Quantum chemical investigation of enzymatic activity in DNA polymerase β.: A mechanistic study
Quantum chemical investigation of enzymatic activity in DNA polymerase β.: A mechanistic study
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DOI：
10.1021/jp003629x
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发表时间：
2001-01-11
影响因子：
3.3
通讯作者：
Burt, SK
中科院分区：
文献类型：
--
作者：
Abashkin, YG;Erickson, JW;Burt, SK
最近的实验观察支持这样的假设，即所有多核苷酸聚合酶家族都具有通用的“双金属离子”核苷酸加成机制。该机制提供了核苷酸转移反应的一般情况。然而，详细的反应途径仍然是一个有争议的问题。我们使用密度泛函理论研究了DNA聚合酶β的两种潜在反应途径。我们的模型由聚合酶活性位点的67个原子组成，包括被认为对催化重要的所有主要特征。我们研究的第一种机制涉及PO 3中间体的形成。该中间体被认为参与溶液中的磷酸盐反应，并且可以容纳在聚合酶β活性位点中。然而，该中间体形成的障碍是37.0 kcal/mol，我们不认为这种机制是在酶中发生的机制。第二种机制，导致五配位的中间体似乎是可行的。这种分步机理具有相对低的势垒，并且在亲核攻击之后，反应的每一步都是放热的。反应的限速步骤是亲核进攻，需要13 kcal/mol的活化能。我们预测，相应的过渡态，这是离子的，可以进一步降低的障碍，考虑到静电稳定来自其余的蛋白质。
Recent experimental observations support the assumption that all families of polynucleotide polymerases have a universal "two-metal-ion" mechanism of nucleotide addition. This mechanism provides a general picture of the nucleotidyl transfer reaction. However, the detailed reaction pathway is still a matter of debate. We investigated two potential reaction pathways for DNA polymerase beta using density-functional theory. Our model consists of 67 atoms of the polymerase active site and includes all major features thought to be important for catalysis. The first mechanism we investigated involves the formation of a PO3 intermediate. This intermediate is thought to be involved in phosphate reactions in solution and could be accommodated in the polymerase beta active site. However, the barrier to formation of this intermediate is 37.0 kcal/mol, and we do not expect that this mechanism is the one that occurs in the enzyme. The second mechanism that leads to a pentacoordinated intermediate appears to be feasible. This stepwise mechanism has relatively low barriers and, after the nucleophilic attack, every step of the reaction is exothermic. The rate-limiting step of the reaction is the nucleophilic attack, which needs 13 kcal/mol of activation energy. We predict that the barrier of the corresponding transition state, which is ionic, can be further lowered by taking into account electrostatic stabilization coming from the rest of the protein.