Activation of androgen biosynthesis and drug metabolism by cytochrome b5

所有的19碳雄激素都是由21碳类固醇通过连续的17 <$-羟化酶产生的 和细胞色素P450 c17（CYP 17 A1）的17，20-裂解酶活性。复杂化学 的17，20-裂解酶反应被细胞色素b5（b5）选择性刺激高达10倍 体外与b5与其他一些细胞色素P450的相互作用相反， 我们的数据表明，b5是变构的， 激活CYP 17 A1的17，20-裂解酶活性。我们已经确定了b5的一个特定区域 这对于刺激17，20-裂解酶活性至关重要， 这种刺激是底物依赖性的。我们现在建议阐明 B5在体内对17，20-裂解酶活性和雄激素合成的这种作用， 比较b5-CYP 17 A1与b5相互作用的机制和结构特征 对其他P450介导的反应的作用，准备详细询问 b5对CYP 17 A1的作用机制。 为了首先证明b5对雄激素生物合成是必需的，我们将产生和 表征睾丸中缺乏b5的小鼠的目的1。我们将描述生育能力， 激素分泌和睾丸组织学。的酶活性 将对睾丸进行详细研究。在目标2中，我们将确定b5上的残基是否是 刺激17，20-裂解酶活性所需的蛋白质也是调节 其他细胞色素P450和开始的活动，以确定是否相同 所有P450酶都涉及微观步骤。因此，我们将定义 b5对CYP 17 A1作用在哺乳动物生理学中的重要性，我们将开始 确定其作用机制是否与b5对其他细胞色素P450的作用相似。 这一知识将提供一种新的方法来抑制雄激素的产生， 靶向CYP 17 A1-b5相互作用。

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 vitro. 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 importance of this action of b5 on 17,20-lyase activity and androgen synthesis in vivo and to compare the mechanistic and structural features of the b5-CYP17A1 interaction with b5 action on other P450-mediated reactions, in preparation for a detailed interrogation of the mechanism of b5 action on CYP17A1. To first prove that b5 is essential for androgen biosynthesis, we will generate and characterize mice lacking b5 in the testis for Aim 1. We will characterize the fertility, hormone production, and testicular histology of these mice. The enzymatic activity of the testes will be studied in detail. In Aim 2, we will determine if residues on b5 that are required for stimulating 17,20-lyase activity are also essential for modulating the activities of other cytochromes P450 and begin to determine whether the same microscopic steps are involved for all P450 enzymes. We thus will define the importance of b5 action on CYP17A1 in mammalian physiology, and we will begin to ascertain if the mechanism of action is similar to b5 action on other cytochromes P450. This knowledge will provide a novel approach for suppressing androgen production, by targeting the CYP17A1-b5 interaction.