Time-resolved X-ray crystallographic studies of enzyme reaction

曲皮酮还原酶-II(Trp-II，EC1.1.1.236)催化托品酮的3-羰基在NADPH作用下还原为β-羟基，生成伪托品(ψ-托品)。谷氨酰半胱氨酸合成酶(γ-Glutaylcysteine Synthetase，γ)是谷胱甘肽生物合成的限速酶，它催化L-谷氨酸和L-半胱氨酸依赖的偶联反应，形成谷胱甘肽前体γ-L-谷氨酸-半胱氨酸。为了在反应前捕捉到它们的结构，我们尝试确定了它们的结构，并应用了两种时间分辨结晶学技术，流动池和劳埃衍射。本文报道了(1)TR-II的时间分辨结构分析和(2)大肠杆菌B的γ-GCS的X射线结晶学研究。(1)将用于X射线衍射实验的TR-II晶体固定在直径为0.7 mm的流动池中。在数据采集过程中，含有0.1M HEPES-Na，pH 7.5，2M Li_2SO_4，4 mM NADPH，100 mM托品酮，1 mM DTT的溶液以1ml/min的速度通过晶体。劳厄衍射数据是在光子工厂的BL-18B站收集的，使用曝光30毫秒的大型成像板。尽管晶体的晶胞较大，但仍能产生良好的劳厄衍射图。通过差分傅立叶分析，LAUE数据显示了与底物和辅因子相对应的清晰的电子密度。TrII-NADP^+-托品酮络合物的结构表明，在反应开始前，活性中心的托品酮结合方向发生了变化，有利于氢化物阴离子对底物的亲核攻击。(2)γGCS天然晶体生长到最大尺寸为0.2×0.2×0.1 mm。这些晶体的质量很好，衍射率达到2.8Å或更高。γ晶体的晶胞参数为a=b=326.7，c=103.9Å。该晶体的空间群被确定为R3。进一步的数据分析目前正在进行中。

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.