Promoting Vibrations in LDH and PNP

本项目的研究将探讨在乳酸脱氢酶和嘌呤核苷磷酸化酶这两个酶系统中，蛋白质的动态运动是催化活动的中心的可能性。这项研究的总体假设是，通过分子动力学、混合量子力学、分子力学和量子化学计算，我们可以识别这样的重要运动，我们称之为‘促进振动’。初步研究表明，乳酸脱氢酶中NAD辅因子的这种周期性运动是由蛋白质振动在亚皮秒时间尺度上造成的。将确定产生促进振动所涉及的特定残留物。该计划项目的化学核心将合成突变蛋白质。然后，在卡伦德实验室进行的实验测试将与速率变化的理论预测进行比较，以评估促进蛋白质运动在催化中的重要性。理论方法将包括在PI小组中开发的Quantum Kramers方法，用于计算#年的利率 复杂系统，用于分子动力学分析以定位促进振动的光谱密度计算，以及钱德勒的跃迁路径采样，以了解哪些原子运动是目标系统中反应坐标的一部分。如果实验证据表明，有助于催化的运动比目前理论上可用的时间尺度更慢，这些时间尺度将被用来指导进一步的理论发展。 嘌呤核苷磷酸化酶已在实验和初步计算中被证明通过三原子氧堆的运动来促进离开基团的释放，该运动破坏了过渡态的成键电子的稳定。这是一种新型的激振--电子激振。基于Schramm过渡态结构的全QM/MM计算将研究这一概念。与乳酸脱氢酶一样，我们将提出突变体，对这一假说进行实验验证，施拉姆实验室将进行光谱和直接测量。这一计划的实施将允许在原子水平上确定蛋白质动力学如何在相对罕见的事件中导致催化。

The research in this project will investigate the possibility that in two enzyme systems, lactate dehydrogenase and purine nucleoside phosphorylase, dynamic motions of the protein are central to the catalytic event. The overall hypothesis for this investigation is that through molecular dynamics, mixed quantum mechanical molecular mechanical, and quantum chemistry calculations we may identify such important motions, which we have called 'promoting vibrations'. Preliminary investigations show such periodic motion in the NAD cofactor in lactate dehydrogenase imposed by protein vibration on a subpicosecond timescale. Specific residues involved in generating the promoting vibration will be identified. The chemistry core of this Program Project will synthesize mutant proteins. Then experimental tests conducted in the Callender lab will be compared with theoretical predictions of changes in rate to evaluate the importance of the promoting protein motion in catalysis. Theoretical approaches will include the Quantum Kramers methodology developed in the PI's group for the calculation of rates in complex systems, spectral density computations for the analysis of molecular dynamics to locate promoting vibrations, and Chandler's transition path sampling to understand what atomic motions are part of the reaction coordinate in the target systems. Should experimental evidence suggest motions that contribute to catalysis more slowly than the currently available theoretically accessible timescales, these timescales will be used to guide further theoretical development. Purine nucleoside phosphorylase has been shown experimentally and in preliminary computations to facilitate explusion of the leaving group via motion of a three-atom oxygen stack that destabilizes bonding electrons at the transition state. This is a new type of promoting vibration - an electronic promoting vibration. Full QM/MM computations based on Schramm's transition state structure will investigate this concept. As in lactate dehydrogenase, we will propose mutants, that will experimentally test this hypothesis, and the Schramm lab will perform spectroscopic and direct measurments. Implementation of this program will allow the determination on an atomic level, how protein dynamics contributes in relatively rare events to cause catalysis.