Muto N, Tan L
Biochem Biophys Res Commun. 1986 Apr 29;136(2):454-62. doi: 10.1016/0006-291x(86)90462-6.
According to the literature, the multistep reaction mechanism of estrogen biosynthesis proceeds with stereospecific loss of the equatorial 1 beta-, and axial 2 beta-protons. These results were deduced from experiments carried out, either with crude microsomes, or at best with impure enzyme extracts. However, when [1,2- 3H]4-androstene-3,17-dione of known absolute 3H-label distribution was incubated with a reconstituted enzyme system, consisting of homogeneous NADPH-cytochrome P-450 reductase and highly purified aromatase, we obtained results that can only be logically explained by a trans- and antiparallel elimination reaction of both the axially oriented C-2 beta-, and C-1-alpha protons. We further demonstrate that the reconstituted enzyme has an aromatase activity optimum at pH 7.2, and an apparent Km of 0.66 microM for NADPH and of 0.24 microM for 4-androstene-3,17-dione. Also, the enzyme requires 3 nmoles of NADPH for each nmole of estrogen that is formed.
根据文献记载，雌激素生物合成的多步反应机制伴随着赤道面1β-质子和轴向2β-质子的立体定向损失而进行。这些结果是从使用粗微粒体或充其量使用不纯酶提取物进行的实验中推导出来的。然而，当将已知绝对3H-标记分布的[1,2-3H]4-雄烯-3,17-二酮与由均一的NADPH-细胞色素P-450还原酶和高度纯化的芳香化酶组成的重组酶系统一起孵育时，我们得到的结果只能通过轴向取向的C-2β-和C-1-α质子的反式和平行消除反应进行合理解释。我们进一步证明，重组酶在pH 7.2时具有最佳芳香化酶活性，对NADPH的表观Km为0.66微摩尔，对4-雄烯-3,17-二酮的表观Km为0.24微摩尔。此外，每形成1纳摩尔雌激素，该酶需要3纳摩尔NADPH。
