Department of Chemistry , Marquette University , Milwaukee , Wisconsin 53233 , United States.
Department of Biochemistry , University of Illinois at Urbana-Champaign , 505 S. Goodwin , Urbana , Illinois 61801 , United States.
J Am Chem Soc. 2018 Jun 13;140(23):7324-7331. doi: 10.1021/jacs.8b03901. Epub 2018 Jun 5.
The multifunctional enzyme, cytochrome P450 (CYP17A1), plays a crucial role in the production of androgens, catalyzing two key reactions on pregnenolone (PREG) and progesterone (PROG), the first being a 17-hydroxylation to generate 17-OH PREG and 17-OH PROG, with roughly equal efficiencies. The second is a C-C bond scission or "lyase" reaction in which the C17-C20 bond is cleaved, leading to the eventual production of powerful androgens, whose involvement in the proliferation of prostate cancer has generated intense interest in developing inhibitors of CYP17A1. For humans, the significance of the C-C bond cleavage of 17-OH PROG is lessened, because it is about 50 times less efficient than for 17-OH PREG in terms of k/K. Recognizing the need to clarify relevant reaction mechanisms involved with such transformations, we first report studies of solvent isotope effects, results of which are consistent with a Compound I mediated PROG hydroxylase activity, yet exclude this intermediate as a participant in the formation of androstenedione (AD) via the lyase reaction. This finding is also supported by a combination of cryoreduction and resonance Raman spectroscopy that traps and structurally characterizes the key hemiketal reaction intermediates. Adding to a previous study of PREG and 17-OH PREG metabolism, the current work provides definitive evidence for a more facile protonation of the initially formed ferric peroxo-intermediate for 17-OH PROG-bound CYP17A1, compared to the complex with 17-OH PREG. Importantly, Raman characterization also reveals an H-bonding interaction with the terminal oxygen of the peroxo fragment, rather than with the proximal oxygen, as is present for 17-OH PREG. These factors would favor a diminished lyase activity of the sample with 17-OH PROG relative to the complex with 17-OH PREG, thereby providing a convincing structural explanation for the dramatic differences in activity for these lyase substrates in humans.
多功能酶细胞色素 P450（CYP17A1）在雄激素的生成中起着关键作用，催化 pregnenolone（PREG）和 progesterone（PROG）上的两个关键反应，第一个是 17-羟化生成 17-OH PREG 和 17-OH PROG，效率大致相等。第二个是 C-C 键的断裂或“裂解”反应，其中 C17-C20 键被切断，最终生成强大的雄激素，其在前列腺癌的增殖中的作用引起了人们极大的兴趣，开发 CYP17A1 的抑制剂。对于人类来说，17-OH PROG 的 C-C 键断裂的意义较小，因为它的 k/K 比 17-OH PREG 低约 50 倍。为了阐明这些转化涉及的相关反应机制，我们首先报告了溶剂同位素效应的研究结果，这些结果与 Compound I 介导的 PROG 羟化酶活性一致，但排除了这种中间体作为通过裂解反应生成雄烯二酮（AD）的参与者。低温还原和共振拉曼光谱的结合也支持了这一发现，该方法捕获并结构表征了关键的半缩酮反应中间体。除了之前对 PREG 和 17-OH PREG 代谢的研究外，目前的工作为 CYP17A1 与 17-OH PROG 结合时，与 17-OH PREG 结合时相比，铁过氧中间体能更易质子化提供了明确的证据。重要的是，拉曼表征还揭示了与过氧片段末端氧的氢键相互作用，而不是与过氧片段近端氧的氢键相互作用，如 17-OH PREG 中存在的氢键相互作用。这些因素有利于 17-OH PROG 结合物的裂解活性相对于 17-OH PREG 结合物降低，从而为这些裂解底物在人类中的活性差异提供了令人信服的结构解释。
