Identification of the folate binding sites on the Escherichia coli T- protein of the glycine cleavage system

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 K(m) values for both folate substrates, but that of Lys-81 affected greatly the K(m) 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 α-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 α-carboxyl group of the second glutamate residue.