Probing DNA Base-Dependent Leaving Group Kinetic Effects on the DNA Polymerase Transition State

Keriann Oertell, Boris A. Kashemirov, Amirsoheil Negahbani, Corinne Minard, Pouya Haratipour, Khadijeh S. Alnajjar, Joann B. Sweasy, Vinod K. Batra, William A. Beard, Samuel H. Wilson, Charles E. McKenna, Myron F. Goodman

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

We examine the DNA polymerase β (pol β) transition state (TS) from a leaving group pre-steady-state kinetics perspective by measuring the rate of incorporation of dNTPs and corresponding novel β,γ-CXY-dNTP analogues, including individual β,γ-CHF and -CHCl diastereomers with defined stereochemistry at the bridging carbon, during the formation of right (R) and wrong (W) base pairs. Brønsted plots of log kpol versus pKa4 of the leaving group bisphosphonic acids are used to interrogate the effects of the base identity, the dNTP analogue leaving group basicity, and the precise configuration of the C-X atom in R and S stereoisomers on the rate-determining step (kpol). The dNTP analogues provide a range of leaving group basicity and steric properties by virtue of monohalogen, dihalogen, or methyl substitution at the carbon atom bridging the β,γ-bisphosphonate that mimics the natural pyrophosphate leaving group in dNTPs. Brønsted plot relationships with negative slopes are revealed by the data, as was found for the dGTP and dTTP analogues, consistent with a bond-breaking component to the TS energy. However, greater multiplicity was shown in the linear free energy relationship, revealing an unexpected dependence on the nucleotide base for both A and C. Strong base-dependent perturbations that modulate TS relative to ground-state energies are likely to arise from electrostatic effects on catalysis in the pol active site. Deviations from a uniform linear Brønsted plot relationship are discussed in terms of insights gained from structural features of the prechemistry DNA polymerase active site.

Original languageEnglish (US)
Pages (from-to)3925-3933
Number of pages9
JournalBiochemistry
Volume57
Issue number26
DOIs
StatePublished - Jul 3 2018
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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