The terminal steps of cortisol and aldosterone biosynthesis

人肾上腺皮质产生过量的醛固酮和皮质醇通常会导致 高血压、心力衰竭、肥胖、葡萄糖耐受不良和低骨量。细胞色素P450 11B 2和 P450 11B 1分别催化醛固酮和皮质醇生物合成的最后步骤。区域- 这些酶的特定调节维持盐和水平衡或碳水化合物代谢， 对生理压力的反应这两种酶有95%的序列相似性，都催化11β- 11-脱氧皮质酮和11-脱氧皮质醇羟基化。相反，P450 11B 1的18-羟化酶是 与P450 11 B2相比，P450 11 B2较差，并且只有P450 11 B2具有18-氧化酶活性，其将18-羟基- 皮质酮到醛固酮。P450 11 B2具有高的持续合成能力，因为中间体主要不 在额外的醛固酮周转之前解离，但是这种持续合成的原因以及这种持续合成是否 醛固酮生产所需的性质尚不清楚。其微观步骤与 P450 11B 1的那些提供了了解醛固酮产生的独特生物化学的机会。 我们的长期目标是阐明生物化学和物理特性，赋予高18- 羟化酶和中度18-氧化酶活性与P450 11B 2有关，但与P450 11B 1无关。我们的核心假设 11-脱氧皮质酮生成的新生皮质酮位于活性部位 P450 11B 2的构象与皮质酮结合时的复合物性质不同， P450 11B 2的持续合成能力反映了缓慢的解离速率 与中间体的偶联效率高。因此，本申请的目的是 表征结合常数、结合和解离速率、预稳态（单转换）动力学， 以及P450 11B 1和P450 11B 2与几个衬底的耦合效率，并推导出 参数与持续合成能力相关性最好。作为这些研究的工具，我们将采用改变基因的突变， P450 11B 1和P450 11B 2以及具有特定化学性质的替代底物的活性。 在目的1中，我们将剖析P450 11B 1和P450 11B 2、野生型和非野生型的18-羟化酶活性。 突变。在目标2中，我们将重点关注18-氧化酶活性，并进行一系列平行研究。在Aim中 3，我们将重点关注外在因素，特别是膜环境的脂质组成， 调节P450 11B 2的持续合成能力和后两种活性。我们将使用纳米盘来生成人造的 酶-膜系统，并确认转染的HEK-293或V79细胞中的关键结果。这项工作将利用 奥胡斯和瓦斯凯尔实验室用来研究细胞色素的专用仪器 P450酶多年和最先进的串联质谱在密歇根大学。在 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 11B 1和P450 11B 2。这些研究将为筛选更好的药物以安全地抑制关键步骤提供信息 用于治疗人类疾病的醛固酮和皮质醇生产。

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.