Crystal structure of calf spleen purine nucleoside phosphorylase in a complex with hypoxanthine at 2.15 angstrom resolution
Crystal structure of calf spleen purine nucleoside phosphorylase in a complex with hypoxanthine at 2.15 angstrom resolution
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DOI：
10.1006/jmbi.1996.0730
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发表时间：
1997-01-17
影响因子：
5.6
通讯作者：
Bzowska, A
中科院分区：
文献类型：
--
作者：
Koellner, G;Luic, M;Bzowska, A
三聚体小牛脾嘌呤核苷磷酸化酶在磷酸盐存在下，通过肌苷的磷酸化与次黄嘌呤形成络合物。由此得到的‘’Michaelis‘’络合物(每个三聚体有三个次黄嘌呤分子)在这些条件下形成，结晶在立方空间群P2(1)3中，晶胞尺寸a=94.11 Angstrom，一个不对称晶体单元中的单体；生物活性三聚体位于晶体结构的三重轴上。高分辨率X射线衍射数据是使用同步辐射(EMBL子站，汉堡，c/o DESY)收集的。通过分子置换确定了晶体结构，并在2.15埃分辨率下精炼到X=0.18。在次黄嘌呤结合部位，观察到Asn243与Lys244之间存在顺式多肽键。Glu201和Asn243的侧链以及位于碱基结合位置的一个完整的水分子与次黄嘌呤N-1H、N-7H和O-6形成氢键。第二个水分子将碱基位置N-3和N-9与邻近的口袋相连，该口袋可能是磷酸盐结合部位。这个口袋完全被一团六个水分子填满了。因此，次黄嘌呤的所有可能的供体/受体位置都被蛋白质侧链或整体水分子的氢键饱和。从人体组织中分离的精氨酸核苷磷酸化酶是化疗干预的主要靶点，更稳定的小牛酶具有相似的物理化学性质和动力学性质，以及对抑制剂的反应。因此，本文提出的高分辨结构可用于设计具有潜在药理应用的抑制剂。(C)1997年学术出版社有限公司
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 Angstrom 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 Angstrom resolution to an X-value of 0.18.In the hypoxanthine binding site, a cis-peptide bond between Asn243 and Lys244 is observed. Side-chains of Glu201 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. (C) 1997 Academic Press Limited