Time-resolved crystallography of enzyme-catalyzed reactions

酶催化反应的时间分辨晶体学

• 批准号: RGPIN-2020-06867
• 负责人: Pai, Emil
• 金额: $ 3.5万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=741469
• 关键词: Time; resolved; crystallography; enzyme; catalyzed

I will focus exclusively on time-resolved crystallography (TRX) of irreversible enzyme reactions by pursuing 3 specific areas, each represented by a protein: 1) improving the time-resolution of TRX of fluoroacetate dehalogenase (FAcD), focusing on bond formation and breakage, 2) exploring new triggering mechanisms with glutathione reductase (GR), 3) investigating the photolysis of caged substrates with H-ras P21. All 3 proteins are active in crystals while preserving crystal integrity. We will use a chip that delivered many data sets in TRX experiments. Projects 1) and 2) need access to high-end synchrotron beamlines, project 3) requires an X-ray Free Electron Laser (XFEL). 18 TRX crystal structures, from 30 ms to 30 s, covering 4 full catalytic cycles of the irreversible FAcD reaction, provide the first visual proof of half-of-the-sites reactivity and reveal correlations between the catalytic steps and molecular breathing motions, the structure of a water network and small changes of the protein mold. The results also set rather narrow boundaries for when bond formation and breakage occur. We will investigate the processes of SN2 substitution and ester hydrolysis with much improved time resolution. The broad absorption spectra of flavoproteins forbid triggering reactions by photolysis of caged precursors. I propose 2 methods to start such TRX reactions: 1) use of an ultrasound-based mixing technique and 2) fast pH shifts caused by temperature (T) jumps. Changes in reaction rates based on accessible T shifts alone are too small, however, for some buffers, 20 °C shifts change pH by 2 units, taking a dormant system to catalysis. T changes can both lower or increase the pH value. While compounds exist that lower pH quickly (`caged protons') no `caged bases' react sufficiently fast upon illumination. GR catalyzes two successive half-reactions, NADPH oxidation and glutathione reduction, which can also be stopped at intermediate states. Applying pL mixing and pH shifts, we will analyze GR's catalytic steps and correlate them to in-crystal VIS spectra and changes to the electron density maps. 3) The third sub-project is the most ambitious one, the structural investigation of the dark reaction following the photolysis of the 2-nitrobenzyl- and 1-(2-nitrophenyl)ethyl- caging groups. While the former has been used successfully in quite a number of TRX studies, e.g. attached to the ?-phosphate of GTP investigating GTP hydrolysis by H-ras P21, the exact mechanism of transformation of aci-nitro- and bicyclic intermediates to nitrosoketone and free compound are still discussed in the literature. TR-spectroscopy indicates time ranges of ?s to ms for the various steps for the dark reactions of these caging groups, making them accessible to TRX experiments. Earlier work has shown that a complex of H-ras P21 protein with caged GTP can be crystallized and the crystals will tolerate the decaging and GTP hydrolysis processes without disintegration.

我将专注于不可逆酶反应的时间分辨晶体学（TRX），研究3个特定区域，每个区域以一个蛋白质为代表：1)提高氟乙酸脱卤酶（FAcD） TRX的时间分辨，重点研究键的形成和断裂；2)利用谷胱甘肽还原酶（GR）探索新的触发机制；3)研究H-ras P21对固定底物的光解作用。这三种蛋白在保持晶体完整性的同时在晶体中都有活性。我们将在TRX实验中使用一种可以提供许多数据集的芯片。项目1)和2)需要高端同步加速器光束线，项目3)需要x射线自由电子激光器（XFEL）。18个TRX晶体结构，从30 ms到30 s，覆盖了不可逆FAcD反应的4个完整催化循环，提供了半位点反应活性的第一个视觉证据，并揭示了催化步骤与分子呼吸运动、水网络结构和蛋白质模具微小变化之间的相关性。结果还为键形成和断裂的发生设定了相当窄的边界。我们将以更高的时间分辨率研究SN2取代和酯水解的过程。黄酮类蛋白的宽吸收光谱阻止了笼状前体光解引发反应。我提出了两种方法来启动这种TRX反应：1)使用基于超声波的混合技术和2)由温度(T)跳变引起的快速pH变化。然而，仅基于可达T位移的反应速率变化太小，对于某些缓冲液，20°C位移使pH值改变2个单位，使休眠系统进行催化。T的变化既可以降低pH值，也可以增加pH值。虽然存在快速降低pH值的化合物（“笼状质子”），但没有“笼状碱”在光照下反应足够快。GR催化两个连续的半反应，NADPH氧化和谷胱甘肽还原，也可以在中间状态停止。利用pL混合和pH位移，我们将分析GR的催化步骤，并将它们与晶体内VIS光谱和电子密度图的变化联系起来。3)第三个子项目是最雄心勃勃的一个，对2-硝基苯-和1-（2-硝基苯）乙基-笼化基团光解后暗反应的结构研究。虽然前者已在相当多的TRX研究中成功使用，例如附属于？研究了H-ras P21对GTP的水解作用，文献中仍在讨论酸性硝基和双环中间体转化为亚硝基酮和游离化合物的确切机制。红外光谱显示时间范围为？对于这些笼化基团的暗反应的各个步骤，可以使用s到ms，使它们可以用于TRX实验。早期的研究表明，H-ras P21蛋白与笼状GTP的复合物可以结晶，并且晶体可以耐受老化和GTP水解过程而不会分解。