The amino-terminal region of the Escherichia coli T-protein of the glycine cleavage system is essential for proper association with H-protein

被引:15
|
作者
Okamura-Ikeda, K [1 ]
Fujiwara, K [1 ]
Motokawa, Y [1 ]
机构
[1] Univ Tokushima, Inst Enzyme Res, Tokushima 7708503, Japan
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1999年 / 264卷 / 02期
关键词
glycine cleavage system; T-protein; cross-linking; protein interaction;
D O I
10.1046/j.1432-1327.1999.00637.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. Our previous work on Escherichia coli T-protein (ET) showed that the lack of the N-terminal 16 residues caused a loss of catalytic activity [Okamura-Ikeda, K., Ohmura, Y., Fujiwara, K. and Motokawa, Y. (1993) Eur: J. Biochem. 216, 539-548]. To define the role of the N-terminal legion of ET, a series of deletion mutants were constructed by site-directed mutagenesis and expressed in E. coli. Deletions of the N-terminal 4, 7 and 11 residues led to reduction in the activity to 42, 9 and 4%, respectively, relative to the wild-type enzyme (wtET). The mutant with 7-residue deletion (ET Delta 7) was purified and analyzed. ET Delta 7 exhibited a marked increase in K-m (25-fold) for E. coli H-protein (EH) accompanied by a 10-fold decrease in k(cat) compared with wtET, indicating the importance of the N-terminal region in the interaction with EH. The role of this region in the ET-EH interaction was investigated by cross-linking of wtET-EH or ET Delta 7-EH complex with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker, in the presence of folate substrates. The resulting tripartite cross-linked products were cleaved with lysylendopeptidase and V8 protease. After purification by reversed-phase HPLC, the cross-linked peptides were subjected to Edman sequencing. An intramolecular cross-linking between Asp34 and Lys216 of wtET which was not observed in wtET alone and an intermolecular cross-linking between Lys288 of wtET and Asp-43 of EH were identified. In contrast, no such cross-linking was detected from the cross-linked product of ET Delta 7. These results suggest that EH. when it interacts with ET. causes a change in conformation of ET and that the N-terminal region of ET is essential for the conformational change leading to the proper interaction with EH.
引用
收藏
页码:446 / 452
页数:7
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