Structural investigation of reaction intermediates in glycolytic aldolases

糖酵解醛缩酶反应中间体的结构研究

• 批准号: RGPIN-2016-04898
• 负责人: Sygusch, Jurgen
• 金额: $ 2.4万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=685045
• 关键词: Structural; investigation; reaction; intermediates; glycolytic

A wealth of information has been obtained about catalysis and thermodynamics of enzyme reactions. However, much less is known about the kinetics and energetics of conformational processes in the enzyme. How enzymes progress their reaction intermediates through the catalytic cycle remains an open question in enzymology. The challenge is to unravel the intimate linkage between protein motion and enzymatic function. Protein crystallography conducted at room temperature (RT) is a tool that can be used to analyze conformational motions of enzymes. We will analyze the electron density obtained from crystallographic studies in terms of one or more conformational states for each amino acid (aa) in the protein sequence. If one aa conformation overlaps with a neighboring aa conformation, it can represent a conduit for transmitting conformational motion in the enzyme. These networks can extend across the entire enzyme and correspond to dynamics-driven allostery that does not change the equilibrium positions of atoms. The goal will be to discern which contact networks interact with the enzyme reaction intermediates to understand how these interactions facilitate the chemical transformation of one intermediate into another. We have extensively investigated the glycolytic enzyme aldolase to unravel active site interactions with reaction intermediates and now wish to understand how conformational motions progress the reaction intermediates through the catalytic cycle. Aldolases are enzymes responsible for the generation of stored biological energy and metabolic precursors.***We will approach this objective experimentally by investigating contact networks using RT protein crystallography in aldolases, inhibited or inactivated distant from the active site. Would interruption/loss of contact networks explain these allosteric blockages? Can appropriate mutations rescue activity by re-establishing appropriate contact networks? To assess changes in contact networks due to active site events, aldolase mutants will be used that populate consecutive intermediates in the active site. Would changes be confined to a single contact network that reorganizes locally as a function of the reaction intermediate, or are there large scale changes in contact networks for each intermediate, extending to even subunit interfaces? Finally, we will implement a time-resolved crystallographic method using a highly intense micron sized X-ray beam at the NSLS-II synchrotron to collect data from microcrystals over the course of an enzyme reaction. The information captured enables a complete description of the conformational motions during catalysis including actual chemical bond breakage taking place, never seen before on an enzyme! Numerous applications are possible in drug design, biosensors, biomimetics; and offers fundamental insight into cellular processes. **

关于酶反应的催化作用和热力学，已经获得了大量的信息。然而，对酶中构象过程的动力学和能量学知之甚少。酶如何通过催化循环来处理它们的反应中间体仍然是酶学中的一个未解决的问题。挑战在于解开蛋白质运动和酶功能之间的密切联系。室温下进行的蛋白质晶体学是一种可用于分析酶构象运动的工具。我们将根据蛋白质序列中每个氨基酸（aa）的一个或多个构象状态，分析从晶体学研究中获得的电子密度。如果一个aa构象与相邻的aa构象重叠，它可以代表一个传递酶构象运动的管道。这些网络可以延伸到整个酶，并对应于不改变原子平衡位置的动力学驱动的变构。我们的目标是辨别哪些接触网络与酶反应中间体相互作用，以了解这些相互作用如何促进一种中间体向另一种中间体的化学转化。我们已经广泛地研究了糖酵解酶醛缩酶，以解开活性位点与反应中间体的相互作用，现在希望了解构象运动如何通过催化循环的反应中间体的进展。醛缩酶是负责产生储存的生物能量和代谢前体的酶。我们将接近这个目标的实验研究接触网络使用RT蛋白质晶体学在醛缩酶，抑制或灭活远离活性位点。接触网络的中断/丧失能否解释这些变构阻滞？适当的突变能否通过重建适当的接触网络来挽救活动？为了评估由于活性位点事件引起的接触网络的变化，将使用在活性位点中填充连续中间体的醛缩酶突变体。变化是否局限于一个单一的接触网络，作为反应中间体的功能，局部重组，或者每个中间体的接触网络都有大规模的变化，甚至延伸到亚基界面？最后，我们将实施一个时间分辨的晶体学方法，使用高度密集的微米大小的X射线束在NSLS-II同步加速器收集数据的过程中的酶反应的微晶。所捕获的信息能够完整描述催化过程中的构象运动，包括发生的实际化学键断裂，这在酶上是前所未有的！在药物设计，生物传感器，仿生学中有许多应用是可能的;并提供了对细胞过程的基本见解。**