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

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，我们测定了RNase Rh的衍生物RNAP-Rh及其突变酶(RNAP-Rh、RNAP-Rh+2‘-AMP复合体、RNAP-Rh+2’-GMP复合体、Y57W、Y57W+2‘-AMP复合体、Y57W+3’-AMP复合体和Y57W+2‘-GMP复合体)的7种晶体结构。晶体的衍射数据是用振荡相机或魏森伯格相机收集的。根据RNaseRh的原子坐标确定了晶体结构，并用X-PLOR程序进行了精修，精修成功，所有的R值都覆盖到0.20以下。(1)发现该酶的碱基识别位点由Trp49、Tyr57和Asp51组成。底物的碱基夹在前两个残基之间，氢键连接到Asp51的侧链上。(2)该酶是腺嘌呤优先型核糖核酸酶，但我们可以通过浸泡技术将2‘-GMP引入核糖核酸酶RNAP-Rh和Y57W的晶体中。鸟嘌呤碱与腺嘌呤的结合方式相似，但也存在一定的差异。

项目成果

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M.Irie 等人：“来自根霉的 RNase Rh 动力学的 pH 曲线”J.Biochem.115。

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K.Nakamura：“X射线结构分析简介” Kagakutoyakugakuno-kyousitsu。

H.Kurihara,et al.: "The Crystal Structure of Ribonuclease Rh" J.Mol.Biol.255. 310-320 (1996)

H.Kurihara 等人：“核糖核酸酶 Rh 的晶体结构”J.Mol.Biol.255。

H. kurihara et al.: "The Crystal Structure of Ribonuclease Rh from Rhizopus niveus" J. Mol. Biol.255. 310-320 (1996)

H. kurihara 等人：“来自雪根霉的核糖核酸酶 Rh 的晶体结构”J. Mol.

M.Irie et al.: "pH Profile of Kinetics of RNase Rh from Rhizopus niveus and Its Mutant Enzymes towards Upv, and Possible Mechanism of RNase Rh" J.Biochem.Vol.115. 1083-1087 (1994)

M.Irie 等人：“来自雪根霉的 RNase Rh 动力学的 pH 曲线及其突变酶对 Upv 的影响，以及 RNase Rh 的可能机制”J.Biochem.Vol.115。