The Role of fs-ps Dynamics in Enzymatic H-Transfer

描述(申请人提供)：当代酶学的圣杯之一是识别和表征飞秒时间尺度上的酶运动及其与决定化学步骤速率的重组和距离采样运动的关系。这项应用的目的是表征飞秒到皮秒时间尺度上的酶活性中心动力学(使用2D IR振动光谱)，并将它们与催化的氢转移反应(使用本征动力学同位素效应的温度依赖性-Kies)联系起来。中心假设是，光谱测量的酶动态运动和KIE的温度依赖关系可以在Marcus类模型的框架内关联，从而产生一个统一的模型，该模型将酶的动力学和功能联系起来。我们计划验证我们的中心假设，并以甲酸脱氢酶(FDH)作为模型系统，通过追求以下三个特定目标来实现这一应用的目标：1)建立优化的隧道就绪配置的动态签名。这一目标的工作假设是，我们最近的发现，在过渡态-模拟复合体中，FDH的活性中心动力学是异常刚性的，其本征Kie与温度无关，反映了形成了一种组织良好、准备隧道的构型。我们将通过测量FDH位点特定突变体的过渡态模拟络合物中叠氮阴离子的反对称伸展的本征Kie和频率-频率时间关联函数(FFCF)的温度依赖性来验证这一假设。2)表征反映供体-受体距离采样的活动部位运动的时间尺度。工作假设是，与温度相关的KIE引发的促进振动发生在数百飞秒的时间尺度上。我们将通过使用2D IR光谱测量酶动力学的温度依赖性并将这种温度依赖性与内在键的相关性来验证这一假说。3)确定外佣的活动场所动态是局部性的还是集体性的。我们的工作假设是，有助于供体受体距离采样的酶的动态运动是活性部位的集体运动。我们将通过测量FDH与叠氮化物三元络合物中的第二个振动发色团azo-NAD的活性部位的动力学来验证这一假设，以比较在第二个位置测得的动力学与叠氮化物的动力学。这项拟议的研究将确定飞秒到皮秒时间尺度上的活性中心动力学的各个分量与用偶氮-NAD测量的本征Kie之间的关系。这些结果有望产生重大的整体影响，因为确定活性中心动力学和催化反应的动力学性质之间的关系将使我们能够利用这种关系来解决围绕这种动力学在酶催化的氢转移反应中的作用的争议。 与公众健康相关：从这项研究中获得的见解有望澄清酶运动对化学步骤的影响，有助于建立一个全面的酶催化反应理论。这项研究的结果将使人们能够努力将酶运动在基于结构的合理药物设计工作中的作用纳入其中，从而提高成功开发治疗一系列疾病的新药的潜力。

DESCRIPTION (provided by applicant): One of the holy grails in contemporary enzymology is to identify and characterize enzyme motions at the femtosecond time scale and their relationship to the reorganization and distance sampling motions that determine the rate of the chemical step. The objective of this application is to characterize the enzyme active site dynamics at the femtosecond to picosecond time scale (using 2D IR vibrational spectroscopy) and relate them to the catalyzed H-transfer reaction (using temperature dependence of the intrinsic kinetic isotope effects - KIEs). The central hypothesis is that the spectroscopically measured enzyme dynamic motions and the temperature dependence of KIEs can be correlated within the framework of the Marcus-like models, yielding a unified model that relates the enzyme's dynamics and functionality. We plan to test our central hypothesis and accomplish the objective of this application using the enzyme formate dehydrogenase (FDH) as a model system by pursuing the following three specific aims: 1) Establish the dynamic signatures of an optimized tunneling-ready configuration. The working hypothesis for this aim is that our recent discoveries that the active- site dynamics of FDH in a transition-state-analog complex are unusually rigid and its intrinsic KIEs are temperature independent reflect the formation of a well organized, tunneling-ready configuration. We will test this hypothesis by measuring the temperature dependence of the intrinsic KIEs and the frequency- frequency time correlation function (FFCF) for the antisymmetric stretch of the azide anion in transition state analog complexes of site-specific mutants of FDH. 2) Characterize the time scales for active-site motions that reflect donor-acceptor distance sampling. The working hypothesis is that the promoting vibrations that have been invoked in connection with temperature dependent KIEs occur on the time scale of hundreds of femtoseconds. We will test this hypothesis by measuring the temperature dependence of the enzyme dynamics using 2D IR spectroscopy and correlating that temperature dependence with that of the intrinsic KIEs. 3) Determine whether the active site dynamics of FDH are localized or collective. Our working hypothesis is that the dynamic motions of the enzyme that contribute to donor acceptor distance sampling are collective motions of the active site. We will test this hypothesis by measuring the dynamics of the active site using a second vibrational chromophore, azo-NAD+, in the ternary complex of FDH with azide to compare the dynamics measured at this second location with those for the azide. The proposed research will identify the relationships between the various components of the active site dynamics at the femtosecond to picosecond time scale and the intrinsic KIEs measured with the azo-NAD+. These outcomes are expected to have significant overall impact because identifying the relationship between active-site dynamics and the kinetic properties of the catalyzed reaction will allow us to exploit this relationship to address the controversy surrounding the role of such dynamics in enzyme catalyzed H-transfer reactions. PUBLIC HEALTH RELEVANCE: There is the promise that the insights gained from this research will clarify the influence of enzyme motions on the chemical step contributing to a comprehensive theory of enzyme-catalyzed reactions. The outcomes of this research will enable efforts to incorporate an understanding of the role of enzyme motions in structure-based rational drug design efforts improving the potential for success in developing new pharmaceuticals to treat an array of diseases.