Evidence for the existence of covalent nucleotide-thymidylate synthase complexes, identification of site of attachment, and enhancement by folates.
The formation of covalent binary complexes of thymidylate synthase and its nucleotide substrate dUMP, product dTMP, and inhibitor, 5-fluorodeoxyuridylate (FdUMP) was investigated using the trichloroacetic acid precipitation method. It was observed that, in addition to FdUMP, both dUMP and dTMP were capable of covalent interactions with the enzyme in the absence of added folates. The presence of folate, dihydrofolate, or tetrahydrofolate (H4folate) was found to produce substantial enhancements in the covalent binding of both FdUMP and dUMP to the enzyme with H4folate being the most effective agent. Further, covalent binary complexes of the enzyme with the three radiolabeled nucleotides were isolated by trichloroacetic acid precipitation and subjected to CNBr cleavage. The active-site CNBr peptide was isolated by reverse phase high performance liquid chromatography, and the first five N-terminal amino acid residues were sequenced by the dansyl-Edman procedure. Each active site peptide obtained from the covalent binary complexes as well as that from the covalent inhibitory ternary complex formed from enzyme, FdUMP, and 5,10-methylene-H4folate exhibited an identical sequence of Ala-Leu-Pro-Pro-(X)-, and the 5th amino acid was found to be associated with radiolabeled nucleotide ligand. Dansyl-Edman sequence analysis of the active site CNBr peptide, derived from enzyme which had been treated with iodoacetic acid, gave a sequence of Ala-Leu-Pro-Pro-CmCys (where CmCys is carboxymethylcysteine), thus confirming the fact that the fifth residue from the N terminus is Cys-198. In all the cases, the active site Cys-198 residue was found to be covalently linked to the nucleotides. These results provide unequivocal proof that the covalent binary complexes of enzyme with dUMP and dTMP predicted in the catalytic reaction mechanism actually exist.
D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D005468:Fluorodeoxyuridylate / Q000494:pharmacology; D005492:Folic Acid / Q000031:analogs & derivatives / Q000494:pharmacology*; D013763:Tetrahydrofolates / Q000494:pharmacology; D013938:Thymidine Monophosphate / Q000378:metabolism; D013940:Thymidylate Synthase / Q000378:metabolism*
D003856:Deoxyuracil Nucleotides; D013763:Tetrahydrofolates; D005468:Fluorodeoxyuridylate; D013938:Thymidine Monophosphate; C010920:dihydrofolate; C030371:5,6,7,8-tetrahydrofolic acid; D005492:Folic Acid; C007267:2'-deoxyuridylic acid; D013940:Thymidylate Synthase