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.

我将专门关注不可逆酶反应的时间分辨晶体学 (TRX)，通过追求 3 个特定区域，每个区域由一个蛋白质代表：1) 提高氟乙酸脱卤酶 (FAcD) 的 TRX 时间分辨率，重点关注键的形成和断裂，2) 探索谷胱甘肽还原酶 (GR) 的新触发机制，3) 研究 用 H-ras P21 笼住底物。所有 3 种蛋白质在晶体中均具有活性，同时保持晶体完整性。我们将使用在 TRX 实验中提供许多数据集的芯片。项目 1) 和 2) 需要使用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，提供了半位点反应性的第一个视觉证据，并揭示了催化步骤和分子呼吸运动、水网络结构和蛋白质模具的微小变化之间的相关性。结果还为键形成和断裂发生的时间设定了相当窄的界限。我们将研究 SN2 取代和酯水解的过程，并大大提高时间分辨率。 黄素蛋白的宽吸收光谱禁止通过笼状前体的光解来触发反应。我提出了两种启动此类 TRX 反应的方法：1) 使用基于超声波的混合技术，2) 由温度 (T) 跳跃引起的快速 pH 变化。仅基于可达到的 T 位移的反应速率变化太小，然而，对于某些缓冲液来说，20 C 位移会使 pH 值改变 2 个单位，从而使休眠系统发挥催化作用。 T 的变化可以降低或升高 pH 值。虽然存在能快速降低 pH 值的化合物（笼中质子），但笼中碱基在光照下的反应速度不够快。 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。应用 pL 混合和 pH 值变化，我们将分析 GR 的催化步骤，并将其与晶内 VIS 光谱和电子密度图的变化相关联。 3）第三个子项目是最雄心勃勃的一个，2-硝基苄基-和1-(2-硝基苯基)乙基-笼蔽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，例如附着在GTP的α-磷酸盐上，研究H-ras P21对GTP的水解作用，酰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仍在文献中讨论。 TR 光谱显示了这些笼养组暗反应各个步骤的时间范围为 ?s 到 ms，从而使它们可以进行 TRX 实验。早期的工作表明，H-ras P21 蛋白与笼状 GTP 的复合物可以结晶，并且晶体能够耐受脱笼和 GTP 水解过程而不会分解。