title: Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase.
abstract: Arginines R23, R178, R179 and R218 in thymidylate synthase (TS, EC 2. 1.1.45) are hydrogen bond donors to the phosphate moiety of the substrate, dUMP. In order to investigate how these arginines contribute to enzyme function, we prepared complete replacement sets of mutants at each of the four sites in Lactobacillus casei TS. Mutations of R23 increase K:(m) for dUMP 2-20-fold, increase K:(m) for cofactor 8-40-fold and decrease k(cat) 9-20-fold, reflecting the direct role of the R23 side chain in binding and orienting the cofactor in ternary complexes of the enzyme. Mutations of R178 increase K:(m) for dUMP 40-2000-fold, increase K:(m) for cofactor 3-20-fold and do not significantly affect k(cat). These results are consistent with the fact that this residue is an integral part of the dUMP-binding wall and contributes to the orientation and ordering of several other dUMP binding residues. Kinetic parameters for all R179 mutations except R179P were not significantly different from wild-type values, reflecting the fact that this external arginine does not directly contact the cofactor or other ligand-binding residues. R218 is essential for the structure of the catalytic site and all mutations of this arginine except R218K were inactive.
authors.affiliation: Department of Biochemistry and Biophysics and Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94143-0448, USA.
authors.forename: S
authors.initials: S
authors.lastname: Kawase
authors.forename: S W
authors.initials: SW
authors.lastname: Cho
authors.forename: J
authors.initials: J
authors.lastname: Rozelle
authors.forename: R M
authors.initials: RM
authors.lastname: Stroud
authors.forename: J
authors.initials: J
authors.lastname: Finer-Moore
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
mesh_terms: D001120:Arginine / Q000378:metabolism*; D005816:Genetic Complementation Test; D007700:Kinetics; D016254:Mutagenesis, Insertional; D010710:Phosphates / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010710:Phosphates; D001120:Arginine; D013940:Thymidylate Synthase