Protein Dynamics and Substrate Specificity in Cytochrome P450s

细胞色素 P450 中的蛋白质动力学和底物特异性

• 批准号: 8025968
• 负责人: Megan Corrine Thielges
• 金额: $ 5.13万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-15 至 2013-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8025968
• 关键词: Active Sites; Adamantane; Address; Affect; Anabolism; Azides; Binding; Binding Sites; Biochemical Process; Biological Availability; Biological Process; Camphor; Camphor 5-Monooxygenase; Catalysis; Complex; Cytochrome P450; Cytochrome P450 3A4; Cytochromes; Distal; Drug usage; Enzymes; Erythromycin; Extravasation; Family; Fatty Acids; Foundations; Future; Health; Heme; Homologous Gene; Human; Hydrogen Peroxide; Hydroxylation; Investigation; Ketoconazole; Label; Ligands; Measurement; Measures; Medicine; Methodology; Motion; NMR Spectroscopy; Nature; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Phenylalanine; Physiological; Play; Process; Production; Progesterone; Protein Binding; Protein Dynamics; Proteins; Research; Resolution; Role; Side; Site; Solvents; Specificity; Spectrum Analysis; Structure; Substrate Specificity; Superoxides; Techniques; Testing; Testosterone; Theoretical Studies; Therapeutic; Time; Toxic effect; Toxin; Xenobiotic Metabolism; analog; chemical addition; design; drug metabolism; flexibility; heme a; hormone biosynthesis; molecular recognition; norcamphor; protein function; public health relevance; research study; two-dimensional

DESCRIPTION (provided by applicant): The proposed research will examine how dynamics influence molecular recognition and catalysis in cytochrome (cyt) P450s. While this family of enzymes catalyzes the hydroxylation of a wide variety of substrates, different cyt P450 homologs show varying degrees of substrate specificity. Understanding this specificity is critical for human health as cyt P450s are involved in many biochemical processes, such as the metabolism of xenobiotics and the biosynthesis of hormones and fatty acids. Importantly, cyt P450s also metabolize the majority of clinically used drugs, so their activity contributes directly to bioavailability and toxicity. While dynamics have been implicated in the substrate specificity of cyt P450s, previous experimental studies have lacked the time resolution to measure the fastest motions that make critical contribution to substrate recognition. However, the newly developed techniques of two dimensional infrared vibrational echo spectroscopy have the potential to directly measure the fast dynamics of cyt P450s and how they might contribute to substrate specificity. The proposed experiments will test the hypothesis that cyt P450s are highly dynamic and that the binding of different substrates is associated with varying changes in dynamics. First, the dynamics at the active site of the relatively substrate-specific cyt P450cam will be characterized using a heme-bound CO probe in the unbound protein and in the protein bound to its substrate, camphor, and several substrate analogs. These experiments will be extended to specific sites throughout the tertiary structure of cyt P450cam by the use of site-specifically incorporated azidophenylalanine probes. Finally, to explore the dynamics of the medically important human cyt P450s, homebound CO will be used to measure the active site dynamics of cyt P450 3A4, one of the most promiscuous cyt P450s that metabolizes a large variety of drug molecules. With both cyt P450s, correlations between the observed dynamics and the presence and nature of bound substrates will support the hypothesis that protein dynamics are important for controlling activity. This would have important ramifications for our understanding of drug metabolism. Thus, the proposed research will further our understanding of dynamics of cyt P450s and how they might contribute to their critical biological functions. PUBLIC HEALTH RELEVANCE: Two dimensional infrared vibrational echo spectroscopy will be used to study how protein motions affect the substrate repertoires of cytochrome P450s. These enzymes are required to process metabolites and toxins, and they play an essential role in drug bioavailability and toxicity. Thus, understanding their activity would be of direct utility in the design of therapeutics with increased efficacy and reduced toxicity.

描述（由申请人提供）：拟议的研究将研究动力学如何影响细胞色素（cyt）P450 中的分子识别和催化。虽然该酶家族催化多种底物的羟基化，但不同的 cyt P450 同系物表现出不同程度的底物特异性。了解这种特异性对于人类健康至关重要，因为 cyt P450 参与许多生化过程，例如外源物质的代谢以及激素和脂肪酸的生物合成。重要的是，cyt P450 还代谢大多数临床使用的药物，因此它们的活性直接影响生物利用度和毒性。虽然动力学与 cyt P450 的底物特异性有关，但之前的实验研究缺乏时间分辨率来测量对底物识别做出关键贡献的最快运动。然而，新开发的二维红外振动回波光谱技术有可能直接测量 cyt P450 的快速动态以及它们如何促进底物特异性。拟议的实验将检验以下假设：细胞色素 P450 具有高度动态性，并且不同底物的结合与动力学的不同变化相关。首先，相对底物特异性细胞色素 P450cam 活性位点的动态将是 在未结合的蛋白质和与其结合的蛋白质中使用血红素结合的 CO 探针进行表征 底物、樟脑和几种底物类似物。通过使用位点特异性掺入的叠氮基苯丙氨酸探针，这些实验将扩展到 cyt P450cam 三级结构中的特定位点。最后，为了探索医学上重要的人类细胞色素 P450 的动力学，Homebound CO 将用于测量细胞色素 P450 3A4 的活性位点动力学，细胞色素 P450 3A4 是代谢多种药物分子的最混杂的细胞色素 P450 之一。对于这两种细胞色素 P450，观察到的动力学与结合底物的存在和性质之间的相关性将支持蛋白质动力学对于控制活性很重要的假设。这将对我们理解药物代谢产生重要影响。因此，拟议的研究将进一步加深我们对 cyt P450 动态以及它们如何促进其关键生物功能的理解。 公共健康相关性：二维红外振动回波光谱将用于研究蛋白质运动如何影响细胞色素 P450 的底物库。这些酶是处理代谢物和毒素所必需的，它们在药物的生物利用度和毒性中发挥着重要作用。因此，了解它们的活性将直接有助于设计提高疗效和降低毒性的疗法。