Ultrafast dynamics in enzyme catalysis

项目总结 酶动力学跨越了广泛的时间尺度，从毫秒和微秒到 皮秒和飞秒。生物反应的催化作用与这些运动有关，但 飞秒和皮秒范围内的超快动力学仍然存在争议。因为在不同的领域 生物科学，如新陈代谢和药物设计的研究，依赖于对 对于酶的功能，解决这一争议可能会对我们理解许多 人类疾病。我们使用传统的蛋白质化学技术和 二维红外光谱将动力学测量与生物相关功能相关联。在这项提案中，我们 描述我们目前和未来的努力，以表征几组酶的超快运动的差异 具有不同活动程度的突变体。 这个项目的长期目标是发现蛋白质序列的变化，包括致病 突变，可以通过与键形成类似的时间尺度上的运动来影响酶的催化功能 并打入酶的活性部位。我们在这项研究中的直接目标是开发实验 需要工具来观察酶的活性部位和支架之间的通信，两者都是通过突变 和光谱学。我们假设，突变体活动的趋势也将反映在它们的超快 由于反应过程中势垒跨越的调制而产生的动力学。 我们将利用蛋白质合成和标记技术来制备一套具有活性位点的模型酶 振动标签，并将使用2D红外光谱来表征局部电场动态。我们将利用 振动标记的底物类似物以及基于蛋白质的标记与泵浦-RPBE的变化相一致 等待时间来描述这些动态。使用随机突变和活动筛选，我们将识别 这些酶的突变体具有改变的催化速率。活动站点标签的动态测量将是 与酶活性和其他性质相关，如折叠稳定性。 我们的研究计划还包括旨在测量非平衡动力学的实验，这些实验可能 影响催化作用。我们还将使用我们的标记系统来调查不可交换重的来源 同位素对催化效率的影响。稍后，我们将研究有源电池内部能量转移的效率 利用二维红外光谱的变化，研究活性中心和支架之间的相互作用。我们将使用双重- 频率2D IR光谱分析探测标记对，以寻找振动能量传递路径的证据。 最后，我们将使用瞬时2D IR光谱来检查通过蛋白质的能量松弛路径 活性中心部分的电子光激发后的支架，我们将其作为松弛的代理 反应步骤。在所有情况下，我们都将在突变背景下进行这些实验，在突变背景下，催化 被更改了。

PROJECT SUMMARY Enzyme dynamics span a broad range of timescales, from milliseconds and microseconds down to picoseconds and femtoseconds. Catalysis of biological reactions is linked to these motions, but the role of ultrafast dynamics in the femtosecond and picosecond range remains controversial. Because diverse fields in the biological sciences, such as the study of metabolism and drug design, depend on a detailed understanding of enzyme function, resolving this controversy could have important implications for our understanding of many human diseases. We approach this problem using a combination of traditional protein chemistry techniques and 2D IR spectroscopy to correlate dynamics measurements with biologically-relevant function. In this proposal, we describe our current and future efforts to characterize differences in the ultrafast motions of enzymes in sets of mutants with different degrees of activity. The long-term goal of this project is to discover how protein sequence variations, including disease-causing mutations, can influence the catalytic function of enzymes via motions on a similar timescale as bond formation and breaking in an enzyme’s active site. Our direct objectives in this research are to develop the experimental tools needed to observe communication between an enzyme’s active site and scaffold, both via mutagenesis and spectroscopy. We hypothesize that trends in the activities of mutants will also be reflected in their ultrafast dynamics due to modulation of barrier crossing during reactions. We will use protein synthesis and labeling techniques to prepare a set of model enzymes with active site vibrational labels, and will use 2D IR spectroscopy to characterize local electric field dynamics. We will utilize vibrationally-labeled substrate analogs as well as protein-based labels in concert with variations in pump-rpbe waiting time to characterize these dynamics. Using random mutagenesis and activity screens, we will identify mutants of these enzymes with altered catalytic rates. Dynamics measurements of active site labels will be correlated to enzyme activities and other properties such as fold stability. Our research program also includes experiments designed to measure non-equilibrium dynamics that may influence catalysis. We will also use our labeled systems to investigate the origins of non-exchangeable heavy isotope effects on catalytic efficiency. Later, we will examine the efficiency of energy transfer within the active site, and between the active site and scaffold, using variations on 2D IR spectroscopy. We will use dual- frequency 2D IR spectroscopy to probe pairs of labels for evidence of vibrational energy transfer pathways. Finally, we will use transient 2D IR spectroscopy to examine pathways for energy relaxation through the protein scaffold after electronic photoexcitation of active site moieties, which we use as a proxy for relaxation after a reaction step. In all cases, we will perform these experiments against mutant backgrounds in which catalysis is altered.