Betts L, Frick L, Wolfenden R, Carter C W
Department of Biochemistry, University of North Carolina, Chapel Hill 27599-7260.
J Biol Chem. 1989 Apr 25;264(12):6737-40.
We have used an incomplete factorial design (Carter, C. W., and Carter, C. W., Jr. (1979) J. Biol. Chem. 254, 12219-12223) to find conditions for growing high quality crystals of Escherichia coli cytidine deaminase (EC 3.5.4.5). Crystals grow at pH 6.0 in hanging or sitting drops with either 1.6 M ammonium sulfate or 2.4-2.5 M sodium phosphate as precipitant. Both conditions produce crystals with identical morphologies and unit cell constants. The space group is P3(1)21 (or its enantiomorph P3(2)21), and the unit cell constants are a = b = 120.3 A, c = 78.4 A. The asymmetric unit is most reasonably one dimer of 66,000 Mr. The crystal size is very dependent on the supersaturation ratio, S = [initial protein concentration]/[equilibrium protein concentration], exhibiting a maximum at S = 7.7. The largest crystals diffract to at least 2.5 A and have a lifetime of 4 to 5 days in the x-ray beam at room temperature. The enzyme in these crystals is complexed with the transition state analog inhibitor 1-(beta-D-ribofuranosyl)-5-fluoropyrimidin-2-one (5-fluoropyrimidin-2-one riboside). We have collected data from parent crystals and from a heavy atom derivative in which the transition state analog is replaced by the active site directed inhibitor 5-(chloromercuri)cytidine.
我们采用了不完全析因设计（Carter, C. W., 和 Carter, C. W., Jr. (1979) J. Biol. Chem. 254, 12219 - 12223）来寻找培养高质量大肠杆菌胞苷脱氨酶（EC 3.5.4.5）晶体的条件。晶体在pH 6.0的悬滴或坐滴中生长，沉淀剂为1.6 M硫酸铵或2.4 - 2.5 M磷酸钠。这两种条件下产生的晶体具有相同的形态和晶胞常数。空间群为P3(1)21（或其对映体P3(2)21），晶胞常数为a = b = 120.3 Å，c = 78.4 Å。不对称单元最合理的是一个66,000 Mr的二聚体。晶体大小非常依赖于过饱和度比S = [初始蛋白质浓度]/[平衡蛋白质浓度]，在S = 7.7时达到最大值。最大的晶体衍射能力至少达到2.5 Å，在室温下于X射线束中的寿命为4至5天。这些晶体中的酶与过渡态类似物抑制剂1-(β-D-呋喃核糖基)-5-氟嘧啶-2-酮（5-氟嘧啶-2-酮核糖苷）复合。我们已从原始晶体和一种重原子衍生物收集了数据，在该重原子衍生物中，过渡态类似物被活性位点导向抑制剂5-(氯汞基)胞苷取代。
