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，并且 天然的和人工设计的酶中。多年来，我们已经确定了这样的 运动在各种酶催化反应，也发现他们失踪，在至少一个。我们的目标 从长远来看，是要破译大自然如何将这种方法作为一种工程原理，以及它如何 作为功能的一部分。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。 降低有效绝热势垒。我们还研究了酶的两种看似二分法的观点 功能-静电视图和动态视图。完全有可能电场环境 对催化作用有很大的贡献，在某些情况下，它受到与催化作用相同类型的运动的调制。 实施例控制施主受主距离。因为我们的方法可以让我们收获反应性的 轨迹，正是这样的机制可以研究。最后，存在自然发生的情况， 酶表现出简单和直接的化学反应，而单个突变会导致动力学上的显着复杂性。 分析.只是不清楚这是如何发生的。为了进行这项研究，我们建议 实现以下三个具体目标： 目的1：我们将研究合成酶化学中比较成功的尝试之一-人工合成酶。 逆向醛缩酶的产生。我们将确定理论设计是否与实验室进化相结合， 这些人工蛋白质催化剂引起蛋白质动力学与反应耦合的变化。 目的2：我们将研究电场如何在酶的活性部位变化，因为反应的进行， 反应物通过过渡态转化为产物。儿茶酚-O-甲基转移酶（COMT）是一种酶，其中 蛋白质动力学和静电预组织都已被其他研究小组强调， 产生催化作用。特别是我们将确定是否有一个促进振动作为反应的一部分 协调这种酶，以及它如何有助于控制现场环境。 目的3：我们将分析由知之甚少的“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以及单点突变产生多重反应的能力 具有高度发散的动力学行为的“构型”。 经过几十年的研究，看似简单的酶如何工作的问题仍然是争论的温床。经由 我们在这里提出的这些研究有助于基础知识和最终的实际控制 应用程序.

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.