Thymidylate synthase is an important chemotherapeutic drug target that utilizes N⁵,N¹⁰-methylene-5,6,7,8-tetrahydrofolate (CH₂H₄folate) to catalyze the formation of 2'-deoxythymidine-5'-monophosphate (dTMP) using 2'-deoxyuridine-5'-monophosphate (dUMP) as a substrate. The product of interest in this reaction is very important for the synthesis of DNA and cellular division, because it is the precursor of one of the DNA’s building blocks: thymidine. Several studies propose that the role of the residue E58 in E. coli is to coordinate a charge stabilizing hydrogen bond network in the active site of thymidylate synthase for proton transfer step, the promotion of ring opening of CH₂H₄ folate, and stabilization of cationic iminium intermediate of the cofactor. In this study, we changed both the charge (E58Q) and length (E58D) of this important active site residue. We investigated the impact of the mutation on catalysis using initial velocity and kinetic isotope effect studies. Improved understanding of the role of this residue might unveil hidden catalytic steps in the reaction and could potentially be instrumental for the development of mechanism based inhibitor.