How Conformational Dynamics of DNA Polymerase Select Correct Substrates: Experiments and Simulations

Abstract

Summary Nearly every enzyme undergoes a significant change in structure after binding it’s substrate. New experimental and theoretical analyses of the role of changes in HIV reverse transcriptase structure in selecting a correct substrate are presented. Atomically detailed simulations using the Milestoning method predict a rate and free energy profile of the conformational change commensurate with experimental data. A large conformational change occurring on a ms timescale locks the correct nucleotide at the active site, but promotes release of a mismatched nucleotide. The positions along the reaction coordinate that decide the yield of the reaction are not determined by the chemical step. Rather, the initial steps of weak substrate binding and protein conformational transition significantly enrich the yield of a reaction with a correct substrate, while the same steps diminish the reaction probability of an incorrect substrate.

Why nature really chose phosphate

“…It might also be useful here to mention a very recent work (Kirmizialtin et al 2012) that performs elegant Milestone simulations of the conformational change in HIV reverse transcriptase, implying that DNA polymerases select the correct substrate by means of conformational dynamics. Now despite the technical accomplishments involved in the Miletone approach (noting, however, that our renormalization approach has also provided a very good estimate of the conformational barrier, Prasad & Warshel, 2011), we find the dynamical and conformational view to be problematic.…”

“…Second, although the simulations reproduce the experimental conformational barriers, they have not considered the chemical barrier, and thus have not established the origin of the fidelity. We would also like to clarify (as was done in Prasad & Warshel, 2011) that even if the rate-determining barrier for R is the conformational step, this has nothing to do with the dynamical view that ' rapid motions in the specificity domain allow the enzyme to explore the substrate at the active site, probing for key interactions indicating a correctly bound substrate ', presented in (Kirmizialtin et al 2012). Rather, all we have here is being entirely determined by the (conformation and chemical) activation barriers, and the depth of the binding free energy, which fully determines the kinetics of the system.…”

Prechemistry barriers and checkpoints do not contribute to fidelity and catalysis as long as they are not rate limiting

“…However, since the main issues with Johnson's proposal were already discussed in Ref. [6], we will examine here a recent work [57] that performed elegant ''Milestone'' simulations of the conformational change in HIV reverse transcriptase and implied that DNA polymerases select the correct substrate by means of conformational dynamics. Now despite the technical accomplishments involved in the Milestone approach (noting, however, that our renormalization approach [58] has also provided a very good estimate of the conformational barrier [6]), we find the dynamical and conformational view to be problematic.…”

“…Now despite the technical accomplishments involved in the Milestone approach (noting, however, that our renormalization approach [58] has also provided a very good estimate of the conformational barrier [6]), we find the dynamical and conformational view to be problematic. That is, Johnson, Elber and their coworkers [57] examined a case that might involve a ratedetermining conformational barrier for the formation of the correct base pair (Fig. 4, presents an identical case in T7 polymerase [44]).…”

“…Furthermore, the calculated conformational barriers could not be correlated with the observed fidelity. We would also like to clarify (as was done in [6]) that even if the rate-determining barrier for the insertion of the correct dNTP substrate is the conformational step, this has nothing to do with the dynamical view that ''rapid motions in the specificity domain allow the enzyme to explore the substrate at the active site, probing for key interactions indicating a correctly bound substrate'', presented in [57]. Rather, all we have here is being entirely determined by the (conformation and chemical) activation barriers, and the depth of the binding free energy, which fully determines the kinetics of the system.…”