Amino acid sequence at the FdUMP binding site of thymidylate synthetase.

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Abstract
CNBr treatment of thymidylate synthetase of Lactobacillus casei, which was converted to a ternary complex with [2-14C]Fd[5-fluorodeoxy]UMP and 5,10-methylenetetrahydrofolate followed by S-carboxymethylation, yielded at least 4 visible peptide bands, the largest with a MW of .apprx. 13,000 on polyacrylamide gel electrophoresis in sodium dodecyl sulfate-urea. Identical results were obtained with enzyme that had all 4 of its cysteinyl residues S-carboxymethylated with iodo[1-14C]acetate in the absence of FdUMP and cofactor. In each case only the 2nd band from the top of the gel (CN2), with an approximate MW of 10,000, was labeled. Analysis of CN2 that was labeled with [2-14C]FdUMP and nonradioactive iodoacetate and of that labeled only with iodo[1-14C]acetate revealed that their amino-acid contents were almost identical except for the presence of 2 S-carboxymethyl(Cm)-cysteinyl residues in the latter peptide and only 1 in FdUMP-CN2. A nonapeptide was isolated from (Cm)2-CN2 after chymotrypsin digestion that contained the following sequence by dansyl-Edman analysis: Ala-Leu-Pro-Pro-[Cm-Cys]-His-Thr-Leu-Tyr. This peptide was located on the NH2-terminal end of CN2. Automatic sequence analysis of the first 13 residues of (Cm)2-CN2 and of the FdUMP-containing CN2 yielded identical results except for the 5th, or cysteinyl, residue, which could not be identified in the latter peptide. These findings strongly suggest that FdUMP is linked to a cysteinyl residue in thymidylate synthetase that has been inactivated irreversibly by this nucleotide.