Protein Dynamics and Substrate Specificity in Cytochrome P450s

描述(申请人提供)：拟议的研究将考察动力学如何影响细胞色素(Cyt)P450的分子识别和催化。虽然这一家族的酶催化多种底物的羟基化，但不同的细胞色素P450同源物表现出不同程度的底物专一性。了解这种特异性对人类健康至关重要，因为细胞色素P450参与了许多生化过程，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，细胞色素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到生物利用度和毒性。虽然动力学与细胞色素P450的底物特异性有关，但以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细胞色素P450的快速动力学以及它们如何有助于底物专一性。拟议的实验将检验这样的假设，即细胞色素P450是高度动态的，不同底物的结合与动力学的不同变化有关。首先，相对底物特异的CyT P450 cam的活性部位的动力学将为 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探针来表征的 底物、樟脑和几种底物类似物。这些实验将通过使用特定部位掺入的叠氮苯丙氨酸探针扩展到细胞色素P450 cam的整个三级结构中的特定部位。最后，为了探索对医学具有重要意义的人类细胞色素P450的动力学，家庭结合的CO将被用来测量细胞色素P450 3A4的活性位点动力学，细胞色素P450是最混杂的细胞色素P450，可以代谢多种药物分子。对于这两个细胞色素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重要的假设。这将对我们理解药物新陈代谢产生重要的影响。因此，拟议的研究将进一步加深我们对细胞色素P450的动力学以及它们如何对其关键生物学功能做出贡献的理解。 与公众健康相关：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底物酶系。这些酶是处理代谢产物和毒素所必需的，它们在药物的生物利用度和毒性方面发挥着至关重要的作用。因此，了解它们的活性将直接有助于设计具有更高疗效和更低毒性的疗法。

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.