title: 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 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. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemistry, University of Southern California, 418 SGM Building, 3620 McClintock Avenue, Los Angeles, California, 90089-1062.
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Matute
authors.affiliation: Department of Chemistry, University of Southern California, 418 SGM Building, 3620 McClintock Avenue, Los Angeles, California, 90089-1062.
authors.forename: Hanwool
authors.initials: H
authors.lastname: Yoon
authors.affiliation: Department of Chemistry, University of Southern California, 418 SGM Building, 3620 McClintock Avenue, Los Angeles, California, 90089-1062. warshel@usc.edu.
authors.forename: Arieh
authors.initials: A
authors.lastname: Warshel
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011522:Protons; D011789:Quantum Theory; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D011522:Protons; D001224:Aspartic Acid; D018698:Glutamic Acid; D019951:DNA Polymerase beta; D000409:Alanine
keywords: enzyme catalysis; phosphoryl transfer; proton transfer