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

基本信息

批准号：
7806852
负责人：
Megan Corrine Thielges
金额：
$ 4.76万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
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/7806852
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

项目摘要

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的分子识别和催化。尽管这种酶家族催化了多种底物的羟基化，但不同的Cyt P450同源物显示出不同程度的底物特异性。由于Cyt P450参与许多生化过程，例如异种生物的代谢以及激素和脂肪酸的生物合成，因此了解这种特异性对于人类健康至关重要。重要的是，CYT P450还代谢了大多数临床使用的药物，因此它们的活性直接促进了生物利用度和毒性。尽管动力学与CYT P450的底物特异性有关，但以前的实验研究缺乏时间分辨率，无法测量对底物识别产生关键贡献的最快动作。然而，新开发的二维红外振动回声光谱的技术具有直接测量Cyt P450的快速动力学以及它们如何对底物特异性有效的潜力。提出的实验将测试Cyt P450具有高度动态性的假设，并且不同底物的结合与动力学变化的变化有关。首先，相对底物特异性CYT P450CAM的活动位点的动力学将是在未结合的蛋白质中使用血红素结合的CO探针和与其结合的蛋白质进行表征底物，樟脑和几个基板类似物。这些实验将通过使用特定于特定的偶氮苯胺探针将CYT P450CAM的三级结构扩展到特定位点。最后，为了探索医学上重要的人类CYT P450的动力学，Homebound CO将用于测量Cyt P450 3A4的活动位点动力学，这是代谢各种药物分子的最混杂的CYT P450之一。对于两个Cyt 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以下假设：蛋白质动力学对于控制活性很重要。这将对我们对药物代谢的理解产生重要的影响。因此，拟议的研究将进一步了解CYT P450的动力学以及它们如何对其关键生物学功能做出贡献。公共卫生相关性：二维红外振动回声光谱将用于研究蛋白质运动如何影响细胞色素P450的底物曲目。这些酶需要处理代谢产物和毒素，并且它们在药物生物利用度和毒性中起着至关重要的作用。因此，了解它们的活性将是直接效用在治疗剂时具有提高和毒性降低的治疗方法。