Energetic contributions of four arginines to phosphate-binding in thymidylate synthase are more than additive and depend on optimization of "effective charge balance"

In thymidylate synthase, four conserved arginines provide two hydrogen bond s each to the oxygens of the phosphate group of the substrate, 2'-deoxyurid ine-5'-monophosphate. Of these, R23, R178, and R179 are far removed from th e site of methyl transfer and contribute to catalysis solely through bindin g and orientation of ligands, These arginines can be substituted by other r esidues, while still retaining more than 1% activity of the wild-type enzym e. We compared the kinetics and determined the crystal structures of dUMP c omplexes of three of the most active, uncharged single mutants of these arg inines, R23I, R178T, and R179T, and of double mutants (R23I, R179T) and (R1 78T, R179T). The dramatically higher K-m for R178T compared to the other tw o single mutants arises from the effects of R178 substitution, on the orien tation of dUMP; 10-15-fold increases in k(m)(CH2H4folate) for R23I and R178 T reflect the role of these residues in stabilizing the closed conformation of TS in ternary complexes. The free energy for productive dUMP binding, D elta Gs, increases by at least 1 kcal/mol for each mutant, even when dUMP o rientation and mobility in the crystal structure is the same as in wild-typ e enzyme. Thus, the four arginines do not contribute excess positive charge to the PO4-2 binding site; rather, they ideally complement the charge and geometry of the phosphate moiety. More-than-additive increases in Delta Gs seen in the double mutants are consistent with quadratic increases in Delta Gs predicted for deviations from ideal electrostatic interactions and may also reflect cooperative binding of the arginines to the phosphate oxygens.