Quantum Mechanical Analysis of Nonenzymatic Nucleotidyl Transfer Reactions: Kinetic and Thermodynamic Effects of β–γ Bridging Groups of dNTP Substrates

Abstract

Rate (k) and equilibrium (K) constants for the reaction of tetrahydrofuranol with a series of Mg2+ complexes of methyl triphosphate analogues, CH3O-P(O2)-O-P(O2)-X-PO34–, X = O, CH2, CHCH3, C(CH3)2, CFCH3, CHF, CHCl, CHBr, CFCl, CF2, CCl2, and CBr2, forming phosphate diester and pyrophosphate or bisphosphonate in aqueous solution were evaluated by B3LYP/TZVP//HF/6-31G* quantum chemical calculations and Langevin dipoles and polarized continuum solvation models. The calculated log k and log K values were found to depend linearly on the experimental pKa4 of the conjugate acid of the corresponding pyrophosphate or bisphosphonate leaving group. The calculated slopes of these Brønsted linear free energy relationships were βlg = −0.89 and βeq = −0.93, respectively. The studied compounds also followed the linear relationship Δlog k = 0.8Δlog K, which became less steep, Δlog k = 0.6Δlog K, after the range of studied compounds was extended to include analogues that were doubly protonated on γ-phosphate, CH3O-P(O2)-O-P(O2)-X-PO3H22–. The scissile Pα–Olg bond length in studied methyl triphosphate analogues slightly increases with decreasing pKa of the leaving group; concomitantly, the CH3OPα(O2) moiety becomes more positive. These structural effects indicate that substituents with low pKa can facilitate both Pα–Olg bond breaking and the Pα–Onuc bond forming process, thus explaining the large negative βlg calculated for the transition state geometry that has significantly longer Pα–Onuc distance than the Pα–Olg distance.

Uniform Free-Energy Profiles of the P–O Bond Formation and Cleavage Reactions Catalyzed by DNA Polymerases β and λ

“…76 The EVB reaction free energy of 19.5 kcal mol –1 for the IS1 → IS2 step was obtained as followswhere Δ g EVB,water ‡ = 23.1 kcal mol –1 is the activation free energy of the IS1 → IS2 step (eq 5), ΔΔ g 0 ‡ = −2.6 kcal mol –1 is the difference between the reaction and activation free energies used by Florián et al, 57 and c = −1.0 kcal mol –1 is the correction introduced in this study to make the intermediate observable on the EVB free-energy surface and to change the rate-limiting step in aqueous solution from the IS2 → PS to the IS1 → IS2 step, in accordance with ref (69). …”

“…Recent ab initio QM calculations of a model reaction between tetrahydrofuranol and methyl triphosphate have suggested that IS2 does not exist, 69 which means that the reaction scheme of the uncatalyzed reaction between 2′-deoxyribose (dRib) and dNTP can be simplified intoWe removed IS2 in the FEP/EVB free-energy profiles by increasing the reaction free energy (Δ g 0 ) of the IS1 → IS2 step by about 3 kcal mol –1 , which resulted in the free energy of IS2 being almost equal to TS2 and TS3. When we applied the same free-energy shift to IS2 in the free-energy profiles of the reactions catalyzed by Polβ and Polλ, we also obtained a flat free-energy surface from TS2 to TS3The absence of IS2 in the water, Polβ, and Polλ systems means that, using the terminology offered by Lassila et al, 83 the O nuc –P α bond formation and the P α –O lg bond cleavage reactions are concerted, 30,44,48,49,75,84 and, unlike T7 DNA polymerase, 57 the studied enzymes do not change the concertedness of the reactions.…”

“…The atomic charges of IS2b were based on IS2a and adjusted to account for the difference between PCM/B3LYP/TZVP//HF/6-31G* Mulliken atomic charges in QM models of the compact and loose associative-like TS between IS1 and IS2. 69 To achieve net zero charge of the reactive region in IS1, IS2, and PS in the SB pathway, the atomic charge of C 3′ in 2′-deoxyribose (dRib; water systems) and dC (Polβ and Polλ systems) was further adjusted (cf. Tables S6 and S7).…”

Catalytic Mechanism of Mammalian Adenylyl Cyclase: A Computational Investigation

“…The activation energy, however, turns out to be 42.6 kcal/mol, suggesting that the enzyme significantly reduces the activation energy of the reaction. In contrast, on the basis of DFT and Hatree Fock calculations, Zhang et al estimated the activation free energy for DNA polarization in solution to be 30.5 kcal/mol 46 . Similar to our results, in their calculated RDTS the P–O nuc and P–O lg are 2.06 Å and 1.88 Å, respectively.…”

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

“…The proton transfer from the nucleophilic water to the Asp83 immediately follows the phosphorous bond forming and bond breaking steps in a concerted mechanism ( Figure 5). The reaction is initiated by the attack of the nucleophilic water, leading to an associative pathway for the phosphate cleavage that has been identified in solution and in several enzymes that carry out phosphate cleavage or transfer (see e.g., Florián et al [53][54] and Klahn et al 55 ). This mechanism is also consistent with the experimentally observed pH change that is used to directly follow the reaction kinetics, 56 and with the lack of observed intermediates for the phosphate hydrolysis.…”