Thymidylate synthase (TSase), an enzyme responsible for the de novo biosynthesis of 2'-deoxythymidine 5'-monophosphate (thymidylate, dTMP) necessary for DNA synthesis, has been a drug target for decades. TSase is a highly conserved enzyme across species ranging from very primitive organisms to mammals. Among the many conserved active site residues, an asparagine (N177, using Escherichia coli residues numbering) appears to make direct hydrogen bonds with both the C4=O4 carbonyl of the 2'-deoxyuridine 5'-monophosphate (uridylate, dUMP) substrate and its pyrimidine ring's N3. Recent studies have reassessed the TSase catalytic mechanism, focusing on the degree of negative charge accumulation at the O4 carbonyl of the substrate during two critical H-transfers - a proton abstraction and a hydride transfer. To obtain insights into the role of this conserved N177 on the hydride transfer, we examined its aspartic acid (D) and serine (S) mutants - each of which is expected to alter hydrogen bonding and charge stabilization around the C4=O4 carbonyl of the 2'-deoxyuridine 5'-monophosphate (uridylate, dUMP) substrate. Steady-state kinetics, substrate binding order studies and temperature-dependency analysis of intrinsic KIEs for the hydride transfer step of the TSase catalytic cycle suggest the active site of N177D is not precisely organized for that step. A smaller disruption was observed for N177S, which could be rationalized by partial compensation by water molecules and rearrangement of other residues toward preparation of the system for the hydride transfer under study. These experimental findings are qualitatively mirrored by QM/MM computational simulations, thereby shedding light on the sequence and synchronicity of steps in the TSase-catalyzed reaction. This information could potentially inform the design of mechanism-based drugs targeting this enzyme.
Keywords: Free energy surfaces; Kinetic Isotope Effects; QM/MM calculations; Steady-state kinetics; Thymidylate synthase; temperature-dependency KIEs.