Protein Dynamics and Substrate Specificity in Cytochrome P450s

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。虽然该酶家族催化多种底物的羟基化，但不同的cyt P450同源物表现出不同程度的底物特异性。了解这种特异性对人类健康至关重要，因为cyt p450参与许多生化过程，如异种生物的代谢、激素和脂肪酸的生物合成。重要的是，cyt p450也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的快速动力学以及它们如何有助于底物特异性。所提出的实验将测试假设，即cyt p450是高度动态的，不同底物的结合与不同的动力学变化有关。首先，相对底物特异性cyt P450cam活性部位的动力学将是

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.