Activation of Two Sequential H-transfers in the Thymidylate Synthase Catalyzed Reaction

Abstract

Thymidylate synthase (TSase) catalyzes the de novo biosynthesis of thymidylate, a precursor for DNA, and is thus an important target for chemotherapeutics and antibiotics. Two sequential C-H bond cleavages catalyzed by TSase are of particular interest: a reversible proton abstraction from the 2'-deoxy-uridylate substrate, followed by an irreversible hydride transfer forming the thymidylate product. QM/MM calculations of the former predicted a mechanism where the abstraction of the proton leads to formation of a novel nucleotide-folate intermediate that is not covalently bound to the enzyme (Wang, Z.; Ferrer, S.; Moliner, V.; Kohen, A. Biochemistry2013, 52, 2348-2358). Existence of such intermediate would hold promise as a target for a new class of drugs. Calculations of the subsequent hydride transfer predicted a concerted H-transfer and elimination of the enzymatic cysteine (Kanaan, N.; Ferrer, S.; Marti, S.; Garcia-Viloca, M.; Kohen, A.; Moliner, V. J. Am. Chem. Soc.2011, 133, 6692-6702). A key to both C-H activations is a highly conserved arginine (R166) that stabilizes the transition state of both H-transfers. Here we test these predictions by studying the R166 to lysine mutant of E. coli TSase (R166K) using intrinsic kinetic isotope effects (KIEs) and their temperature dependence to assess effects of the mutation on both chemical steps. The findings confirmed the predictions made by the QM/MM calculations, implicate R166 as an integral component of both reaction coordinates, and thus provide critical support to the nucleotide-folate intermediate as a new target for rational drug design.

Keywords: C-H bond activation; Donor and acceptor distances; Kinetic Isotope Effect; Phenomenological models; QM/MM calculations; Thymidylate Synthase; Tunneling ready state.

Figure 1

Phenomenological models for activated H-tunneling. Left column represents three stages of the reaction along the H-transfer coordinate. Motion of the protein, solvent, and reactants modulate the potential energy surfaces for the H-transfer. At the reactant state (A), the H-wavefunction is localized in the donor well. The motion of the heavy atoms transiently brings the donor and the acceptor wells into the tunneling ready state (TRS, B), where isotopically sensitive H-transfer from donor to acceptor occurs. Further rearrangement in the heavy atoms interrupts the TRS, resulting in entrapment of the hydrogen in the product well (C). The right column demonstrates the contributing factors that modulate H-transfer probability at the TRS. The panel B1 shows the transmission probabilities of light (l, green) and heavy (h, purple) hydrogen isotopes as a function of DAD. Panel B2 represents a potential energy surface (PES) for the DAD fluctuations. Panel B3 shows the product of DADs and transmission probabilities.

Figure 2

Arrhenius plots of intrinsic H/T KIEs on the proton abstraction (PA, red) and the hydride transfer (HT, blue)^, for the WT (Squares) and R166K (Diamonds) TSase.

Figure 3

Illustration of potential energy surfaces (PESs) along the donor-acceptor distance (DAD) coordinate in TRS for WT (red) and R166K (blue) TSase catalyzed proton abstraction (PA) and hydride transfer (HT) reactions. The relative changes in the distribution of DADs are demonstrated.

Figure 4

Presentation of the interrelation between isotope effects on activation parameters in the two H-transfers catalyzed by the WT and R166K TSases.

Figure 5

Active site structure of TSase covalently bound with 5-fluoro dUMP and CH₂H₄folate (PDB ID 1TLS). The ligands (dUMP, CH₂H₄folate) and residues close to H-transfers’ site are shown in sticks, and atoms that were predicted to interact during the catalytic cycle are connected by dashed lines.

Scheme 1

The principle mechanism of TSase.

Scheme 2

Traditional (A) and calculated^, (B) mechanisms for the proton abstraction and the hydride transfer in TSase. In the QM/MM calculations, the abstraction of C5_U proton induces the cleavage of S_C146-C6_U, leading to the formation of the nucleotide-folate intermediate. Following the protonation of the N5 of folate, the departed C146 then re-attacks the nucleotide-folate intermediate, assisting in the succeeding β-elimination of the cofactor, generating the exocyclic methylene intermediate. Then, the concerted hydride transfer and elimination of C146 leads to the product dTMP.