Trapping and partial characterization of an adduct postulated to be the covalent catalytic ternary complex of thymidylate synthase.
The proposed mechanism of action of thymidylate synthase envisages the formation of a covalent ternary complex of the enzyme with the substrate dUMP and the cofactor 5,10-methylenetetrahydrofolate (CH2H4folate). The proposed structure of this adduct has been based by analogy on that of the covalent inhibitory ternary complex thymidylate synthase-FdUMP-CH2H4folate. Our recent success in using the protein precipitant trichloroacetic acid to trap the latter complex and covalent binary complexes of the enzyme with FdUMP, dUMP, and dTMP led to the use of this technique in attempts to trap the transient putative covalent catalytic ternary complex. Experiments performed with [2-14C]dUMP and [3',5',7,9-3H]CH2H4folate show that both the substrate and the cofactor remained bound to the protein after precipitation with trichloroacetic acid. The trapped putative covalent catalytic complex was subjected to CNBr fragmentation, and the resulting peptides were fractionated by reverse-phase high-pressure liquid chromatography. The isolated active site peptide was shown to retain the two ligands and was further characterized by a limited sequence analysis using the dansyl Edman procedure. The inhibitory ternary complex, which was formed with [14C]FdUMP and [3H]CH2H4folate, served as a control. The active site peptide isolated from the CNBr-treated inhibitory ternary complex was also subjected to sequence analysis. The two peptides exhibited identical sequences for the first four residues from the N-terminus, Ala-Leu-Pro-Pro, and the fifth amino acid residue was found to be associated with the labeled nucleotides and the cofactor.(ABSTRACT TRUNCATED AT 250 WORDS)
D002250:Carbon Radioisotopes; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D013940:Thymidylate Synthase / Q000378:metabolism*; D014316:Tritium
D002250:Carbon Radioisotopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014316:Tritium; D013940:Thymidylate Synthase; D003488:Cyanogen Bromide