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

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.