Ultrafast dynamics in enzyme catalysis

项目摘要 酶动力学跨越了很宽的时间尺度，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是有争议的。因为不同的领域 生物科学，如新陈代谢和药物设计的研究，依赖于对药物的详细了解。 解决这一争议可能对我们理解许多酶的功能具有重要意义。 人类疾病。我们使用传统的蛋白质化学技术和 2D IR光谱将动态测量与生物相关功能相关联。在本提案中，我们 描述了我们目前和未来的努力，以表征酶在超快运动中的差异， 具有不同程度活性的突变体。 该项目的长期目标是发现蛋白质序列变异，包括致病基因， 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 破坏酶的活性位点我们在这项研究中的直接目标是开发实验性的 观察酶的活性位点和支架之间的通讯所需的工具，两者都是通过诱变进行的 和光谱学。我们推测，突变体的活动趋势也将反映在它们的超快 动力学由于反应过程中的势垒跨越的调制。 我们将利用蛋白质合成和标记技术制备一套具有活性位点的模型酶 振动标签，并将使用二维红外光谱来表征局部电场动力学。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 等待时间来描述这些动态。使用随机诱变和活性筛选，我们将鉴定 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 与酶活性和其他性质如折叠稳定性相关。 我们的研究计划还包括旨在测量非平衡动力学的实验， 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的重金属的来源。 同位素对催化效率的影响。稍后，我们将研究活性物质内能量转移的效率。 位点，以及活性位点和支架之间，使用2D IR光谱的变化。我们将使用双- 频率2D IR光谱以探测标记对，以证明振动能量转移途径。 最后，我们将使用瞬态二维红外光谱来检查通过蛋白质的能量弛豫途径 支架后，电子光激发的活性位点部分，我们使用作为代理松弛后， 反应步骤。在所有情况下，我们将对突变体背景进行这些实验，其中催化作用是 改变了

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.