Time-Resolved Crystal Structure Analysis of Glutathione Synthetase

谷胱甘肽合成酶的时间分辨晶体结构分析

• 批准号: 04044103
• 负责人: KATUBE Yukiteru
• 金额: $ 5.18万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04044103/
• 关键词: Time Resolve X-ray Structure Analysis; Glutatuione Synthetase; Laue Method in Time Resolution; Kinetic Protein-Crystallography; 時間分割結晶構造解析; ラウエ法; 放射光; フローセル; フラッシュ光反応; 変異体酵素; ATP

One of the major goals of structural biology should perhaps be to achieve 4-dimensional structure determination (with time being the 4th dimension).In order to apply time resolved crystallography to glutathione synthetase from E.coli, we determined a crystal structure of the enzyme by X-ray diffraction method. The enzyme is a tetramer with four identical Subunits of 316 amino acid residues. The loop from Ile-226 to Arg-241 in the enzyme is rich in glycine and alanine and too flexible to take a fixed conformation. The apparent function of the loop is to serve as a lid that shields the reaction intermediate, gamma-glutamylcysteinylphosphate, from the solvent water.Kinetic diffraction studies require the fast measurement of a sequence of 3-dimensional sets of structure factors with an accuracy and completeness that is sufficient to describe the structures during the reaction in the crystal. This is not easy requirement because of fast catalytic reaction-rate of the enzyme, msec-mu sec. To restrict the catalytic reaction-rate, we prepared some mutants by site-directed mutagenesis. The Km value of R210K mutant was similar to that of the wild-type, but its kcat value drastically decreased. Laue data of R210K by synchrotron radiation were processed using the program LAUEMAD. The power and limitations of time resolved X-method by using Laue diffraction were investigated. In particular, the effects of crystalline disorder, imperfect wavelength scaling, and a systematic lack of completeness in Laue structure factor sets at low and medium resolution were analyzed.As results of the above investigation, we learned that R210K would be a good target of time resolved X-ray structure study combined Laue and monochromatic diffraction techniques.

结构生物学的主要目标之一可能是实现四维结构测定（时间是第四维），为了将时间分辨晶体学应用于大肠杆菌谷胱甘肽合成酶，我们用X射线衍射方法测定了该酶的晶体结构。该酶是一个四聚体，具有316个氨基酸残基的四个相同亚基。酶中从Ile-226到Arg-241的环富含甘氨酸和丙氨酸，并且太灵活而不能采取固定构象。环的明显功能是作为一个盖子，屏蔽反应中间体，γ-谷氨酰半胱氨酰磷酸，从溶剂water.Kinetic衍射研究需要快速测量的序列的三维集的结构因子的准确性和完整性，足以描述在晶体中的反应过程中的结构。这是不容易的要求，因为酶的快速催化反应速率，毫秒-μ秒。为了限制催化反应速率，我们通过定点突变制备了一些突变体。R210 K突变体的Km值与野生型相似，但其kcat值显著降低。用LAUEMAD程序处理了R210 K的同步辐射劳厄数据。研究了劳厄衍射时间分辨X射线方法的能力和局限性。特别是，我们分析了晶体无序、波长标度不完善以及劳厄结构因子集在中低分辨率下系统性不完整等因素对R210 K的影响，并由此得出结论：R210 K是劳厄和单色衍射技术相结合的时间分辨X射线结构研究的理想靶点。

项目成果

M.SATO,Y.KATSUBE,M.KATAI,J.KATAKAWA,T.TETSOMI: "Crystcal and Molecular Structare of 3,6-Dioxograyanotoxin I" Bull.Cheu.Soc,JAPAN. 65. 2836-2838 (1992)

M.SATO、Y.KATSUBE、M.KATAI、J.KATAKAWA、T.TETSOMI：“3,6-二氧木芥毒素 I 的晶体和分子结构”Bull.Cheu.Soc，日本。

Takuji Tanaka,Hiroshi Yamaguchi,Hiroaki Kato,Takaaki Nishioka,Yukiteru Katsube,Jun'ichi Oda: "Flexibility Imparired by Mutantions Revealed the Multifunctional Roles of the Loop in Glutathione Synthetase" Biochemistry. 32. 12398-12404 (1993)

Takuji Tanaka、Hiroshi Yamaguchi、Hiroaki Kato、Takaaki Nishioka、Yukiteru Katsube、Junichi Oda：“突变损害的灵活性揭示了环在谷胱甘肽合成酶中的多功能作用”生物化学。

Takuji Tanaka, et al.: "Flexibility Imparired by Mutation Revealed the Multifunctional Roles of the Loop in Glutathione Synthetase" Biochemistry. 32. 12398-12404 (1993)

Takuji Tanaka 等人：“突变导致的灵活性受损揭示了环在谷胱甘肽合成酶中的多功能作用”生物化学。

M.SAKIRAI,K.ONODERA,H.MORIYAMA,O.MATSUMOTO,N.TANAKA,M.SATO,Y.KATSUBE,T.OSHIMA: "Crstallization and Preliminary X-ray Studies of A B.sicdtilis and T.ther mophilus HB8 Chimeric 3-IPMDH and Thermostable Mutants of It." J.Biochem.112. 173-174 (1992)

M.SAKIRAI、K.ONODERA、H.MORIYAMA、O.MATSUMOTO、N.TANAKA、M.SATO、Y.KATSUBE、T.OSHIMA：“A.sicdtilis 和 T.thermophilus 的结晶和初步 X 射线研究

C.Branden,J.Tooze著 監修 勝部幸輝.松原央.松原謙一 翻訳 勝部幸輝.竹中章郎.福山恵一.松原央: "タンパク質の構造入門(Introduction to Protein Structure)" 教育社, 285 (1992)

作者：C. Branden、J. Tooze 监督：Koki Katsabe、Nao Matsubara、Kenichi Matsubara 翻译：Koki Katsabe、Akio Takenaka、Keiichi Fukuyama 和 Nao Matsubara：“蛋白质结构导论” Kyoikusha，285 (1992)