Time-resolved X-ray crystallographic studies of enzyme reaction

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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.