PMID:10364177
| Citation |
Okamura-Ikeda, K, Fujiwara, K and Motokawa, Y (1999) Identification of the folate binding sites on the Escherichia coli T-protein of the glycine cleavage system. J. Biol. Chem. 274:17471-7 |
|---|---|
| Abstract |
T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. To determine the folate-binding site on the enzyme, 14C-labeled methylenetetrahydropteroyltetraglutamate (5,10-CH2-H4PteGlu4) was enzymatically synthesized from methylenetetrahydrofolate (5, 10-CH2-H4folate) and [U-14C]glutamic acid and subjected to cross-linking with the recombinant Escherichia coli T-protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker between amino and carboxyl groups. The cross-linked product was digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase high performance liquid chromatography. Amino acid sequencing of the labeled peptides revealed that three lysine residues at positions 78, 81, and 352 were involved in the cross-linking with polyglutamate moiety of 5, 10-CH2-H4PteGlu4. The comparable experiment with 5,10-CH2-H4folate revealed that Lys-81 and Lys-352 were also involved in cross-linking with the monoglutamate form. Mutants with single or multiple replacement(s) of these lysine residues to glutamic acid were constructed by site-directed mutagenesis and subjected to kinetic analysis. The single mutation of Lys-352 caused similar increase (2-fold) in Km values for both folate substrates, but that of Lys-81 affected greatly the Km value for 5,10-CH2-H4PteGlu4 rather than for 5,10-CH2-H4folate. It is postulated that Lys-352 may serve as the primary binding site to alpha-carboxyl group of the first glutamate residue nearest the p-aminobenzoic acid ring of 5,10-CH2-H4folate and 5,10-CH2-H4PteGlu4, whereas Lys-81 may play a key role to hold the second glutamate residue through binding to alpha-carboxyl group of the second glutamate residue. |
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| Keywords |
Amino Acid Sequence; Aminomethyltransferase; Binding Sites; Cross-Linking Reagents; Escherichia coli/enzymology; Ethyldimethylaminopropyl Carbodiimide/analogs & derivatives; Ethyldimethylaminopropyl Carbodiimide/chemistry; Hydroxymethyl and Formyl Transferases/chemistry; Hydroxymethyl and Formyl Transferases/genetics; Kinetics; Metalloendopeptidases; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Peptide Fragments/analysis; Pteroylpolyglutamic Acids/chemistry; Sequence Alignment |
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