Identification of the folate binding sites on the Escherichia coli T-protein of the glycine cleavage system.
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.
Okamura-Ikeda
K
K
Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan.
Fujiwara
K
K
Motokawa
Y
Y
D000595:Amino Acid Sequence; D050960:Aminomethyltransferase; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000201:enzymology*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000031:analogs & derivatives / Q000737:chemistry; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments / Q000032:analysis; D011624:Pteroylpolyglutamic Acids / Q000737:chemistry*; D016415:Sequence Alignment
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D011624:Pteroylpolyglutamic Acids; C024898:methylenetetrahydropteroylpolyglutamate; C041829:1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide methiodide; D019877:Hydroxymethyl and Formyl Transferases; D050960:Aminomethyltransferase; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase; D005022:Ethyldimethylaminopropyl Carbodiimide