Activation of androgen biosynthesis and drug metabolism by cytochrome b5

所有19碳的雄激素都来自21碳的类固醇，通过顺序的17？- 细胞色素P450c17(CyP17A1)的羟基酶和17，20-裂解酶活性。这个 17，20-裂解酶反应的复杂化学被选择性地刺激高达10倍 细胞色素b5。与b5与其他一些细胞色素的相互作用不同 P450，显然涉及b5的电子转移，我们的数据认为b5 变构激活CYP17A1的17，20-裂解酶活性。我们已经确定了一个 B5的特定区域，对刺激17，20-裂解酶活性至关重要，并已显示出 这种刺激的大小取决于底物。我们现在建议 阐明b5对17，20-裂解酶活性的作用机制，并比较 B5-CyP17A1与b5相互作用的机制和结构特征 其他由P450介导的反应。 在目标1中，我们将确定关键残基的空间和电子要求 在激活17，20-裂解酶活性所必需的CYP17a1和b5上。在目标2中，我们将 推导出b5促进的17，20-裂解酶反应的微观步骤 快速和稳态前的动力学实验。在目标3中，我们将确定是否相同 刺激17，20-裂解酶活性的重要的b5表面也调节活性。 以及同样的机制原理是否适用于这些 其他反应。在目标4中，我们将设计b5的可溶性形式，它也可以刺激17，20- 裂解酶活性，为未来对CYP17a1-b5复合体的结构研究奠定了基础。 因此，我们将系统地定义b5对细胞色素P17A1的作用机制， 潜在地确定抑制雄激素产生的新方法，通过 靶向CYP17a1-b5相互作用。 涉及雄激素过多的疾病(如多囊卵巢 综合症)和需要雄激素的疾病(如前列腺癌) 通过抑制雄激素合成来治疗的。目前可用的代理有 不太理想。通过定义17-羟基酶/17，20-裂解酶如何使 雄激素，我们希望建立治疗雄激素依赖的新方法 人类疾病。我们还将确定这些原则是否适用于其他类似的 代谢药物的酶。抑制雄激素合成是目前治疗前列腺癌等人类疾病的方法 癌症和多囊卵巢综合征。这种方法不是最优的。通过定义如何使用 负责合成这些雄激素的酶，我们希望建立新的 治疗方法。我们的研究将适用于其他代谢药物的酶。

All 19-carbon androgens derive from 21-carbon steroids via sequential 17¿- hydroxylase and 17,20-lyase activities of cytochrome P450c17 (CYP17A1). The complex chemistry of the 17,20-lyase reaction is selectively stimulated up to 10-fold by cytochrome b5 (b5). In contrast to the interactions of b5 with some other cytochromes P450, which apparently involve electron transfer from b5, our data argue that b5 allosterically activates the 17,20-lyase activity of CYP17A1. We have identified a specific region of b5 that is critical for stimulation of 17,20-lyase activity and have shown that the magnitude of this stimulation is substrate-dependent. We now propose to elucidate the mechanism of action of b5 on 17,20-lyase activity and to compare the mechanistic and structural features of the b5-CYP17A1 interaction with b5 action on other P450-mediated reactions. In Aim 1, we will determine the steric and electronic requirements of key residues on CYP17A1 and b5 necessary to stimulate 17,20-lyase activity. In Aim 2, we will deduce the microscopic steps of the 17,20-lyase reaction that are enhanced by b5 using rapid and pre-steady state kinetics experiments. In Aim 3, we will determine if the same surface of b5 important for stimulating 17,20-lyase activity also modulates the activities of other cytochromes P450 and whether the same mechanistic principles apply to these other reactions. In Aim 4, we will engineer soluble forms of b5 that also stimulate 17,20- lyase activity, setting the stage for future structural studies of the CYP17A1-b5 complex. We thus will systematically define the mechanism of action of b5 on CYP17A1, potentially identifying novel approaches for suppressing androgen production, by targeting the CYP17A1-b5 interaction. Diseases that involve androgen overproduction (such as polycystic ovary syndrome) and diseases that require androgens (such as prostate cancer) can be treated by inhibition of androgen synthesis. Currently available agents are suboptimal. By defining how the enzyme 17-hydroxylase/17,20-lyase makes androgens, we hope to establish new approaches to treating androgen-dependent human diseases. We will also determine if these principles apply to other similar enzymes that metabolize drugs. Inhibition of androgen synthesis is the current treatment of human diseases like prostate cancer and polycystic ovary syndrome. This approach is suboptimal. By defining how the enzyme responsible synthesizes these androgens, we hope to establish new approaches to treatment. Our study will apply to other enzymes that metabolize drugs.