Koellner Gertraud, Bzowska Agnieszka, Wielgus-Kutrowska Beata, Luić Marija, Steiner Thomas, Saenger Wolfram, Stepiński Janusz
Freie Universität Berlin, Institut für Chemie-Kristallographie, Takustrasse 6, D-14195 Berlin, Germany.
J Mol Biol. 2002 Jan 18;315(3):351-71. doi: 10.1006/jmbi.2001.5211.
The crystal structure of the ternary complex of hexameric purine nucleoside phosphorylase (PNP) from Escherichia coli with formycin A derivatives and phosphate or sulphate ions is determined at 2.0 A resolution. The hexamer is found as a trimer of unsymmetric dimers, which are formed by pairs of monomers with active sites in different conformations. The conformational difference stems from a flexible helix (H8: 214-236), which is continuous in one conformer, and segmented in the other. With the continuous helix, the entry into the active site pocket is wide open, and the ligands are bound only loosely ("open" or "loose binding" conformation). By segmentation of the helix (H8: 214-219 and H8': 223-236, separated by a gamma-turn), the entry into the active site is partially closed, the pocket is narrowed and the ligands are bound much more tightly ("closed" or "tight binding" conformation). Furthermore, the side-chain of Arg217 is carried by the moving helix into the active site. This residue, conserved in all homologous PNPs, plays an important role in the proposed catalytic mechanism. In this mechanism, substrate binding takes place in the open, and and the catalytic action occurs in the closed conformation. Catalytic action involves protonation of the purine base at position N7 by the side-chain of Asp204, which is initially in the acid form. The proton transfer is triggered by the Arg217 side-chain which is moved by the conformation change into hydrogen bond distance to Asp204. The mechanism explains the broad specificity of E. coli PNP, which allows 6-amino as well as 6-oxo-nucleosides as substrates. The observation of two kinds of binding sites is fully in line with solution experiments which independently observe strong and weak binding sites for phosphate as well as for the nucleoside inhibitor.
测定了来自大肠杆菌的六聚体嘌呤核苷磷酸化酶（PNP）与间型霉素A衍生物以及磷酸根或硫酸根离子形成的三元复合物的晶体结构，分辨率为2.0埃。发现该六聚体是不对称二聚体的三聚体，不对称二聚体由具有不同构象活性位点的单体对形成。构象差异源于一条柔性螺旋（H8：214 - 236），在一种构象中它是连续的，而在另一种构象中是分段的。在连续螺旋的情况下，进入活性位点口袋的入口是敞开的，配体仅松散结合（“开放”或“松散结合”构象）。通过螺旋的分段（H8：214 - 219和H8'：223 - 236，由一个γ-转角隔开），进入活性位点的入口部分关闭，口袋变窄，配体结合得更紧密（“关闭”或“紧密结合”构象）。此外，Arg217的侧链随着移动的螺旋进入活性位点。该残基在所有同源PNP中都保守，在提出的催化机制中起重要作用。在该机制中，底物在开放构象下结合，催化作用在关闭构象下发生。催化作用涉及由最初呈酸性形式的Asp204的侧链使嘌呤碱基的N7位质子化。质子转移由Arg217侧链触发，该侧链通过构象变化移动到与Asp204形成氢键距离的位置。该机制解释了大肠杆菌PNP的广泛特异性，它允许6-氨基以及6-氧代核苷作为底物。观察到两种结合位点与溶液实验完全一致，溶液实验独立观察到了磷酸根以及核苷抑制剂的强结合位点和弱结合位点。
