Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase

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, reflec ting the direct role of the R23 side chain in binding and orienting the cof actor in ternary complexes of the enzyme. Mutations of R178 increase K-m fo r dUMP 40-2000-fold, increase K-m for cofactor 3-20-fold and do not signifi cantly 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 p arameters for all R179 mutations except R179P were not significantly differ ent from wildtype values, reflecting the fact that this external arginine d oes not directly contact the cofactor or other ligand-binding residues. R21 8 is essential for the structure of tbe catalytic site and all mutations of this arginine except R218K were inactive.