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

fs-ps 动力学在酶 H 转移中的作用

基本信息

批准号：
8727580
负责人：
CHRISTOPHER M CHEATUM
金额：
$ 28.29万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
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

项目摘要

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.

描述（由申请人提供）：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。此应用的目的是表征飞秒到皮秒时间尺度的酶活性位点动力学（使用 2D IR 振动光谱），并将其与催化氢转移反应联系起来（使用固有动力学同位素效应的温度依赖性 - KIE）。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 Marcus 模型的框架内相关联，从而产生一个与酶的动态和功能相关的统一模型。我们计划通过追求以下三个具体目标来测试我们的中心假设并使用甲酸脱氢酶（FDH）作为模型系统来实现该应用的目标：1）建立优化的隧道就绪配置的动态特征。这一目标的工作假设是，我们最近的发现是，过渡态模拟复合物中 FDH 的活性位点动力学异常刚性，并且其内在的 KIE 与温度无关，反映了组织良好、可隧道化的构型的形成。我们将通过测量FDH位点特异性突变体的过渡态类似复合物中叠氮化物阴离子反对称拉伸的固有KIE的温度依赖性和频频时间相关函数（FFCF）来检验这一假设。 2）表征反映供体-受体距离采样的活性位点运动的时间尺度。工作假设是，与温度相关的 KIE 相关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并将该温度依赖性与内在 KIE 的温度依赖性相关联来检验这一假设。 3) 确定FDH的活性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。我们将通过使用 FDH 与叠氮化物三元复合物中的第二个振动发色团偶氮-NAD+ 测量活性位点的动力学来测试这一假设，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。拟议的研究将确定飞秒到皮秒时间尺度的活性位点动力学的各个组成部分与使用偶氮-NAD+测量的内在 KIE 之间的关系。这些结果预计将产生重大的总体影响，因为确定活性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来解决围绕此类动力学在酶催化氢转移反应中的作用的争议。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Line shape analysis of two-dimensional infrared spectra.

二维红外光谱的线形分析。

DOI：
10.1063/1.4918350
发表时间：
2015-06-07
期刊：
The Journal of chemical physics
影响因子：
0
作者：
Q. Guo;Philip Pagano;Yun;A. Kohen;C. Cheatum
通讯作者：
C. Cheatum

Oscillatory Enzyme Dynamics Revealed by Two-Dimensional Infrared Spectroscopy.

二维红外光谱揭示的振荡酶动力学。

DOI：
发表时间：
2016-07-07
期刊：
影响因子：
0
作者：
Pagano, Philip;Guo, Qi;Kohen, Amnon;Cheatum, Christopher M
通讯作者：
Cheatum, Christopher M

Oscillatory Active-site Motions Correlate with Kinetic Isotope Effects in Formate Dehydrogenase.

振荡活性位点运动与甲酸脱氢酶中的动力学同位素效应相关。

DOI：
发表时间：
2019-12-06
期刊：
影响因子：
12.9
作者：
Pagano, Philip;Guo, Qi;Ranasinghe, Chethya;Schroeder, Evan;Robben, Kevin;Häse, Florian;Ye, Hepeng;Wickersham, Kyle;Aspuru;Major, Dan T;Gakhar, Lokesh;Kohen, Amnon;Cheatum, Christopher M
通讯作者：
Cheatum, Christopher M

2D IR spectroscopy using four-wave mixing, pulse shaping, and IR upconversion: a quantitative comparison.

使用四波混合、脉冲整形和红外上转换的 2D 红外光谱：定量比较。