PMID:10491090
| Citation |
Okamura-Ikeda, K, Fujiwara, K and Motokawa, Y (1999) The amino-terminal region of the Escherichia coli T-protein of the glycine cleavage system is essential for proper association with H-protein. Eur. J. Biochem. 264:446-53 |
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| Abstract |
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 region 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 (ETDelta7) was purified and analyzed. ETDelta7 exhibited a marked increase in Km (25-fold) for E. coli H-protein (EH) accompanied by a 10-fold decrease in kcat 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 ETDelta7-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 ETDelta7. 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. |
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| Keywords |
Amino Acid Oxidoreductases; Amino Acid Sequence; Aminomethyltransferase; Carrier Proteins/chemistry; Cross-Linking Reagents/chemistry; Escherichia coli/enzymology; Glycine Decarboxylase Complex H-Protein; Glycine Dehydrogenase (Decarboxylating); Hydroxymethyl and Formyl Transferases/chemistry; Hydroxymethyl and Formyl Transferases/genetics; Molecular Sequence Data; Mutation; Peptide Mapping; Protein Binding; Protein Conformation; Recombinant Proteins; Sequence Analysis; Tetrahydrofolates/chemistry |
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