Thymidylate synthase (TSase) catalyzes the intracellular de novo formation of thymidylate (a DNA building block) in most living organisms, making it a common target for chemotherapeutic and antibiotic drugs. Two mechanisms have been proposed for the rate-limiting hydride transfer step in TSase catalysis: a stepwise mechanism in which the hydride transfer precedes the cleavage of the covalent bond between the enzymatic cysteine and the product and a mechanism where both happen concertedly. Striking similarities between the enzyme-bound enolate intermediates formed in the initial and final step of the reaction supported the first mechanism, while QM/MM calculations favored the concerted mechanism. Here, we experimentally test these two possibilities using secondary kinetic isotope effect (KIE), mutagenesis study, and primary KIEs. The findings support the concerted mechanism and demonstrate the critical role of an active site arginine in substrate binding, activation of enzymatic nucleophile, and the hydride transfer studied here. The elucidation of this reduction/substitution sheds light on the critical catalytic step in TSase and may aid future drug or biomimetic catalyst design.

Thymidylate Synthase (TSase) is a highly conserved enzyme that catalyzes the production of the DNA building block thymidylate. Structurally, functionally and mechanistically, bacterial and mammalian TSases share remarkable similarities. Because of this closeness, bacterial enzymes have long been used as model systems for human TSase. Furthermore, while TSase inhibitors have long served as chemotherapeutic drugs, no TSase inhibitor serves as an antibiotic. Despite their high resemblance, the mammalian TSases are distinct in a few known aspects, such as having a N-terminal tail and two insertions in the primary sequence and active/inactive conformations. Here, we aim to comprehensively characterize human (hs) TSase and delineate its contrasts and the similarities to the well-studied Escherichia coli (ec) TSase. We found that, in contrast to ecTSase, Mg2+ does not enhance reaction rates for hsTSase. The temperature dependence of intrinsic kinetic isotope effects (KIEs), on the other hand, suggests that Mg2+ has little or no impact on the transition state of hydride transfer in either enzyme, and that the transition state for the hydride transfer in hsTSase is looser than in ecTSase. Additionally, the substrates’ binding order is strictly ordered for ecTSase but slightly less ordered for hsTSase. The observed kinetic and functional differences between bacterial and human enzymes may aid in the development of antibiotic drugs with reduced toxicity.

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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.