The reaction mechanism and base recognition of RNase Rh

RNase Rh的反应机理及碱基识别

• 批准号: 06672154
• 负责人: NAKAMURA Kazuo
• 金额: $ 1.28万
• 依托单位: Showa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06672154/
• 关键词: Ribonuclease; X-ray structure analysis; Enzyme-substrate complex; Ribonuclease; X-ray structure analysis; Enzyme-substrate complex

During the present study we have determined seven crystal structures of RNase RNAP-Rh, a derivative of RNase Rh, and its mutant enzymes (RNAP-Rh, RNAP-Rh+2'-AMP complex, RNAP-Rh+2'-GMP complex, Y57W, Y57W+2'-AMP complex, Y57W+3'-AMP complex and Y57W+2'-GMP complex) . Diffraction data of the crystals were collected using an oscillation camera or a Weissenberg camera. The crystal structures were determined based on the atomic coordinates of RNase Rh, and refined usingthe program X-PLOR.The refinements were done successfully, and all the R-values were coverged to under 0.20. (1) We found out that the base recognition site of this enzyme consists of Trp49, Tyr57 and Asp51. The base of substrate is sandwiched in between the two former residues, and hydrogen-boned to the side chain of Asp51. (2) This enzyme is an adenine preferential RNase, but we could introduce 2'-GMP into the crystals of RNase RNAP-Rh and Y57W by soaking technique. The binding mode of guanine base is similar to that of adenine, but there are some differences between them.

在本研究中，我们确定了RNase RNAP-RH的七个晶体结构，RNase RH的衍生物及其突变酶（RNAP-RH，RNAP-RH，RNAP-RH+2'-AMP复合物，RNAP-RH+2'-GMP复合物，Y57W，Y57W+2'-Amp Complex，Y57W+2'-Amp Complex，Y57W+3'-amp＆y577-＆y577-gm and y577-gM+2”使用振荡摄像头或Weissenberg摄像头收集晶体的衍射数据。基于RNase RH的原子坐标确定晶体结构，并使用程序X-Aplor进行了完善。修改成功完成，所有R值覆盖为0.20。 （1）我们发现该酶的基础识别位点由TRP49，Tyr57和Asp51组成。底物的碱基夹在两个以前的残基之间，并氢有氢，并在ASP51的侧链上。 （2）该酶是一种腺嘌呤的优先RNase，但我们可以通过浸泡技术将2'-GMP引入RNase RNAP-RH和Y57W的晶体。鸟嘌呤碱的结合模式与腺嘌呤的结合模式相似，但是它们之间存在一些差异。