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

描述（由申请人提供）：当代酶学的圣杯之一是在飞秒时间尺度上识别和表征酶的运动，以及它们与决定化学步骤速率的重组和距离采样运动的关系。本应用程序的目的是表征酶活性位点在飞秒到皮秒时间尺度上的动力学（使用二维红外振动光谱），并将它们与催化的h转移反应联系起来（使用本征动力学同位素效应- KIEs的温度依赖性）。核心假设是，光谱测量的酶动力学运动和KIEs的温度依赖性可以在马库斯模型的框架内相关联，从而产生一个统一的模型，将酶的动力学和功能联系起来。我们计划使用甲酸脱氢酶（FDH）作为模型系统，通过追求以下三个具体目标来验证我们的中心假设并实现本应用的目标：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，我们最近发现，过渡态模拟复合体中FDH的活性位点动力学异常刚性，其固有的key与温度无关，反映了组织良好的隧道准备结构的形成。我们将通过测量FDH位点特异性突变体过渡态类似物中叠氮化物阴离子的反对称拉伸的固有KIEs的温度依赖性和频率-频率时间相关函数（FFCF）来验证这一假设。2)表征反映供体-受体距离采样的主动部位运动的时间尺度。工作的假设是，与温度相关的KIEs所引起的促进振动发生在数百飞秒的时间尺度上。我们将通过使用二维红外光谱测量酶动力学的温度依赖性来检验这一假设，并将这种温度依赖性与内在KIEs的温度依赖性联系起来。3)确定外佣的活动位点动态是局部的还是集体的。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位点的集体运动。我们将通过在FDH与叠氮化物的三元配合物中使用第二个振动发色团azo-NAD+来测量活性位点的动力学来验证这一假设，并将在第二个位置测量的动力学与叠氮化物的动力学进行比较。提出的研究将确定在飞秒到皮秒时间尺度上活性位点动力学的各个组成部分与偶氮- nad +测量的内在ky之间的关系。这些结果预计将产生重大的整体影响，因为确定活性位点动力学与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h转移反应中的作用的争议。

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.