title: Evidence from chemical degradation studies for a covalent bond from 5-fluoro-2'-deoxyuridylate to N-5 of tetrahydrofolate in the ternary complex of thymidylate synthetase-5-fluoro-2'-deoxyuridylate-5,10-methylenetetrahydrofolate.
abstract: In the ternary complex of thymidylate synthetase, 5-fluoro-2'-deoxyuridylate (FdUMP), and 5,10-methylenetetrahydrofolate (5,10-CH2H4folate), the 5-fluorouracil moiety is covalently bound to the enzyme by a sulfide linkage from C-6 and to either N-5 or N-10 of H4folate by a methylene bridge from C-5. In an effort to establish the site by which H4folate is attached to FdUMP, the ternary complex was subjected to reagents that cleave the C-9, N-10 bond of folate derivatives. The complex was stable to zinc dust in hydrochloric acid, a reagent that cleaves N-10-substituted but not N-5-substituted folates. The conditions of the Bratton-Marshall reaction, which involve the use of nitrous acid, were found to cleave N-5-substituted folates in yields ranging from 5 to 50%. Exposure of the double-labeled thymidylate synthetase-FdUMP-[2-14C,7,9,3',5'-3H]5,10-CH2H4folate complex to the Bratton-Marshall reaction resulted in 16% cleavage of the C-9, N-10 bond with release solely of p-aminobenzoylglutamate, whereas all of the carbon-14-labeled pterin residue remained covalently bound to the protein. These results demonstrate that in the ternary complex, the 5-fluorouracil residue is connected by a covalent bond to N-5 of H4folate.
mesh_terms: D002250:Carbon Radioisotopes; D003124:Colorimetry / Q000379:methods; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D005468:Fluorodeoxyuridylate / Q000378:metabolism*; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008780:Methyltransferases / Q000378:metabolism*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000378:metabolism*; D014316:Tritium
chemical_list: D002250:Carbon Radioisotopes; D003856:Deoxyuracil Nucleotides; D013763:Tetrahydrofolates; C013123:5,10-methylenetetrahydrofolic acid; D014316:Tritium; D005468:Fluorodeoxyuridylate; C030371:5,6,7,8-tetrahydrofolic acid; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D008780:Methyltransferases; D013940:Thymidylate Synthase