The Gaussian Electrostatic Model (GEM) is a density-based force field that employs Hermite Gaussian auxiliary basis sets (ABSs) to represent molecular electroni

Enzymes are critical biological catalysts involved in maintaining the intricate balance of metabolic processes within living organisms. Mutations in enzymes can result in disruptions to their functio...

DNA polymerases are essential enzymes responsible for accurate genome replication and repair, with divalent metal cofactors playing a crucial role in their catalytic function. Polymerase gamma (Pol γ) is the primary DNA polymerase in mitochondria, ensuring the faithful replication of mitochondrial DNA. The choice of metal cofactor, typically magnesium (Mg²⁺) or manganese (Mn²⁺), influences its structural stability, enzymatic activity, and fidelity. In this study, we employed molecular dynamics (MD) simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations to investigate how Mg²⁺ and Mn²⁺ affect the flexibility, active site stabilization, and catalytic efficiency of Pol γ. It is seen that Mn²⁺ increases overall protein flexibility, whereas Mg²⁺ provides greater active site stabilization. Intermolecular interaction analysis of individual residues are consistent with experimental mutagesis reports, and highlight the importance of specific residues, many of which are evolutionarily conserved, and some are involved in pathogenic mutations. Despite this, Mn²⁺ enhances catalytic efficiency, exhibiting higher exoergicity (-3.65 kcal/mol vs. -1.61 kcal/mol for Mg²⁺) and a lower activation barrier. Inter-molecular interaction analysis reveals that Mn²⁺ provides a larger stabilization of the transition state and product complex, favoring reaction progression. Investigation of the effects of electric field in the active site suggest that the O3’ atom on the DNA primer base experiences a larger polarization in the system with Mn2+ ions when compared to Mg2+, with dipole directions consistent with the catalytic reaction progress. Our findings highlight a trade-off between structural stability and catalytic efficiency, providing insights into the role of metal ions in mitochondrial polymerase function and their implications for mutagenesis and mitochondrial disorders.