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

Research Project

Project/Area Number	07670146
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	General medical chemistry
Research Institution	THE UNIVERSITY OF TOKUSHIMA
Principal Investigator	OKOMURA Kazuko THE UNIVERSITY OF TOKUSHIMA,INSITUTE FOR ENZYME RESEARCH,RESEARCH ASSOCIATE, 酵素科学研究センター, 助手 (10108863)
Project Period (FY)	1995 – 1996
Project Status	Completed (Fiscal Year 1996)
Budget Amount *help	¥2,300,000 (Direct Cost: ¥2,300,000) Fiscal Year 1996: ¥600,000 (Direct Cost: ¥600,000) Fiscal Year 1995: ¥1,700,000 (Direct Cost: ¥1,700,000)
Keywords	GLYCINE CLEAVAGE SYSTEM / T-PROTEIN / H-PROTEIN / NONKETOTIC HYPERGLYCINEMIA / METHYLENETETRAHYDROPTEROYLTETRAGLUTAMATE / CROSS-LINKING / methylenetetrahydropteroyltetraglutamate / methylenetetrahydrofolate / methylenetetrahydropteroylpolyglutamate
Research Abstract	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