S. Kawase et al., Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase, PROTEIN ENG, 13(8), 2000, pp. 557-563
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.