TY - JOUR
T1 - Exploring the Reaction Mechanism of HIV Reverse Transcriptase with a Nucleotide Substrate
AU - Wang, Hao
AU - Huang, Nathan
AU - Dangerfield, Tyler
AU - Johnson, Kenneth A.
AU - Gao, Jiali
AU - Elber, Ron
AU - Elber, Ron
N1 - Funding Information:
This research is supported by grants from The National Institute of Health, GM 59796, and the Welch Foundation, F-1896
Publisher Copyright:
Copyright © 2020 American Chemical Society.
PY - 2020/5/28
Y1 - 2020/5/28
N2 - Enzymatic reactions consist of several steps: (i) a weak binding event of the substrate to the enzyme, (ii) an induced fit or a protein conformational transition upon ligand binding, (iii) the chemical reaction, and (iv) the release of the product. Here we focus on step iii of the reaction of a DNA polymerase, HIV RT, with a nucleotide. We determine the rate and the free energy profile for the addition of a nucleotide to a DNA strand using a combination of a QM/MM model, the string method, and exact Milestoning. The barrier height and the time scale of the reaction are consistent with experiment. We show that the observables (free energies and mean first passage time) converge rapidly, as a function of the Milestoning iteration number. We also consider the substitution of an oxygen of the incoming nucleotide by a nonbridging sulfur atom and its impact on the enzymatic reaction. This substitution has been suggested in the past as a tool to examine the influence of the chemical step on the overall rate. Our joint computational and experimental study suggests that the impact of the substitution is small. Computationally, the differences between the two are within the estimated error bars. Experiments suggest a small difference. Finally, we examine step i, the weak binding of the nucleotide to the protein surface. We suggest that this step has only a small contribution to the selectivity of the enzyme. Comments are made on the impact of these steps on the overall mechanism.
AB - Enzymatic reactions consist of several steps: (i) a weak binding event of the substrate to the enzyme, (ii) an induced fit or a protein conformational transition upon ligand binding, (iii) the chemical reaction, and (iv) the release of the product. Here we focus on step iii of the reaction of a DNA polymerase, HIV RT, with a nucleotide. We determine the rate and the free energy profile for the addition of a nucleotide to a DNA strand using a combination of a QM/MM model, the string method, and exact Milestoning. The barrier height and the time scale of the reaction are consistent with experiment. We show that the observables (free energies and mean first passage time) converge rapidly, as a function of the Milestoning iteration number. We also consider the substitution of an oxygen of the incoming nucleotide by a nonbridging sulfur atom and its impact on the enzymatic reaction. This substitution has been suggested in the past as a tool to examine the influence of the chemical step on the overall rate. Our joint computational and experimental study suggests that the impact of the substitution is small. Computationally, the differences between the two are within the estimated error bars. Experiments suggest a small difference. Finally, we examine step i, the weak binding of the nucleotide to the protein surface. We suggest that this step has only a small contribution to the selectivity of the enzyme. Comments are made on the impact of these steps on the overall mechanism.
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U2 - 10.1021/acs.jpcb.0c02632
DO - 10.1021/acs.jpcb.0c02632
M3 - Article
C2 - 32364738
AN - SCOPUS:85085586368
SN - 1520-6106
VL - 124
SP - 4270
EP - 4283
JO - Journal of Physical Chemistry B
JF - Journal of Physical Chemistry B
IS - 21
ER -