SummaryHuman DNA polymerase β (pol β) fills gaps in DNA as part of base excision DNA repair. Due to its small size it is a convenient model enzyme for other DNA polymerases. Its active site contains two Mg 2+ ions, of which one binds an incoming dNTP and one catalyzes its condensation with the DNA primer strand. Simulating such binuclear metalloenzymes accurately but computationally efficiently is a challenging task. Here, we present a magnesium-cationic dummy atom approach that can easily be implemented in molecular mechanical force fields such as the ENZYMIX or the AMBER force fields. All properties investigated in this paper, that is, structure and energetics of both Michaelis complexes and transition state (TS) complexes were represented more accurately using the magnesium-cationic dummy atom model than using the traditional one-atom representation for Mg 2+ ions. The improved agreement between calculated free energies of binding of TS models to different pol β variants and the experimentally determined activation free energies indicates that this model will be useful in studying mutational effects on catalytic efficiency and fidelity of DNA polymerases. The model should also have broad applicability to the modeling of other magnesiumcontaining proteins.

In order to describe possible reaction mechanisms involving amino acids, and the evolution of the protonation state of amino acid side chains in solution, a reactive force field (ReaxFF-based description) for peptide and protein simulations has been developed as an expansion of the previously reported glycine parameters. This expansion consists of adding to the training set more than five hundred molecular systems, including all the amino acids and some short peptide structures, which have been investigated by means of quantum mechanical calculations. The performance of this ReaxFF protein force field on a relatively short time scale (500 ps) is validated by comparison with classical non-reactive simulations and experimental data of well characterized test cases, comprising capped amino acids, peptides, and small proteins, and reaction mechanisms connected to the pharmaceutical sector. A good agreement of ReaxFF predicted conformations and kinetics with reference data is obtained.

The control of the catalytic power and fidelity of DNA polymerases involves the complex combined effect of the protein residues, the Mg2+ ions, and the interaction between the DNA bases. In an attempt to advance the understanding of catalytic control, we analyze the effect of the protein residues, taking human DNA polymerase beta as a model system. Specifically, we examine the ability of different theoretical models to reproduce the effect of ionized residues on the transition state (TS) binding energy and the corresponding k(pol)/KD. We also explore the role of the Mg2+ ions in the binding and catalysis processes. The application of the microscopic linear response approximation (LRA) and the semimacroscopic PDLD/S-LRA methods to a benchmark of mutational studies produces a semiquantitative correlation and indicates that these methods can provide predictive power. However, pre-steady-state and steady-state kinetic studies currently available do not give a unique benchmark, owing principally to widely varying experimental conditions. We believe that a more uniform experimental benchmark is needed for further refinement of the theoretical models. The analysis of the correlation between the results obtained by a rigorous thermodynamic cycle and by simpler approximations indicates that the protein reorganization between the open, i.e., unbound, form and the closed form does not change the magnitude of the calculated mutational effects in a major way for the experimental data used in this study. The use of the PDLD/S-LRA group contributions allows us to construct energy-based correlation diagrams that can help toward understanding the coupling, i.e., transfer of information, between the base-binding and catalytic sites and to gain a deeper insight into the molecular basis of DNA replication fidelity. Our analysis suggests that the allosteric matrix obtained by subtracting the correlation matrix of the correct and incorrect base pairs should prove useful in exploring the information transfer occurring between the base-binding and catalytic sites. This type of treatment should be especially effective when coupled with structural studies of polymerase-DNA-base mispair ternary complexes and studies using polymerase double mutants. We discuss the potential of direct calculations of binding energy of the TS in a rational design of TS analogues and in drug design.

Metallo-␤-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of ␤-lactam antibiotics. The G262S point mutation distinguishing the metallo-␤-lactamase IMP-1 from IMP-6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single-nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y, and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin, and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R 2 electron donors, which may stabilize the substrate intermediate in the binding pocket. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally.