title: Heterodimeric thymidylate synthases with C-terminal deletion on one subunit.
abstract: We have combined site-directed mutagenesis with the technique of reversible unfolding and subunit dissociation to construct heterodimeric thymidylate synthases that lack the C-terminal valine from only one subunit of the dimer. Removal of this residue either from both subunits of the dimer by mutagenesis (V316Am mutation) or from only one subunit by treatment with carboxypeptidase has been reported to result in an inactive enzyme (Carreras, C. W., Climie, S. C., and Santi, D. V. (1992) Biochemistry 31, 6038-6044; Aull, J.L., Loeble, R.B., and Dunlap. R.B. (1974) J. Biol. Chem. 249, 1167-1172). Arg-178 is an essential active site residue of thymidylate synthase that is donated from the opposing subunit of the dimer. The R178F-V316Am heterodimer was formed by the unfolding and refolding of a mixture of inactive R178F and V316Am mutants. This enzyme has one intact active site and was found to have half of the activity and the same Km values as wild-type thymidylate synthase that was unfolded and refolded as a control. We have also formed the V316Am-WT heterodimer and report that this heterodimeric enzyme is also active, has a kcat value that is approximately half of that of the wild-type thymidylate synthase dimer, and binds substrate and cofactor with Km values similar to those of the wild-type enzyme.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0448.
authors.forename: C W
authors.initials: CW
authors.lastname: Carreras
authors.forename: P M
authors.initials: PM
authors.lastname: Costi
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D007780:Lacticaseibacillus casei / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017384:Sequence Deletion; D013940:Thymidylate Synthase / Q000235:genetics / Q000378:metabolism*
chemical_list: D013940:Thymidylate Synthase