) Arch. Biochem. Biophys. 232, 189-196). It is then incorporated into melanin (Jacobsohn et al. (1988) J. Steroid Biochem. 31, 377-385). We now report on the isolation and characterization of the o-quinone as a product of the enzyme reaction from 2-hydroxyestradiol. The o-quinone was isolated from incubates and identified by its FTIR spectrum, in particular, by the appearance of a new band at 1652 cm-1, its migration in HPLC systems, its ultraviolet spectrum, its derivatization with phenylenediamine and comparison of these properties with the periodate oxidation product of the same substrate. The enzyme oxidation of the catechol estrogen was performed at 37 degrees C and did not require an activator; dopa at concentrations higher than 5 microM was inhibitory. At concentrations lower than 5 microM, dopa acted catalytically and was not consumed during the course of reaction. Ascorbic acid inhibited the reaction. The quinone exhibited both reversible and irreversible binding to performed melanin and to melanin actively synthesized by the enzyme. Incubation of 18 microM newly synthesized [4-14C]estradiol-2,3- quinone with mushroom tyrosinase for 45 min at 37 degrees C in presence of 400 microM dopa showed incorporation (irreversible binding) of 6.3 +/- 0.3% of label into melanin produced during the course of reaction. Similar incubations for 45 min of pre-formed melanin prepared from 400 microM dopa showed incorporation of 4.4 +/- 0.2% of the label. Reversible binding was 10-times greater than incorporation for both actively synthesized and preformed melanins. In the absence of dopa or catechol, enzyme incubations of either 2-hydroxy-estradiol or its quinone did not yield melanin. Data suggest that estradiol-2,3-quinone is an intermediate in the incorporation of the catechol estrogen into melanin by tyrosinase.
我们之前曾报道，在儿茶酚存在的情况下，2-羟基雌二醇可被蘑菇酪氨酸酶氧化，反应中对分子氧的化学计量需求为1:1（雅各布松，G.M.和雅各布松，M.K.（1984年）《生物化学与生物物理学文献》232卷，189 - 196页）。然后它会被整合到黑色素中（雅各布松等人（1988年）《类固醇生物化学杂志》31卷，377 - 385页）。我们现在报告关于从2-羟基雌二醇的酶反应产物中分离和鉴定邻醌的情况。邻醌是从孵育物中分离出来的，并通过其傅里叶变换红外光谱进行鉴定，具体而言，是通过在1652 cm-1处出现的新谱带、其在高效液相色谱系统中的迁移情况、其紫外光谱、用苯二胺进行衍生化以及将这些性质与相同底物的高碘酸盐氧化产物进行比较来鉴定的。儿茶酚雌激素的酶氧化反应在37℃下进行，不需要激活剂；浓度高于5 microM的多巴具有抑制作用。在浓度低于5 microM时，多巴起催化作用，并且在反应过程中不会被消耗。抗坏血酸会抑制该反应。醌对预先形成的黑色素以及由该酶主动合成的黑色素都表现出可逆和不可逆的结合。在400 microM多巴存在的情况下，将18 microM新合成的[4-14C]雌二醇-2,3-醌与蘑菇酪氨酸酶在37℃下孵育45分钟，结果显示有6.3±0.3%的标记物不可逆地结合到反应过程中产生的黑色素中。用400 microM多巴制备的预先形成的黑色素进行类似的45分钟孵育，结果显示有4.4±0.2%的标记物被结合。对于主动合成的黑色素和预先形成的黑色素，可逆结合都比结合量大10倍。在没有多巴或儿茶酚的情况下，对2-羟基雌二醇或其醌进行酶孵育都不会产生黑色素。数据表明，雌二醇-2,3-醌是儿茶酚雌激素通过酪氨酸酶整合到黑色素过程中的一个中间产物。
