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

1.将重组大肠杆菌的t蛋白（ET）和h蛋白（EH）与NH_2和COOH的零长度交联剂1-乙基-3-（3-二甲基氨基丙基）碳二亚胺（EDC）交联，得到由ET和EH各1分子组成的共价结合产物。其赖基肽的HPLC图谱显示，ET与EH之间不仅存在分子间交联，而且在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 （ETDELTA7）对EH的亲和力显著降低，也与EH形成交联产物。然而，未观察到ETDELTA7的分子内交联。这些结果表明ET的n端区域对其功能构象的贡献。以亚甲基四氢叶酸和^<14> c -谷氨酸为原料，通过EDC与ET交联，酶促合成了t蛋白的一种生理叶酸底物-甲基四氢丙基四谷氨酸（CH_2-H_4PteGlu_4）。产物由ET和CH_2-H_4PteGlu_4各1个分子组成。HPLC图谱和氨基酸序列分析显示，CH_2-H_4PteGlu_4.3的聚谷氨酸尾与赖氨酸交联的氨基酸残基分别为赖氨酸78、81和352。Lys-352在目前已确定的7种不同物种的t蛋白中保守，通过定点诱变被谷氨酸、谷氨酰胺和精氨酸取代。这些突变体经过过表达、纯化和鉴定。它们对CH_2-H_4PteGlu_4的比活性和Km值与野生型et相似，78和81赖氨酸残基的突变正在进行中。在非酮症高血糖症患者中发现的正常和突变的人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

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