We examined the roles of Mg2+ ions in DNA polymerization by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase and by molecular dynamics simulation of Mg2+ binding. Binding of the Mg-nucleotide complex induces a conformational change of the enzyme from open to closed states in a process that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds weakly to the closed state of the enzyme-DNA-Mg.dNTP complex with an apparent Kd = 3.7 mM and facilitates the catalytic reaction. This weak binding of the catalytic Mg2+ is important to maintain fidelity in that the Mg2+ samples the correctly aligned substrate without perturbing the equilibrium at physiological Mg2+ concentrations. The binding of the catalytic Mg2+ increases nucleotide specificity (kcat/Km) by increasing the rate of the chemistry and decreasing the rate of enzyme opening allowing nucleotide release. Changing the free Mg2+ concentration from 0.25 to 10 mM increased nucleotide specificity (kcat/Km) by 12-fold. Mg2+ binds very weakly to the open state of the enzyme in the absence of nucleotide (Kd ~ 34 mM) and competes with Mg.dNTP. Analysis based on publish crystal structures showed that HIV RT binds only two metal ions during incorporation of a correct base-pair. MD simulations support the kinetic studies suggesting weak binding of the catalytic Mg2+ in open and closed states. They also support the two-metal ion mechanism, although the polymerase may bind a third metal ion in the presence of a mismatched nucleotide.

中文翻译：

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动力学和热力学分析定义了两种金属离子在DNA聚合酶特异性和催化作用中的作用

我们检查了Mg2 +离子在DNA聚合中的作用，方法是通过HIV逆转录酶催化的单核苷酸掺入的动力学分析以及Mg2 +结合的分子动力学模拟来分析Mg2 +离子在DNA聚合中的作用。Mg-核苷酸复合物的结合在不依赖于游离Mg2 +浓度的过程中诱导酶的构象变化，从开放状态变为闭合状态。随后，第二个Mg2 +弱结合到酶-DNA-Mg.dNTP复合物的闭合状态，其表观Kd = 3.7 mM，并促进催化反应。催化Mg2 +的这种弱结合对于保持保真度很重要，因为Mg2 +会采样正确对齐的底物，而不会干扰生理Mg2 +浓度下的平衡。催化性Mg2 +的结合通过增加化学反应速率和降低允许核苷酸释放的酶开放速率来增加核苷酸特异性（kcat / Km）。将游离Mg2 +浓度从0.25更改为10 mM，可使核苷酸特异性（kcat / Km）提高12倍。在没有核苷酸（Kd〜34 mM）的情况下，Mg2 +与酶的开放状态非常弱地结合，并与Mg.dNTP竞争。根据公开的晶体结构进行的分析表明，在掺入正确的碱基对期间，HIV RT仅结合两个金属离子。MD模拟支持动力学研究，表明在打开和关闭状态下催化Mg2 +的弱结合。它们也支持两种金属离子的机制，尽管聚合酶可能在核苷酸错配的情况下结合第三种金属离子。