Ray W J, Puvathingal J M, Liu Y W
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Biochemistry. 1991 Jul 16;30(28):6875-85. doi: 10.1021/bi00242a011.
Crystals of phosphoglucomutase, grown in 2.1 M ammonium sulfate, "desalted", and suspended in a 30% polyoxyethylene-8000/1 M glycine solution as described in the accompanying paper [Ray, W. J., Jr., Puvathingal, J. M., Bolin, J. T., Minor, W., Liu, Y., & Muchmore, S. W. (1991) Biochemistry 30 (preceding paper in this issue)], were treated with glucose phosphates to form an equilibrium mixture of the catalytically active substrate/product complexes. However, this treatment extensively fractured the crystals, even when very dilute solutions of glucose phosphates were used. But formation of the desired complexes was achieved, without fracturing, by introducing the glucose phosphates at high salt concentration, where they do not bind significantly to the enzyme, and maintaining their presence during subsequent sulfate-removal steps, in order to obtain essentially uniform binding throughout the crystal at all times. Although this procedure produced unfractured crystals of the catalytically active complexes, an adjustment in water activity was required to prevent the crystals from slowly liquefying in the presence of the added glucose phosphates. After this adjustment, the quality of diffraction-grade crystals subjected to this treatment was not significantly altered. An even larger adjustment in water activity was required to stabilize crystals that had been largely converted into a mixture of vanadate-based transition-state analogue complexes [cf. Ray, W. J., Jr., & Puvathingal, J. M. (1990) Biochemistry 29, 2790-2801] by means of an analogous procedure. The rationale for, and the implications of, this adjustment of water activity are discussed. The phenomenon of lattice-based binding cooperativity also is discussed together with a possible role for such cooperativity in the fracturing of protein crystals during formation of ligand complexes and possible ways to circumvent such fracturing based on the annealing of crystals at fractional saturation. An assay for quantifying the extent of formation of the vanadate-based transition-state analogue complexes in crystals of phosphoglucomutase is described. A solution to problems associated with producing and maintaining a steady-state in treated crystals is discussed within the context of maximizing the fraction of the crystalline enzyme present as a complex with one such inhibitor, glucose alpha-1-phosphate-6-vanadate. One of these problems, achieving a substantial reduction in sulfate concentration, could not be successfully addressed by employing the desalting procedure used to produce the substrate/product complexes, because of reduced diffusional rates in the final solution.(ABSTRACT TRUNCATED AT 400 WORDS)
磷酸葡萄糖变位酶晶体在2.1 M硫酸铵中生长，“脱盐”后，按照随附论文[Ray, W. J., Jr., Puvathingal, J. M., Bolin, J. T., Minor, W., Liu, Y., & Muchmore, S. W. (1991) Biochemistry 30 (本期前一篇论文)]所述，悬浮于30%聚氧乙烯-8000/1 M甘氨酸溶液中，用葡萄糖磷酸酯处理以形成催化活性底物/产物复合物的平衡混合物。然而，即使使用非常稀的葡萄糖磷酸酯溶液，这种处理也会使晶体大量破碎。但是，通过在高盐浓度下引入葡萄糖磷酸酯（此时它们与酶的结合不显著），并在随后的除硫酸盐步骤中保持其存在，从而在整个晶体中始终获得基本均匀的结合，实现了所需复合物的形成且晶体未破碎。尽管此方法产生了具有催化活性复合物的未破碎晶体，但需要调节水活性以防止晶体在添加葡萄糖磷酸酯的情况下缓慢液化。进行此调节后，经过这种处理的衍射级晶体的质量没有明显改变。对于通过类似方法已大量转化为基于钒酸盐的过渡态类似物复合物混合物的晶体[参见Ray, W. J., Jr., & Puvathingal, J. M. (1990) Biochemistry 29, 2790 - 2801]，需要更大程度地调节水活性以使其稳定。讨论了这种水活性调节的原理及其影响。还讨论了基于晶格的结合协同性现象，以及这种协同性在配体复合物形成过程中蛋白质晶体破碎中的可能作用，以及基于晶体在部分饱和度下退火来规避这种破碎的可能方法。描述了一种用于定量磷酸葡萄糖变位酶晶体中基于钒酸盐的过渡态类似物复合物形成程度的测定方法。在使作为与一种此类抑制剂（葡萄糖α-1-磷酸-6-钒酸盐）的复合物存在的结晶酶的比例最大化的背景下，讨论了与处理后的晶体中产生和维持稳态相关问题的解决方案。其中一个问题，即大幅降低硫酸盐浓度，由于最终溶液中扩散速率降低，无法通过用于产生底物/产物复合物的脱盐程序成功解决。
