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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=709221
• 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.

在催化和酶反应热力学方面获得了大量的信息。然而，对酶的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推进其反应中间体仍然是酶学中的一个悬而未决的问题。挑战在于解开蛋白质运动和酶功能之间的密切联系。在室温下进行的蛋白质晶体学是分析酶的构象运动的一种工具。我们将根据蛋白质序列中每个氨基酸（aa）的一种或多种构象状态来分析晶体学研究获得的电子密度。如果一个aa构象与相邻的aa构象重叠，它可以代表酶中传递构象运动的通道。这些网络可以延伸到整个酶，并与不改变原子平衡位置的动力学驱动的变构相对应。目标将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，以揭示活性位点与反应中间体的相互作用，现在希望了解在催化循环中，构象运动是如何促进反应中间体的。醛缩酶是负责产生储存的生物能和代谢前体的酶。