Höschler K, Hoier H, Hubner B, Saenger W, Orth P, Hahn U
Institut für Biochemie Fakultät für Biowissenschaften Pharmazie und Psychologie, Universität Leipzig, Talstrasse 33, Leipzig, 04103, Germany.
J Mol Biol. 1999 Dec 17;294(5):1231-8. doi: 10.1006/jmbi.1999.3324.
The ribonuclease T1 variant 9/5 with a guanine recognition segment, altered from the wild-type amino acid sequence 41-KYNNYE-46 to 41-EFRNWQ-46, has been cocrystallised with the specific inhibitor 2'-GMP. The crystal structure has been refined to a crystallographic R factor of 0.198 at 2.3 A resolution. Despite a size reduction of the binding pocket, pushing the inhibitor outside by 1 A, 2'-GMP is fixed to the primary recognition site due to increased aromatic stacking interactions. The phosphate group of 2'-GMP is located about 4.2 A apart from its position in wild-type ribonuclease T1-2'-GMP complexes, allowing a Ca(2+), coordinating this phosphate group, to enter the binding pocket. The crystallographic data can be aligned with the kinetic characterisation of the variant, showing a reduction of both, guanine affinity and turnover rate. The presence of Ca(2+) was shown to inhibit variant 9/5 and wild-type enzyme to nearly the same extent.
具有鸟嘌呤识别片段的核糖核酸酶T1变体9/5，其野生型氨基酸序列41-KYNNYE-46改变为41-EFRNWQ-46，已与特异性抑制剂2'-GMP共结晶。晶体结构已在2.3 Å分辨率下精修至晶体学R因子为0.198。尽管结合口袋尺寸减小，使抑制剂向外移动了1 Å，但由于芳香族堆积相互作用增加，2'-GMP仍固定在主要识别位点。2'-GMP的磷酸基团与其在野生型核糖核酸酶T1-2'-GMP复合物中的位置相距约4.2 Å，使得配位该磷酸基团的Ca(2+)能够进入结合口袋。晶体学数据可以与该变体的动力学特征相匹配，显示鸟嘌呤亲和力和周转速率均降低。已表明Ca(2+)的存在对变体9/5和野生型酶的抑制程度几乎相同。
