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）：当代酶学中的圣杯之一是识别和表征在飞秒时间尺度上的酶动作，及其与决定化学步骤速率的重组和距离采样动作的关系。该应用的目的是表征酶的活性位点动力学在飞秒至皮秒时间尺度上（使用2D IR振动光谱法），并将它们与催化的H-转移反应（使用固有的动力学同位素效应的温度依赖性-KIES）相关联。中心假设是，光谱测量的酶动态运动和KIE的温度依赖性可以在类似Marcus模型的框架内关联，从而产生了与酶的动力学和功能相关的统一模型。我们计划通过追求以下三个特定目的来测试我们的中心假设，并使用甲甲酸甲酸盐脱氢酶（FDH）作为模型系统来实现此应用的目标：1）确定可优化的隧道就绪配置的动态签名。这个目标的工作假设是，我们最近的发现，在过渡状态 - 分析络合物中FDH的主动位点动力学异常刚性，其内在的KIES是温度独立的，反映了组织良好的隧道就绪配置的形成。我们将通过测量固有KIE的温度依赖性和FDH位点特异性突变体的过渡状态类似物复合物中的叠氮化阴离子的抗对称拉伸来测量固有KIE的温度依赖性和频率时间相关函数（FFCF）。 2）表征反映供体距离距离采样的主动位点运动的时间尺度。工作假设是，与温度依赖性KIE相关的促进振动发生在数百个飞秒的时间尺度上。我们将通过使用2D IR光谱法测量酶动力学的温度依赖性来检验这一假设，并将该温度依赖性与内在KIE相关联。 3）确定FDH的主动位点动力学是本地化的还是集体的。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。我们将使用第二个振动发色团AZO-NAD+在FDH的三元复合物中测量活性位点的动力学来检验这一假设，以将其在第二个位置测量的动力学与叠氮化叠氮化物测量的动力学进行比较。拟议的研究将确定飞秒至Picsecond Time量表的主动位点动力学各个组件与用Azo-NAD+测量的内在KIE之间的关系。预计这些结果将产生重大的总体影响，因为确定活跃位点动力学与催化反应的动力学特性之间的关系将使我们能够利用这种关系，以解决围绕这种动力学在酶催化的H-Transfer反应中的作用的争议。 公共卫生相关性：有望从这项研究中获得的见解阐明酶动作对化学步骤的影响，从而有助于酶催化反应的全面理论。这项研究的结果将使努力能够纳入对酶动作在基于结构的理性药物设计方面的作用的理解，从而提高了成功开发新药物以治疗一系列疾病的潜力。