Chin C C, Pineda J, Warren J C
J Biol Chem. 1982 Mar 10;257(5):2225-9.
Two affinity-labeling steroids (2-bromo[2'-14C]acetamidoestrone methyl ether and 16 alpha-bromo[2'-14C]acetoxyestradiol 3-methyl ether) which bear their reagent groups on the A- and D-ring of the molecule, respectively, and which are both substrates, have been used to elucidate spatial relationships of steroid and cofactor as they undergo the reversible binding step at the active site of human placental estradiol 17 beta-dehydrogenase. The 2-derivative alkylates its evolutive cofactor (NADH) in the presence of the enzyme but not in the absence of enzyme. The rate of cofactor alkylation increases with increasing quantities of enzyme and is slowed by estrone which competes for the enzyme-active site. To the contrary, the 16 alpha-derivative does not detectably alkylate its evolutive cofactor (NAD+). The product of cofactor alkylation by 2-bromoacetamidoestrone methyl ether was treated to effect hydrolytic removal of the adenine moiety from the remainder of the cofactor, reduction of the steroid 17-keto group, and crystallization. The final crystalline product has been identified as 2-[2'-6N-adenyl]acetamidoestradiol 3-methyl ether (IUPAC name: N-(3-methoxy-17 beta-hydroxy-1,3,5(10)-estratrien-2-yl)-2-(purin-6-ylamino) acetamide) by ultraviolet, infrared, NMR, and mass spectral analysis.
两种亲和标记类固醇（2-溴[2'-¹⁴C]乙酰氨基雌酮甲醚和16α-溴[2'-¹⁴C]乙酰氧基雌二醇3-甲醚），它们的反应基团分别位于分子的A环和D环上，并且都是底物，已被用于阐明类固醇和辅因子在人胎盘雌二醇17β-脱氢酶活性位点进行可逆结合步骤时的空间关系。2-衍生物在酶存在下会烷基化其进化辅因子（NADH），但在没有酶的情况下则不会。辅因子烷基化的速率随着酶量的增加而增加，并被竞争酶活性位点的雌酮所减缓。相反，16α-衍生物不会明显烷基化其进化辅因子（NAD⁺）。对2-溴乙酰氨基雌酮甲醚烷基化辅因子的产物进行处理，以实现从辅因子其余部分水解去除腺嘌呤部分、还原类固醇17-酮基并结晶。通过紫外、红外、核磁共振和质谱分析，最终的结晶产物已被鉴定为2-[2'-6N-腺苷基]乙酰氨基雌二醇3-甲醚（IUPAC名称：N-（3-甲氧基-17β-羟基-1,3,5(10)-雌甾三烯-2-基）-2-（嘌呤-6-基氨基）乙酰胺）。
