Conformational Landscape and the Selectivity of Cytochrome P450cam
Conformational Landscape and the Selectivity of Cytochrome P450cam
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DOI：
10.1021/acs.jpcb.5b03896
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发表时间：
2015-06-04
影响因子：
3.3
通讯作者：
Thielges, Megan C.
中科院分区：
文献类型：
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作者：
Basom, Edward J.;Spearman, James W.;Thielges, Megan C.
构象异质性和动力学可能有助于酶的显着活性，但具有挑战性的实验表征。这些特征在细胞色素P450类单加氧酶中特别令人感兴趣，所述细胞色素P450类单加氧酶具有很大的学术、医学和生物技术兴趣，因为它们识别广泛的底物，例如各种脂质、类固醇前体和异生物质，包括治疗剂。在这里，我们使用线性和二维红外光谱表征的原型P450，细胞色素P450凸轮，绑定到三个不同的基板，樟脑，去甲樟脑，或thiocamphor，这是羟基化高，低，和中间区域选择性，分别。这些数据表明，特定的相互作用与基板驱动人口的两种不同的构象，一个是与高区域选择性和另一个与较低的区域选择性。虽然Y96介导的氢键被认为是必要的，以定向基板的高区域选择性，人口和动态的构象状态在很大程度上是不变的Y96F突变。这项研究表明，构象景观的知识是理解P450活性的核心，这对优化药代动力学的治疗设计，以及P450和可能的其他酶的生物技术应用的操作具有重要的实际影响。
Conformational heterogeneity and dynamics likely contribute to the remarkable activity of enzymes but are challenging to characterize experimentally. These features are of particular interest within the cytochrome P450 class of monooxygenases, which are of great academic, medicinal, and biotechnological interest as they recognize a broad range of substrates, such as various lipids, steroid precursors, and xenobiotics, including therapeutics. Here, we use linear and 2D IR spectroscopy to characterize the prototypical P450, cytochrome P450cam, bound to three different substrates, camphor, norcamphor, or thiocamphor, which are hydroxylated with high, low, and intermediate regioselectivity, respectively. The data suggest that specific interactions with the substrate drive the population of two different conformations, one that is associated with high regioselectivity and another associated with lower regioselectivity. Although Y96 mediates a hydrogen bond thought necessary to orient the substrate for high regioselectivity, the population and dynamics of the conformational states are largely unaltered by the Y96F mutation. This study suggests that knowledge of the conformational landscape is central to understanding P450 activity, which has important practical ramifications for the design of therapeutics with optimized pharmacokinetics, and the manipulation of P450s, and possibly other enzymes, for biotechnological applications.