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

• 批准号: 10058272
• 负责人: STEVEN D SCHWARTZ
• 金额: $ 29.75万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10058272
• 关键词: 3-hydroxybutanal; Active Sites; Address; Alcohol dehydrogenase; Aldehyde-Lyases; Area; Behavior; Biochemical Reaction; Catalysis; Catechol O-Methyltransferase; Charge; Chemicals; Chemistry; Coupled; Couples; Coupling; Cryptosporidium parvum; Dihydrofolate Reductase; Directed Molecular Evolution; Electrostatics; Engineering; Environment; Enzymes; Escherichia coli; Evolution; Exhibits; Family; Goals; Harvest; Kinetics; Knowledge; Laboratories; Lactate Dehydrogenase; Light; Methods; Molds; Motion; Mutation; Natural Selections; Nature; Oxidoreductase; Pathway interactions; Plasmodium falciparum; Point Mutation; Protein Dynamics; Proteins; Publishing; Reaction; Research; Sampling; Spectrum Analysis; System; Techniques; Time; Variant; Work; catalyst; design; electric field; experimental study; improved; mutant; programs; success; theories; vibration

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.

本申请中描述的研究计划的目标是更深入地了解如何快速 采用蛋白质动力学，即所谓的亚皮秒时间尺度上的促进振动， 多年来，我们已经发现了这种酶被纳入天然和人工设计的酶中。 各种酶催化反应中的运动，并且还发现它们至少在一个我们的目标中缺失。 从长远来看，就是要破译大自然如何使用这种方法作为工程原理，以及它是如何发挥作用的。 例如，现在许多研究发现，在氢化物转移中。 酶如乙醇脱氢酶，供体向受体的快速运动促进化学反应 我们还研究了酶的两种看似二分的观点。 函数——静电视图和动态视图 电场环境完全有可能两者兼而有之。 对催化作用有很大贡献，并且在某些情况下受到与催化作用相同类型的运动的调节 示例控制供体受体距离。因为我们的方法允许我们收获反应性的集合。 最后，存在自然发生的情况。 酶表现出简单而直接的化学反应，而单一突变会导致动力学的显着复杂性 根本不清楚这是如何发生的。为了进行这项研究，我们建议这样做。 实现以下三个具体目标： 目标 1：我们将研究合成酶化学中比较成功的尝试之一——人工酶 我们将确定理论设计是否与实验室进化相结合。 这些人造蛋白质催化剂引起蛋白质动力学与反应耦合的变化。 目标 2：我们将研究随着反应的进行，酶活性位点的电场如何变化 儿茶酚-O-甲基转移酶 (COMT) 是一种酶，其中 其他团体已强调蛋白质动力学和静电预组织 特别是，我们将确定是否存在促进振动作为反应的一部分。 这种酶的协调，以及它如何帮助控制现场环境。 目标 3：我们将分析人们知之甚少的“烯还原酶”家族所催化的反应。 蛋白质动力学的重要性以及单点突变产生多个反应的能力 具有高度不同的动力学行为的“配置”。 经过数十年的研究，酶如何发挥作用这个看似简单的问题仍然是争论的温床。 此处提出的此类研究我们有助于基础知识和最终的实际控制 应用。