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Proteins. 2016 Nov;84(11):1644-1657. doi: 10.1002/prot.25106. Epub 2016 Aug 24.

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

Author information

1
Department of Chemistry, University of Southern California, 418 SGM Building, 3620 McClintock Avenue, Los Angeles, California, 90089-1062.
2
Department of Chemistry, University of Southern California, 418 SGM Building, 3620 McClintock Avenue, Los Angeles, California, 90089-1062. warshel@usc.edu.

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 the 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. Proteins 2016; 84:1644-1657.

KEYWORDS:

enzyme catalysis; phosphoryl transfer; proton transfer

PMID:
27488241
PMCID:
PMC5073053
DOI:
10.1002/prot.25106
[Indexed for MEDLINE]
Free PMC Article

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