Trapping and partial characterization of an adduct postulated to be the covalent catalytic ternary complex of thymidylate synthetase

The proposed mechanism of action of thymidylate synthetase envisages the formation of a covalent ternary complex of the enzyme via the active site cysteine with dUMP and 5,10-methylenetetrahydrofolate (CH2H4folate). The authors recent success in using trichloroacetic acid to trap the covalent enzyme-FdUMP binary and ternary (enzyme-FdUMP-CH2H4folate) complexes led to the use of this technique in attempts to trap the transient covalent catalytic ternary complex. Experiments performed with [2-C14]dUMP and 3H-CH2H4folate show that both these ligands remained bound to the enzyme after trichloroacetic acid precipitation. The trapped covalent catalytic ternary complex was subjected to CNBr fragmentation, and the peptides were fractionated by HPLC. The isolated active-site peptide was shown to retain the two ligands and was subjected to a limited sequence analysis by the dansyl-Edman procedure. The inhibitory ternary complex formed with 14C-FdUMP and 3H-CH24folate served as a control. The active-site peptides isolated from the CNBr treated inhibitory ternary complex and the catalytic complex exhibited identical sequences for the first four N-terminal residues, Ala-Leu-Pro-Pro, and the fifth residue was found to be associated with the labeled ligands. Sequence analysis of the active site peptide derived from the carboxymethylated enzyme confirmed this sequence and the 5th residue was shown to be Cm-Cys