Enzyme design and protein dynamics.

这个项目之前的工作已经确定了参与催化功能的运动 酵素。我们在这个续订申请中工作的假设是，通过使用理论和 计算方法我们可以确定产生与催化有关的动力学的结构元素 所有的时间尺度，通过这些手段，我们可以创建两种速率的蛋白质动力学设计范例 增强，和酶抑制。为了完成这个研究计划，我们将在5个不同的方面进行工作 这些方面是：1)我们将确定在所有反应时间尺度上必需的构象运动。就像 我们已经跟踪了形成促进振动的蛋白质动力学的轨迹，并确定了它是 反应坐标，我们将把这些计算扩展到酶周转的时间尺度。2)我们会 确定特定酶系统(乳酸脱氢酶和嘌呤核苷)的结构元素 磷酸化酶)，创造了特定的动态。3)我们将确定变构结合部位并确定 结合对蛋白质动力学的影响。4)我们将确定动力学如何参与过渡状态 抑制剂结合。5)我们将建议重新设计“重型”PnP，以纠正我们存在的动态缺陷 以前发现并恢复了正常的催化功能。 该计划项目中所有工作的总体推动力一直是将动力学识别为 酶功能的中心特征从选择具有催化活性的构象亚态到 快速促进振动。我们现在继续这个程序的下一步--将动力学识别为 酶功能中的设计要素。我们建议开始将这一概念作为一种蛋白质来开发 工程工具。

Previous work in this program project has identified motions that are involved in the catalytic function of enzymes. The hypothesis for our work in this renewal application is that by using theoretical and computational methods we can identify the structural elements that create dynamics related to catalysis on all timescales, and through these means we may create protein dynamics design paradigms for both rate enhancement, and enzyme inhibition. In order to complete this research program we will work on 5 distinct areas that are: 1) we will identify conformational movements necessary on all timescales for reaction. Just as we have followed trajectories and identified protein dynamics that form promoting vibrations and are part of the reaction coordinate, we will extend these calculations to the timescale of enzyme turnover. 2) We will identify structural elements in specific enzyme systems (lactate dehydrogenase and purine nucleoside phosphorylase) that create the specific dynamics. 3) We will identify allosteric binding sites and determine the effect of binding on protein dynamics. 4) We will identify how dynamics is involved in transition state inhibitor binding. 5) we will propose a redesign of "heavy" PNP that corrects dynamic defects we have previously identified and restores normal catalytic function. The overall thrust to all of the work in this program project has been the identification of dynamics as a central feature in enzyme function from the selection of a catalytically competent conformational substate to rapid promoting vibrations. We now proceed to the next step in this program - identification of dynamics as a design element in enzyme function. We propose to begin the process of developing this concept as a protein engineering tool.