Amino acid sequence at the FdUMP binding site of thymidylate synthetase.
Cyanogen bromide treatment of thymidylate synthetase of Lactobacillus casei, which had been converted to a ternary complex with [2-14c] FdUMP and 5,10-methylene-tetrahydrofolate followed by S-carboxymethylation, yielded at least four visible peptide bands, the largest with a molecular weight of about 13,000, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate-urea. Identical results were obtained with enzyme that had all four of its cysteinyl residues S-carboxymethylated with iodo [I-14C] acetate in the absence of FdUMP and cofactor. In each case, only the second band from the top of the gel (CN2), with an approximate molecular weight of 10,000= was labeled. Analysis of CN2 that had been 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 two S-carboxymethyl (Cm)-cysteinyl residues in the latter peptide and only one 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 found to be 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 fifth, 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.
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001665:Binding Sites; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D003545:Cysteine; D008780:Methyltransferases / Q000378:metabolism; D013940:Thymidylate Synthase / Q000378:metabolism; D014500:Uracil Nucleotides / Q000378:metabolism
D000345:Affinity Labels; D014500:Uracil Nucleotides; D008780:Methyltransferases; D013940:Thymidylate Synthase; D002918:Chymotrypsin; D003545:Cysteine; D003488:Cyanogen Bromide