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: 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=718478
• 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.

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用H-RAS P21的笼子底物。所有3种蛋白质都在晶体中活跃，同时保留晶体完整性。我们将使用在TRX实验中传递许多数据集的芯片。项目1）和2）需要访问高端同步器束线，项目3）需要X射线免费电子激光器（XFEL）。 18 TRX晶体结构从30 ms到30 s，涵盖了不可逆的FACD反应的4个完整的催化循环，提供了第一个现场反应性的视觉证明，并揭示了催化步骤与分子呼吸运动，水网络的结构和蛋白质模具的小变化之间的相关性。结果还设定了何时形成键和破裂的范围狭窄的边界。我们将研究SN2取代和酯水解的过程，并以大大改善的时间分辨率进行研究。 黄素蛋白的广泛吸收光谱禁止通过对笼前体的光解会触发反应。我提出了两种启动此类TRX反应的方法：1）使用基于超声的混合技术和2）由温度（T）跳跃引起的快速pH值。仅基于可访问t偏移的反应速率的变化太小了，但是对于某些缓冲液来说，20 C位移将pH值换2个单位，使休眠系统进行催化。 t变化既可以降低或增加pH值。尽管存在较低pH（笼质子的）的化合物，但没有笼子碱在照明时反应得足够快。 GR催化了两种连续的半反应，NADPH氧化和谷胱甘肽还原，也可以在中间状态停止。应用PL混合和pH值，我们将分析GR的催化步骤，并将它们与晶体内的光谱相关联，并将其与电子密度图更改。 3）第三个亚参与是最雄心勃勃的，是对2-硝基苯和1-（2-硝基苯基）乙基囊基组光解的黑暗反应的结构研究。尽管前者已成功地用于许多TRX研究，例如文献中仍讨论了H-RAS P21的GTP的磷酸GTP水解，这在文献中仍在研究中讨论了ACI-硝基和双环状中间体向硝基激素和游离化合物转化的确切机制。 Tr-SpectRoscopicy指示这些笼子组的黑暗反应的各个步骤的时间范围为MS，这使得它们可以通过TRX实验访问。较早的工作表明，具有笼中GTP的H-RAS P21蛋白的复合物可以结晶，并且晶体将耐受分离和GTP水解过程而不会分解。