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

fs-ps 动力学在酶 H 转移中的作用

• 批准号: 8527797
• 负责人: CHRISTOPHER M CHEATUM
• 金额: $ 27.31万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8527797
• 关键词: Accounting; Active Sites; Address; Anions; Azides; Behavior; Biological Models; Chemicals; Complex; Data; Dependence; Disease; Drug Design; Enzymatic Biochemistry; Enzymes; Exhibits; Formate dehydrogenase; Foundations; Frequencies; Goals; Hydrogen; Isotopes; Kinetics; Label; Laboratories; Link; Location; Measurement; Measures; Modeling; Molecular; Motion; Outcome; Outcomes Research; Pharmacologic Substance; Play; Property; Protein Dynamics; Proteins; Reaction; Research; Role; Sampling; Site; Source; Spectrum Analysis; Stretching; Structure; Support System; System; Temperature; Testing; Time; Work; analog; base; chromophore; cofactor; computer studies; human disease; improved; infrared spectroscopy; innovation; insight; mutant; public health relevance; reaction rate; research study; success; theories; two-dimensional; vibration

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.

描述（由申请人提供）：当代酶学的圣杯之一是确定和表征飞秒时间尺度下的酶运动及其与确定化学步骤速率的重组和距离采样运动的关系。本申请的目的是表征酶活性位点在飞秒至皮秒时间尺度下的动力学（使用2D IR振动光谱），并将其与催化的H-转移反应（使用固有动力学同位素效应的温度依赖性-KIE）相关。中心的假设是，光谱测量的酶的动态运动和温度依赖性的KIE可以在马库斯模型的框架内相关，产生一个统一的模型，涉及酶的动力学和功能。我们计划测试我们的中心假设，并通过追求以下三个具体目标，使用甲酸脱氢酶（FDH）作为模型系统来实现本申请的目标：1）建立优化的隧穿就绪配置的动态特征。为此目的的工作假设是，我们最近发现，过渡态类似物复合物中FDH的活性位点动力学异常刚性，其内在KIE与温度无关，这反映了良好组织的隧穿就绪构型的形成。我们将测试这一假设，通过测量的温度依赖性的内在KIE和频率-频率时间相关函数（FFCF）的叠氮阴离子的反对称拉伸的过渡态模拟复合物的特定位点的突变体的FDH。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。工作的假设是，促进振动已被调用与温度相关的KIE发生在数百飞秒的时间尺度。我们将测试这一假设，通过测量温度依赖性的酶动力学使用二维红外光谱和相关的温度依赖性与内在的KIE。3)确定FDH的活性位点动力学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。我们将测试这一假设，通过测量的活性位点的动力学使用第二个振动发色团，偶氮-NAD+，在FDH与叠氮化物的三元复合物中，以比较在第二个位置测量的动力学与叠氮化物。拟议的研究将确定在飞秒到皮秒的时间尺度上的活性位点动力学的各个组成部分之间的关系和用azo-NAD+测量的固有KIE。这些结果预计将有显着的整体影响，因为确定活性位点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争议的酶催化氢转移反应中的作用，这种动力学。 公共卫生关系：有希望从这项研究中获得的见解将澄清酶运动对化学步骤的影响，有助于酶催化反应的综合理论。这项研究的结果将使人们能够努力将对酶运动在基于结构的合理药物设计中的作用的理解纳入其中，从而提高成功开发新药以治疗一系列疾病的潜力。