Exploring the mechanism of DNA polymerases by analyzing the effect of mutations of active site acidic groups in Polymerase β

Abstract

Elucidating the catalytic mechanism of DNA polymerase is crucial for a progress in the understanding of the control of replication fidelity. This work tries to advance the mechanistic understanding by analyzing the observed effect of mutations of the acidic groups in the active site of Polymerase β as well as the pH effect on the rate constant. The analysis involves both empirical valence bond (EVB) free energy calculations and considerations of the observed pH dependence of the reaction. The combined analysis indicates that the proton transfer (PT) from the nucleophilic O3’ has two possible pathways, one to D256 and the second to the bulk. We concluded based on calculations and the experimental pH profile that the most likely path for the wild-type (WT) and D256E and D256A mutants is a PT to the bulk, although the WT may also use a PT to Asp 256. Our analysis highlights the need for very extensive sampling in the calculations of the activation barrier and also clearly shows that ab initio QM/MM calculations that do not involve extensive sampling are unlikely to give a clear quantitative picture of the reaction mechanism.

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

“…The activation free-energy barrier of this step is 9, 6, and 0 kcal mol –1 larger than the assumed Δ g ‡ of the proton transfer in Polβ, tPolλ, and tPolλΔL1. Thus, the P–O bond formation/cleavage reaction is the rate-limiting step in Polβ (in agreement with refs (30, 43, 46, 48, 51) and in disagreement with ref (44)) and tPolλ (in disagreement with ref (49)), and rate-colimiting (with the proton transfer) in tPolλΔL1where k 1 is the rate constant of the RS → IS1 reaction, k –1 is the rate constant of the RS ← IS1 reaction, and k 2 is the rate constant of the IS1 → PS reaction. Because k 2 is a constant in Polβ, tPolλ, and tPolλΔL1, the relative reaction rates among these enzymes depend solely on the p K a of the O nuc Equation 18 describes an intermolecular LFER (not to be confused with the intramolecular Brønsted LFER) between Δ g 0 of the proton transfer step and Δ G ‡ of the overall reaction with a slope of ∼1.…”

“…Neither of these two studies, however, assessed the possibility of the proton transfer to the bulk solution (acceptor a ), which requires calculating p K a of the 3′-OH group. The proton transfer to acceptor a has so far been reported only for Polβ, using FEP or protein-dipoles-Langevin-dipoles linear-response approximation (PDLD/S-LRA) simulations, by Sucato et al, 30 Xiang et al, 46 Ram Prasad and Warshel, 48 and Matute et al: 51 acceptor a (Δ g 0 of 4 kcal mol –1 ) seems to be at least as good as c (Δ g 0 of 6 kcal mol –1 ). 51 How about the remaining proton acceptors?…”

“…Thus, the proton transfer to the bulk solution (via Asp256/490/485, an active site water molecule, or a hydroxide anion) seems to be the proton transfer pathway of the least resistance. 51 Moreover, the subsequent IS1 → IS2 reaction step, which also contributes into the overall free-energy barrier, proceeds with lower Δ g ‡ when preceded by the SB proton transfer. Two questions remain unresolved: (1) What makes the proton transfer to the bulk solution superior over the proton transfer to the active site aspartate in Polβ and tPolλ, but not in tPolλΔL1?…”

“…30,42−44,49,51 (2) Associative and concerted P–O bond formation and cleavage reactions have been suggested. 30,43,48,49 (3) Either the proton transfer or the P–O bond formation/cleavage reaction has been reported as the rate-limiting step.…”

Metal Fluorides: Tools for Structural and Computational Analysis of Phosphoryl Transfer Enzymes

“…In this way, the initial conditions of the experimental crystal structure are not a trap; the computational protocol allows the biomolecule to move freely. A study of the mechanism of DNA polymerase b provides a good example of the application of the EVB methodology [82]. The questions under examination were (i) the destination and timing of PT from the nucleophilic 3 0 -OH, with three aspartates and water as potential acceptors, and (ii) the concerted or stepwise nature of phosphorus migration.…”