Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.1 (chymotrypsin)
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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.
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PMID:Amino acid sequence at the FdUMP binding site of thymidylate synthetase. 106 57

Formiminotransferase-cyclodeaminase, an octameric protein of identical, bifunctional polypeptides of Mr = 62,000, yields a transferase-active fragment of Mr = 80,000 upon proteolysis with chymotrypsin in the presence of the inhibitor folic acid. The purified fragment contains one size of polypeptide, Mr = 39,000, on dodecyl sulfate gels. Cross-linking with the bifunctional reagent dithiobis(succinimidyl propionate) confirmed the dimeric structure of the purified fragment. Reaction of the native octamer with the very short bifunctional reagent difluorodinitrobenzene yields dimer and tetramer in excess of trimer, thereby indicating two types of subunit interaction in the protein. The isolation of a dimeric fragment after proteolysis and the results of cross-linking support a tetramer of dimers structure for the native enzyme. The purified transferase fragment has approximately 68% of the activity of the native enzyme, but has lost specificity for the naturally occurring polyglutamate derivatives of tetrahydrofolate. This is illustrated by an increase in Km for tetrahydropteroylpentaglutamate from 3.4 microM with the native transferase to 89 microM with the fragment transferase. It is suggested that the bifunctional enzyme may have only one polyglutamate binding site/pair of transferase-deaminase sites.
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PMID:The bifunctional enzyme formiminotransferase-cyclodeaminase is a tetramer of dimers. 741 Apr 36