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

本申请中描述的研究计划的目标是更深入地了解 蛋白质动力学，即所谓的在亚皮秒时间尺度上促进振动，被使用，并 加入到天然和人工设计的酶中。多年来，我们已经确定了这样的 在各种酶催化的反应中发现了它们的运动，并且还发现至少有一个没有运动。我们的目标 因为从长远来看，就是要破译大自然是如何将这种方法作为一种工程原理使用的，以及它是如何 已被纳入为该职能的一部分。例如，许多研究现在发现，在氢化物转移中 酶，如酒精脱氢酶，供体到受体的快速运动促进了化学物质的生成 通过降低有效绝热势垒来逐步降低。我们还研究了关于酶的两种看似二分法的观点 功能--静态视图和动态视图。完全有可能电场环境既有可能 对催化有很大的贡献，在某些情况下，它受相同类型的运动的调节， 实例控制供体受体距离。因为我们的方法允许我们收集反应的集合 轨迹，正是这样的机制可以被研究。最后，存在自然发生的情况 酶表现出简单而直接的化学，而单一突变导致动力学上的显著复杂性 分析。目前还不清楚这是如何实现的。为了继续这项研究，我们建议 落实以下三个具体目标： 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 复古缩醛酶的产生。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导致了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，酶活性部位的电场是如何变化的 通过过渡态将反应物转化为产物。儿茶酚-O-甲基转移酶(COMT)是一种 蛋白质动力学和静电预组织都是由其他小组强调的 产生催化作用。特别是，我们将确定是否存在促进振动作为反应的一部分 协调这种酶，以及它可能如何帮助控制田间环境。 目的3：我们将分析由鲜为人知的“烯-还原酶”家族催化的反应。我们将解决 蛋白质动力学的重要性和单点突变产生多重反应的能力 具有高度不同的动力学行为的“构型”。 经过几十年的研究，酶是如何工作的这个看似简单的问题仍然是一个争论的温床。通过 这里提出的这些研究既有助于我们的基础知识，也有助于最终的实际控制 申请。

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.