title: Thymidylate synthetase and 2'-deoxyuridylate form a tight complex in the presence of pteroyltriglutamate.
abstract: Thymidylate synthetases of human and bacterial origin form a tightly bound complex with the substrate dUMP in the presence of pteroyltriglutamate. This complex and the weaker enzyme . dUMP binary complex can be isolated and conveniently assayed by nitrocellulose disc filtration using [6-3H]dUMP as the radioactive ligand. Intact thymidylate synthetase . dUMP . pteroyltriglutamate complex can be obtained by gel filtration chromatography on Sephadex G-25, but the binary enzyme . dUMP complex dissociates under the same conditions. Scatchard plots show the presence of two nonequivalent dUMP binding sites on the enzyme for the pteroyltriglutamate complex, with dissociation constants of 5 and 95 nM compared to 730 nM for the binary complex. The implications of these findings for folate analog inhibition of thymidylate synthetase are discussed.
mesh_terms: D002460:Cell Line; D003856:Deoxyuracil Nucleotides; D005492:Folic Acid / Q000031:analogs & derivatives; D006801:Humans; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D007938:Leukemia; D008780:Methyltransferases / Q000378:metabolism*; D011485:Protein Binding; D011624:Pteroylpolyglutamic Acids; D013940:Thymidylate Synthase / Q000378:metabolism*
chemical_list: D003856:Deoxyuracil Nucleotides; D011624:Pteroylpolyglutamic Acids; D005492:Folic Acid; C007267:2'-deoxyuridylic acid; D008780:Methyltransferases; D013940:Thymidylate Synthase