Catalytic Effects of Mutations of Distant Protein Residues in Human DNA Polymerase beta: Theory and Experiment
Abstract
We carried out free-energy calculations and transient kinetic experiments for the insertion of the right (dC) and wrong (dA) nucleotides by wild-type (WT) and six mutant variants of human DNA polymerase beta (Pol beta). Since the mutated residues in the point mutants, I174S, 1260Q M282L, H285D, E288K, and K289M, were not located in the Pol beta catalytic site, we assumed that the WT and its point mutants share the same dianionic phosphorane transition state structure of the triphosphate moiety of deoxyribonucleotide 5'-triphosphate (dNTP) substrate.
On the basis of this assumption, we have formulated a thermodynamic cycle for calculating relative dNTP insertion efficiencies, Omega = (k(pol)/K-D)(mut)/(k(pol)/K-D)(WT) using free energy perturbation (FEP) and linear interaction energy (LIE) methods. Kinetic studies on five of the mutants have been published previously using different experimental conditions, e.g., primer-template sequences.
We have performed a presteady kinetic analysis for the six mutants for comparison with wild-type Pol beta using the same conditions, including the same primer/template DNA sequence proximal to the dNTP insertion site used for X-ray crystallographic studies. This consistent set of kinetic and structural data allowed us to eliminate the DNA sequence from the list of factors that can adversely affect calculated Omega values.
The calculations using the FEP free energies scaled by 0.5 yielded 0.9 and 1.1 standard deviations from the experimental log Omega values for the insertion of the right and wrong dNTP, respectively. We examined a hybrid FEP/LIE method in which the FEP van der Waals term for the interaction of the mutated amino acid residue with its surrounding environment was replaced by the corresponding van der Waals term calculated using the LIE method, resulting in improved 0.4 and 1.0 standard deviations from the experimental log Omega values.