Structural investigation of reaction intermediates in glycolytic aldolases

关于酶反应的催化和热力学，人们已经获得了丰富的信息。然而，人们对该酶构象过程的动力学和能量学知之甚少。酶如何在催化循环中进行反应中间产物在酶学中仍然是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结晶学是一种可以用来分析酶的构象运动的工具。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来分析从结晶学研究中获得的电子密度。如果一个氨基酸构象与相邻的一个氨基酸构象重叠，它可以代表一个在酶中传递构象运动的管道。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。我们已经对糖酵解酶醛缩酶进行了广泛的研究，以揭示活性部位与反应中间体的相互作用，现在我们希望了解构象运动如何在催化循环中推进反应中间体。醛缩酶是负责产生储存的生物能量和代谢前体的酶。*我们将通过实验研究远离活性部位被抑制或失活的醛缩酶的接触网络。联系网络的中断/丢失可以解释这些变构障碍吗？适当的突变能否通过重新建立适当的联系网络来挽救活动？为了评估由于活性部位事件引起的接触网络的变化，将使用在活性部位填充连续中间体的醛缩酶突变体。变化是否仅限于作为反应中间体的函数在局部重组的单个接触网络，或者每个中间体的接触网络是否存在大规模变化，甚至延伸到亚基界面？最后，我们将在NSLS-II同步加速器上实施一种时间分辨结晶学方法，使用高强度微米大小的X射线束来收集酶反应过程中微晶体的数据。捕捉到的信息能够完整地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、仿生学中有许多可能的应用，并提供了对细胞过程的基本洞察。**

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. **