The terminal steps of cortisol and aldosterone biosynthesis

人类肾上腺皮质产生过多的醛固酮和皮质醇通常会导致 高血压、心力衰竭、肥胖、糖耐量减低和骨量减少。细胞色素P450 11B2和 P450 11B1分别催化醛固酮和皮质醇生物合成的最后步骤。该地带- 这些酶的特定调节维持盐和水的平衡或碳水化合物代谢，并 对生理压力的反应。这两种酶具有95%的序列相似性，并且都催化11β- 11-脱氧皮质酮和11-脱氧皮质醇的羟基化。相比之下，P450 11B1的18-羟基酶是 与P450 11B2相比，P450 11B2的活性较差，并且只有P450 11B2具有18-氧化酶活性，它能将18-羟基-2 从皮质酮到醛固酮。P450 11B2具有很高的加工性，因为中间体主要不 在更多的转化为醛固酮之前解离，但这种过程的原因以及这是否 生产醛固酮所需的属性尚不清楚。它的微观步骤与 这些P450 11B1提供了一个机会，以了解独特的生物化学的醛固酮生产。 我们的长期目标是阐明导致高18岁的生化和物理特性- 羟基酶和中等活性的18-氧化酶对P450 11B2而不是P450 11B1。我们的中心假设 是：(1)11-脱氧皮质酮生成的新生皮质酮在活性部位的定位 P45011B2的构象不同于皮质酮与AS结合时复合体的性质 初始底物；以及(2)P450 11B2的加工性反映了慢解离速率的组合 与中间体的偶联效率高。因此，本应用程序的目标是 表征结合常数、结合和解离速率、稳态前(单周转)动力学、 以及P450 11B1和P450 11B2与几种底物的偶联效率，并推导出 参数与过程性的相关性最好。作为这些研究的工具，我们将使用改变 P450 11B1和P450 11B2以及具有特定化学性质的替代底物的活性。 在目标1中，我们将剖析野生型和野生型P450 11B1和P450 11B2的18-羟基酶活性 突变。在目标2中，我们将与目标2一样，通过一系列平行的研究集中于18-氧化酶的活性。 3、我们将重点关注外在因素，特别是膜环境的脂质成分，在 调节P450 11B2的加工性和后两种活性。我们将使用纳米盘来产生人造 并在HEK-293或V79细胞中证实了关键结果。这项工作将利用 Auchus和Waskell实验室用来研究细胞色素的专门仪器 P450酶和密歇根大学最先进的串联质谱仪。在……里面 通过这种方式，我们将系统地定义P450的反应机理和性质的差异 11B1和P450 11B2。这些研究将为筛选安全抑制关键步骤的更好药物的策略提供依据 用于治疗人类疾病的醛固酮和皮质醇的生产。

Excess aldosterone and cortisol production from the human adrenal cortex commonly contributes to hypertension, heart failure, obesity, glucose intolerance, and low bone mass. Cytochromes P450 11B2 and P450 11B1 catalyze the final steps in the biosynthesis of aldosterone and cortisol, respectively. The zone- specific regulation of these enzymes maintains salt and water balance or carbohydrate metabolism and response to physiologic stress. The enzymes share 95% sequence similarity, and both catalyze the 11β- hydroxylation of 11-deoxycorticosterone and 11-deoxycortisol. In contrast, the 18-hydroxylase of P450 11B1 is poor compared to P450 11B2, and only P450 11B2 has 18-oxidase activity, which converts 18-hydroxy- corticosterone to aldosterone. P450 11B2 has high processivity, in that the intermediates predominantly do not dissociate before additional turnovers to aldosterone, but the reasons for this processivity and whether this property is required for aldosterone production are not known. A comparison of its microscopic steps with those of P450 11B1 offers an opportunity to understand the unique biochemistry of aldosterone production. Our long-term goal is to elucidate the biochemical and physical properties that confers high 18- hydroxylase and moderate 18-oxidase activities to P450 11B2 but not P450 11B1. Our central hypotheses are that: (1) the positioning of nascent corticosterone generated from 11-deoxycorticosterone in the active site and the conformation of P450 11B2 is different that the nature of the complex when corticosterone binds as initial substrate; and (2) that the processivity of P450 11B2 reflects a combination of slow dissociation rates and high coupling efficiency with the intermediates. Consequently, the objectives of this application are to characterize the binding constants, binding and dissociation rates, pre-steady state (single turnover) kinetics, and coupling efficiencies for P450 11B1 and P450 11B2 with several substrates and to deduce which parameters correlate best with processivity. As tools for these studies, we will employ mutations that alter the activities for P450 11B1 and P450 11B2 and alternate substrates that have specific chemical properties. In Aim 1, we will dissect the 18-hydroxylase activity of P450 11B1 and P450 11B2, wild-type and mutations. In Aim 2, we will focus on the 18-oxidase activity with a parallel series of studies as in Aim 2. In Aim 3, we will focus on extrinsic factors, specifically the lipid composition of the membrane environment, in regulating the processivity and latter two activities of P450 11B2. We will use nanodiscs to generate artificial enzyme-membrane systems and confirm key results in transfected HEK-293 or V79 cells. The work will utilize the specialized instrumentation that the Auchus and Waskell laboratories have employed to study cytochrome P450 enzymes for many years and state-of-the art tandem mass spectrometry at the University of Michigan. In this manner, we will systematically define the differences in the reaction mechanisms and properties for P450 11B1 and P450 11B2. These studies will inform strategies to screen for better drugs to safely inhibit key steps of aldosterone and cortisol production for the treatment of human diseases.