Koellner G, Luić M, Shugar D, Saenger W, Bzowska A
Institut für Kristallographie Freie Universität Berlin, Germany.
J Mol Biol. 1997 Jan 17;265(2):202-16. doi: 10.1006/jmbi.1996.0730.
Trimeric calf spleen purine nucleoside phosphorylase has been complexed with hypoxanthine via phosphorolysis of inosine in the presence of phosphate. The resulting, "Michaelis" complex (three hypoxanthine molecules per trimer), presumed to be formed under these conditions, crystallized in the cubic space group P2(1)3, with unit cell dimension a = 94.11 A and one monomer in the asymmetric crystal unit; the biologically active trimer is located on the crystallographic 3-fold axis. High-resolution X-ray diffraction data were collected using synchrotron radiation (EMBL outstation, Hamburg, c/o DESY). The crystal structure has been determined by molecular replacement and refined at 2.15 A resolution to an R-value of 0.18. In the hypoxanthine binding site, a cis-peptide bond between Asn243 and Lys244 is observed. Side-chains of GIu201 and Asn243, as well as one integral water molecule located in the base binding site, form hydrogen bonds with the hypoxanthine N-1 H, N-7 H and O-6. A second water molecule links the base positions N-3 and N-9 with an adjacent pocket, which presumably is the phosphate-binding site. This pocket is filled completely by a cluster of six water molecules. Hence all possible donor/acceptor-positions of hypoxanthine are saturated by hydrogen-bonding to protein side-chains or integral water molecules. Purine nucleoside phosphorylase isolated form human tissues is a primary target for chemotherapeutic intervention, and the more stable calf enzyme has similar physico-chemical and kinetic properties, as well as response to inhibitors. Hence the high-resolution structure presented here may serve for design of inhibitors with potential pharmacological applications.
三聚体小牛脾嘌呤核苷磷酸化酶在磷酸盐存在下通过肌苷的磷酸解作用与次黄嘌呤复合。在这些条件下假定形成的“米氏”复合物（每个三聚体有三个次黄嘌呤分子），结晶于立方空间群P2(1)3中，晶胞参数a = 94.11 Å，不对称晶胞中有一个单体；具有生物活性的三聚体位于晶体学的三重轴上。使用同步辐射（欧洲分子生物学实验室汉堡分站，由德国电子同步加速器研究所代管）收集了高分辨率X射线衍射数据。通过分子置换确定了晶体结构，并在2.15 Å分辨率下进行精修，R值为0.18。在次黄嘌呤结合位点，观察到Asn243和Lys244之间存在一个顺式肽键。Glu201和Asn243的侧链，以及位于碱基结合位点的一个完整水分子，与次黄嘌呤的N-1 H、N-7 H和O-6形成氢键。第二个水分子将碱基的N-3和N-9位置与相邻的口袋相连，该口袋可能是磷酸盐结合位点。这个口袋被六个水分子簇完全填满。因此，次黄嘌呤所有可能的供体/受体位置都通过与蛋白质侧链或完整水分子形成氢键而饱和。从人体组织中分离的嘌呤核苷磷酸化酶是化疗干预的主要靶点，而更稳定的小牛酶具有相似的物理化学和动力学性质，以及对抑制剂的反应。因此，这里呈现的高分辨率结构可用于设计具有潜在药理学应用的抑制剂。
