Comparison of Magnesium and Manganese Ions on the Structural and Catalytic Properties of Human DNA Polymerase Gamma

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 γ (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. Intermolecular interaction analysis of individual residues is consistent with experimental mutagenesis reports and highlights the importance of specific residues, many of which are evolutionarily conserved, and some are involved in pathogenic mutations. It is also observed that Mn2+ enhances catalytic efficiency, exhibiting higher exoergicity (−3.65 kcal mol–1 vs −1.61 kcal mol–1 for Mg2+) and a lower activation barrier. Intermolecular interaction analysis reveals that Mn2+ provides larger stabilization of the transition state and product complex, favoring reaction progression. Investigation of the effects of the electric field in the active site suggests that the O3′ atom on the DNA primer base experiences 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.DNA聚合酶是负责精准基因组复制与修复的必需酶类，二价金属辅因子对其催化功能发挥着关键作用。聚合酶γ（Pol γ）是线粒体中的主要DNA聚合酶，负责保障线粒体DNA的忠实复制。可供选择的金属辅因子通常为镁离子（Mg²+）或锰离子（Mn²+），二者会影响酶的结构稳定性、酶活性与复制保真度。本研究采用分子动力学（MD）模拟与混合量子力学/分子力学（QM/MM）计算方法，探究了Mg²+与Mn²+对Pol γ的柔性、活性位点稳定性及催化效率的影响。针对单个残基的分子间相互作用分析结果与实验诱变研究报道一致，凸显了特定残基的重要性：其中多数为进化保守残基，部分残基与致病性突变相关。研究还观察到，Mn²+可提升催化效率，其表现出更高的放能性（-3.65 kcal mol–1 相较于Mg²+的-1.61 kcal mol–1）与更低的活化能垒。分子间相互作用分析显示，Mn²+可更大程度稳定过渡态与产物复合物，从而助力反应推进。对活性位点电场的研究表明，相较于Mg²+体系，Mn²+体系中DNA引物碱基上的O3'原子极化程度更强，且偶极方向与催化反应进程相符。本研究结果揭示了结构稳定性与催化效率之间的权衡关系，为理解金属离子在线粒体聚合酶功能中的作用，以及其对诱变与线粒体疾病的潜在影响提供了全新见解。