Hawkinson D C, Eames T C, Pollack R M
Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228-5398.
Biochemistry. 1991 Jul 16;30(28):6956-64. doi: 10.1021/bi00242a021.
The putative intermediate dienol (2) in the steroid isomerase (KSI) catalyzed conversion of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (3) has been independently generated and tested as a substrate for KSI. At pH 7, dienol 2 is converted by KSI to a mixture of 1 (46%) and 3 (54%). The apparent second-order rate constant for reaction of 2 with KSI to produce 3 (kappa cat/Km = 2.3 x 10(8) M-1 s-1) is similar to that for reaction of 1 with KSI (kappa cat/Km = 2.1 x 10(8) M-1 s-1), demonstrating that 2 is kinetically competent. Isomerization of 1 by KSI in D2O gives only 5% of solvent deuterium incorporated into the product 3. When 2 reacts with KSI in D2O, and the product 3 is isolated (from direct reaction of 2 and from subsequent conversion of the 1 initially formed), ca. 80 atom % deuterium is located at C-6 beta, confirming that protonation of the dienol by KSI occurs at the same face as the proton transfer in the KSI catalyzed reaction of 1 to 3.
在甾体异构酶（KSI）催化5-雄甾烯-3,17-二酮（1）转化为4-雄甾烯-3,17-二酮（3）的过程中，假定的中间体二烯醇（2）已被独立制备并作为KSI的底物进行测试。在pH 7时，KSI将二烯醇2转化为1（46%）和3（54%）的混合物。2与KSI反应生成3的表观二级速率常数（kappa cat/Km = 2.3×10⁸ M⁻¹ s⁻¹）与1与KSI反应的速率常数（kappa cat/Km = 2.1×10⁸ M⁻¹ s⁻¹）相似，这表明2在动力学上是可行的。在D₂O中，KSI催化1异构化时，只有5%的溶剂氘掺入产物3中。当2在D₂O中与KSI反应，并分离出产物3（从2的直接反应以及最初形成的1的后续转化中得到）时，约80原子%的氘位于C-6β位，这证实了KSI使二烯醇质子化的位置与KSI催化1转化为3的反应中质子转移的面相同。
