Synchronizing enzyme-mediated radical chemistry with domain motion

这项研究概述了两个相对较新和未知的酶学领域：(1)基于自由基的反应机制和(2)蛋白质构象运动在指导催化中的作用。最近基因组测序的热潮和改进的检测技术导致了已知或假设通过基于自由基的机制进行的酶的数量迅速增加。由于这些酶中的许多都是重要的生物过程中的关键角色，我们不仅要了解它们是如何进化来控制自由基的轨迹的，而且要了解这种类型的化学是如何与不同水平和频率的蛋白质运动一起精心设计的，这对我们来说是至关重要的。使用依赖辅酶B12的酶作为模型系统，该研究计划将努力推导出这些类型的酶如何工作的模型。这些研究将比较酶在催化过程中处于不同状态时的原子结构，以确定蛋白质可能在自由基稳定性和/或其动态性质中发挥作用的方面。这些结构特征将被改变，酶的动力学和功能行为将使用光谱(紫外可见和荧光)和快速反应技术进行分析。在UBC-Okanagan与国际公认的无机化学家(专门从事基于自由基催化剂的合成和设计)的合作下，将创造出新的辅酶B12类似物，使我们能够研究分子的反应性质。了解酶是如何部署基于自由基化学的是对它们进行工程设计的第一步，以执行适用于工业和药物设计的所需反应，这一目标最终将提高加拿大人未来的生活质量。改进的基于自由基的催化模型也可以应用于更具医学相关性的系统，这些系统通常不太容易进行研究。最后，这项研究将对酶学领域做出重大贡献，因为它不仅在“结构决定功能”范式的背景下考察酶催化，而且还从构象动力学在其催化行为中发挥不可或缺的作用这一新兴概念出发。

The research outlined in this proposal examines two relatively new and uncharted areas of enzymology: (1) radical-based reaction mechanisms and (2) the role of protein conformational motion in directing catalysis. A recent upsurge in genome sequencing and improved detection techniques has led to a rapid increase in the number of enzymes known or hypothesized to proceed by radical-based mechanisms. As many of these enzymes are key players in important biological processes, it is critical for us to understand, not only how they have evolved to control the trajectories of radicals, but also how this type of chemistry is exquisitely choreographed with varying levels and frequencies of protein motion. Using a coenzyme B12-dependent enzyme as a model system, the research program will strive to derive models for how these types of enzymes operate. The studies will compare the atomic structure of the enzyme "poised" at different states during catalysis, in order to pinpoint aspects of the protein that may play a role in radical stability and/or its dynamic properties. These structural features will be altered and the kinetic and functional behavior of the enzyme will be analyzed using spectroscopic (UV-visible and fluorescence) and fast reaction techniques. In collaboration with internationally recognized inorganic chemists (specializing in synthesis and design of radical-based catalysts) at UBC-Okanagan, novel coenzyme B12 analogs will be created that will allow us to investigate the reactive nature of the molecule. An understanding of how enzymes deploy radical-based chemistry is the first step towards engineering them to perform desired reactions that are applicable to industry and drug design, an objective that will ultimately improve the quality of life for Canadians in the future. Improved models for radical-based catalysis can also be applied to more medically relevant systems, that are often less accessible for investigation. Finally, the research will make a significant contribution to the field of enzymology, as it examines enzyme catalysis, not just in the context of "structure defines function" paradigm, but also from the emerging concept that conformational dynamics plays an integral role in its catalytic behavior.