Time-resolved X-ray crystallographic studies of enzyme reaction

托品酮还原酶-II（TR-II，EC 1. 1. 1. 236)催化托品酮的3-羰基还原为β-羟基的NADPH依赖性还原，并产生假托品碱（β-托品碱）。γ-谷氨酰半胱氨酸合成酶（γ-Glutamylcysteine synthetase，γGCS）是谷胱甘肽生物合成的限速酶，催化谷氨酸（L-Glu）和半胱氨酸（L-Cys）在ATP依赖下偶联生成谷胱甘肽前体γ-L-Glu-Cys。为了在反应发生之前捕捉它们的结构，我们试图确定它们的结构，并应用两种时间分辨晶体学技术，流动池和劳厄衍射。本文报道（1）TR-Ⅱ的时间分辨结构分析和（2）大肠杆菌B γGCS的X射线晶体学研究。(1)A将用于X射线衍射实验的TR-II晶体安装在流动池（直径0.7mm）中。在数据收集期间，含有0.1M HEPES-Na（pH 7.5）、2 M Li_2SO_4、4 mM NADPH、100 mM托品酮、1 mM DTT的溶液以1 ml/min的速率通过晶体。劳厄衍射数据是在光子工厂的BL-18 B站用30毫秒曝光的大成像板收集的。它给出了良好的劳厄衍射图案，尽管大的晶体的晶胞。劳厄数据显示明确的电子密度对应的基板和辅因子的差分傅立叶分析。TR-II-NADP^+-托品酮复合物的结构表明，在反应开始前，托品酮在活性中心的结合方向发生了变化，有利于氢化物阴离子对底物的亲核进攻。（2）γGCS晶体最大尺寸为0.2×0.2× 0.1mm，衍射分辨率达2.8 μ m以上，晶体质量好。γGCS晶体的加工数据显示为六方晶系，晶胞参数a =B =326.7，c =103.9 μ m。该晶体的空间群确定为R3。目前正在进行进一步的数据分析。

Tropinone reductase-II(TR-II, EC 1. 1. 1. 236)catalyzes an NADPH-dependent reduction of the 3-carbonyl group of tropinone to a β-hydroxyl group, and produces pseudotropine(ψ-tropine). γ-Glutamylcysteine synthetase(γGCS), which catalyzes the ATP-dependent coupling of L-Glu and L-Cys to form a glutathione precursor γ-L-Glu-Cys, is the rate-limiting enzyme in glutathione biosynthesis. In order to capture their structures just before the reaction occurs, we tried to determine their structures and apply two techniques for time-resolved crystallography, flow cell and Laue diffraction. Here we report(1)time-resolved structural analysis of TR-II and(2)X-ray crystallographic study on γGCS from Escherichia coli B.(1)A TR-II crystal for X-ray diffraction experiment was mounted in a flow cell(0.7 mm in diameter). A solution containing 0.1 M HEPES-Na, pH 7.5, 2 M Li_2SO_4, 4 mM NADPH, 100 mM tropinone, 1 mM DTT wa passed across the crystal at a rate of 1 ml/min during data collection. Laue diffraction data were collected at BL-18B station, Photon Factory, using large imaging-plates with 30 msec exposure. It gave good Laue diffraction patterns despite of the large unit cell of the crystal. The Laue data showed clear electron densities corresponding to the substrate and cofactor by difference Fourier analysis. The structure of TR-II-NADP^+-tropinone complex indicated that the direction of tropinone binding in the active-site changed just before the reaction started, and it gave an advantage in the nucleophilic attack of hydride anion to the substrate.(2)Native crystals of γGCS grew to maximum dimensions of 0.2×0.2×0.1 mm. These crystal were of good quality, diffracting to 2.8 Å resolution or better. Processing data of γGCS crystals revealed a hexagonal crystal system with unit-cell parameters a =b =326.7, c =103.9 Å. The space group of this crystal was determined to be R3. Further data analysis is now underway.