Rapid protein dynamics and catalysis: modulation by laboratory evolution, designed mutation, and protein control of electric field environment

快速蛋白质动力学和催化：实验室进化调节、设计突变和电场环境的蛋白质控制

基本信息

批准号：
10303036
负责人：
STEVEN D SCHWARTZ
金额：
$ 29.7万
依托单位：
UNIVERSITY OF ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-01 至 2023-11-30
项目状态：
已结题

项目摘要

The goal of the research program described in this application is to obtain a deeper understanding of how rapid protein dynamics, so called promoting vibrations on a sub picosecond timescale, are employed and incorporated into both natural and artificially designed enzymes. Over the years we have identified such motions in a variety of enzymatically catalyzed reactions, and also found them missing in at least one. Our goal for the long term is to decipher how nature has used this approach as an engineering principle and how it has been incorporated as a part of the function. For example, many studies have now found that in hydride transfer enzymes such as alcohol dehydrogenase, rapid motion of the donor to the acceptor facilitates the chemical step by lowering the effective adiabatic barrier. We also study two seemingly dichotomous views of enzyme function – the electrostatic view, and the dynamic view. It is entirely possible that the electric field milieu both contributes strongly to catalysis, and in certain cases is modulated by the same types of motions that for example control donor acceptor distance. Because our methods allow us to harvest ensembles of reactive trajectories, exactly such mechanisms can be studied. Finally, there exist cases in which naturally occurring enzymes exhibit simple and direct chemistry, while single mutations cause significant complexities in kinetic analysis. It is simply not clear how this can come about. In order to pursue this research we propose to implement the following three specific aims: Aim 1: We will study one of the more successful attempts in synthetic enzymatic chemistry – the artificial creation of retro-aldolases. We will ascertain whether theoretical design coupled to laboratory evolution of these artificial protein catalysts caused change in the coupling of protein dynamics to reaction. Aim 2: We will study how electric field varies in the active site of an enzyme as the reaction proceeds from reactants to products through a transition state. Catechol-O-methyltransferase (COMT) is an enzyme in which both protein dynamics and electrostatic preorganization have been stated emphatically by other groups to produce the catalytic effect. In particular we will identify if there is a promoting vibration as part of the reaction coordinate in this enzyme, and how it may help to control the field environment. Aim 3: We will analyze reactions catalyzed by the poorly understood “ene-reductase” family. We will address the importance of protein dynamics and the ability of a single point mutation to create multiple reaction “configurations” with highly divergent kinetic behavior. After decades of study, the deceptively simple question of how enzymes work is still a hotbed of debate. Via such studies as here proposed we contribute to both basic knowledge and eventual practical control application.

本应用程序中描述的研究计划的目标是更深入地了解如何快速蛋白质动力学，所谓的促进振动在次秒时间尺度上，采用了，并且掺入天然和人为设计的酶中。多年来，我们已经确定了这样的各种酶促催化反应的运动，也发现它们至少在一个中缺失。我们的目标从长远来看，是解释自然如何将这种方法用作工程原则及其如何使用被合并为功能的一部分。例如，许多研究现在发现在氢化物转移中诸如酒精脱氢酶之类的酶，供体向受体的快速运动有助于化学物质通过降低有效的绝热屏障来迈进。我们还研究了两种酶的二分法观点功能 - 静电视图和动态视图。电场环境完全有可能对催化有很大贡献，在某些情况下，由相同类型的动作调节示例控制供体受体距离。因为我们的方法使我们能够收获反应性的合奏轨迹，准确的机制可以研究。最后，存在自然发生的情况酶暴露了简单和直接的化学，而单个突变导致动力学显着复杂性分析。根本不清楚如何产生。为了购买这项研究，我们建议实施以下三个特定目标：目标1：我们将研究合成酶学化学的最成功尝试之一 - 艺术创建复古 - 醛固酶。我们将确定理论设计是否与实验室的演变相结合这些人造蛋白催化剂导致蛋白质动力学与反应的偶联变化。 AIM 2：我们将研究反应从反应开始时，电场在酶的活性位点如何变化通过过渡状态对产品的反应物。 Catechol-O-甲基转移酶（COMT）是一种酶蛋白质动力学和静电预组织都被其他组强调产生催化作用。特别是我们将确定是否存在促销振动作为反应的一部分协调该酶，以及如何帮助控制现场环境。 AIM 3：我们将分析较知识的“ ENE-REDUCTase”家族催化的反应。我们将解决蛋白质动力学的重要性以及单点突变产生多重反应的能力具有高度不同动力学行为的“配置”。经过数十年的研究，关于酶的工作方式仍然是辩论的温床的看似简单的问题。通过此处提出的研究我们为基础知识和最终实践控制做出了贡献应用。