STUDIES ON THE MOLECULAR STRUCTURE AND FUNCTION OF THE GLYCINE CLEAVAGE SYSTEM

甘氨酸裂解体系的分子结构和功能研究

• 批准号: 07670146
• 负责人: OKOMURA Kazuko
• 金额: $ 1.47万
• 依托单位: THE UNIVERSITY OF TOKUSHIMA
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670146/
• 关键词: GLYCINE CLEAVAGE SYSTEM; T-PROTEIN; H-PROTEIN; NONKETOTIC HYPERGLYCINEMIA; METHYLENETETRAHYDROPTEROYLTETRAGLUTAMATE; CROSS-LINKING; methylenetetrahydropteroyltetraglutamate; methylenetetrahydrofolate; methylenetetrahydropteroylpolyglutamate

1.Cross-linking of the recombinant E.coli T-protein (ET) and H-protein (EH) of the glysine cleavage system with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), a zero-length cross-linker of NH_2 and COOH,produced covalently bound product consisted of one moleculef eachof ET and EH.HPLC mapping of its lysylpeptides showed not only intermolecular cross-linking between ET and EH,but also intramolecular cross-linking in ET.The latter cross-linking was EH-dependent but not-dependent folate. The ET with 7 amino acid deletion in N-terminus (ETDELTA7) which showed a remarkably reduced affinity for EH compared with wild-type ET also formed a cross-linked product with EH.However the intramolecular cross-linking in ETDELTA7 was not observed. These results suggest the contribution of the N-terminal region of ET to the functional conformation.2. ^<14>C-labeled methylenetetrahydropteroyltetraglutamate (CH_2-H_4PteGlu_4), a physiological folate substrate of T-protein, was enzymatically synthes … More ized from methylenetetrahydrofolate and ^<14>C-glutamic acid, and subjected to cross-linking with ET using EDC.The product also consisted of one molecule each of ET and CH_2-H_4PteGlu_4. HPLC mapping and amino acid sequence of its lysylpeptides revealed that three lysine residues, Lys-78, Lys-81 and Lys-352 were involved in cross-linking with polyglutamate tail of CH_2-H_4PteGlu_4.3. The Lys-352, which is conserved in T-proteins from seven different species so far determined, was replaced by glutamate, glutamine, and arginine by site-directed mutagenesis. The mutants were overexpressed, purified, and characterized. All of them showed similar specific activity and Km values for CH_2-H_4PteGlu_4 to that of wild-type ET.The mutations of the lysine residue at 78 and 81 are now in progress.4. Overexpression of normal and mutant human T-proteins with the point mutations identified in nonketotic hyperglycinemia patients are also underway to elucidate the relationship between the structure and the function of T-protein. Less

1.用1-乙基-3-甲基-1，3-二氧杂环己烷交联甘氨酸裂解系统的重组大肠杆菌T-蛋白（ET）和H-蛋白（EH）。NH_2和COOH的零长交联剂（3-二甲基氨基丙基）碳二亚胺（EDC）可使ET和EH形成一个分子的共价结合物，其赖氨酸肽的HPLC图谱显示ET和EH之间不仅存在分子间交联，但在ET中也存在分子内交联，后者的交联依赖于EH，而不依赖于叶酸。与野生型ET相比，N端缺失7个氨基酸的ET（ETDELTA 7）与EH的亲和力明显降低，但ETDELTA 7与EH形成交联产物，但未观察到分子内交联。这些结果表明ET的N端区域对功能构象的贡献.用<14>酶法合成了T蛋白的生理叶酸底物--~（13）C-亚甲基四氢蝶酰四谷氨酸（CH_2-H_4PteGlu_4）。 ...更多信息 由亚甲基四氢叶酸和~ 4C<14>-谷氨酸制备的β-氨基葡萄糖醛酸酯，经EDC与ET交联，产物也由ET和CH_2-H_4PteGlu_4各一分子组成。HPLC图谱分析和氨基酸序列分析表明，CH_2-H_4PteGlu_4的赖氨酸残基有三个，即Lys-78、Lys-81和Lys-352，它们与聚谷氨酸的尾部交联。赖氨酸-352，这是保守的T-蛋白质从7个不同的物种，到目前为止确定，取代谷氨酸，谷氨酰胺和精氨酸的定点诱变。突变体过表达，纯化，并进行了表征。它们对CH_2-H_4PteGlu_4的比活性和Km值均与野生型ET相似。78和81位赖氨酸残基的突变正在进行中。在非酮症高甘氨酸血症患者中鉴定的点突变的正常和突变的人T蛋白的过表达也正在进行中，以阐明T蛋白的结构和功能之间的关系。少

项目成果

池田 和子: "グリシン開裂酵素系T蛋白質のfolate結合部位" 生化学. 68,7. 1227 (1996)

Kazuko Ikeda：“甘氨酸裂解酶 T 蛋白的叶酸结合位点”生物化学 68,7 (1996)。

池田和子: "グリシン開裂酵素系T蛋白質とH蛋白質の架橋" 生化学. 67. 949- (1995)

Kazuko Ikeda：“甘氨酸裂解酶系统 T 蛋白和 H 蛋白的交联”生物化学 67. 949- (1995)。

池田和子: "グリシン開裂酵素系T蛋白質のfolate結合部位" 生化学. 68. 1227- (1996)

Kazuko Ikeda：“甘氨酸裂解酶系统 T 蛋白的叶酸结合位点”生物化学 68. 1227- (1996)。