Theoretical Study of the Reaction Mechanism of Streptomyces coelicolor Type II Dehydroquinase
Theoretical Study of the Reaction Mechanism of Streptomyces coelicolor Type II Dehydroquinase
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DOI：
10.1021/ct800480d
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发表时间：
2009-05-01
影响因子：
5.5
通讯作者：
Blumberger, Jochen
中科院分区：
文献类型：
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作者：
Blomberg, L. Mattias;Mangold, Martina;Blumberger, Jochen
利用分子动力学模拟和密度泛函理论 (DFT) 计算研究了天蓝色链霉菌 11 型脱氢喹酶 (DHQase) 的反应机制。 DHQase 催化脱氢奎宁 (DHQ) 中水分子的消除，这是细菌、真菌和植物中芳香族氨基酸生物合成的关键步骤。在 DFT 计算中，使用了包含多达 230 个原子的 10 个模型来研究根据晶体结构和动力学数据提出的反应机制的不同建议。通过探索活性位点的灵活性，分子动力学模拟表明，去质子化的Tyr28可以作为催化第一步反应的碱基，即DHQ C2处pro-S质子的质子夺取以及烯醇化物中间体的形成。计算得出的第一过渡态 (TS1) 势垒为 13-15 kcal/mol，仅受所使用的活性位点模型的轻微影响，并且与速率决定步骤的相应实验势垒 13.4 kcal/mol 非常一致。发现先前提出的中间体的烯醇形式的能量明显高于烯醇化物形式，因此在热力学上不具有竞争性。在第二个也是最后一个反应步骤中，C1 处的羟基被 His106 质子化，然后消除水，由于质子从 His106 净转移到 Tyr28，偶极矩显着增加。只有在第二反应步骤中偶极矩的积累至少得到部分补偿的情况下，才能找到与相应实验势垒非常吻合的第二过渡态（TS2）势垒。我们推测，这可以通过 Tyr28 阴离子的再生或在达到 TS2 之前将质子转移到 His106 附近来促进。根据当前计算结果讨论了 11 型 DHQase 的修订机制。
The reaction mechanism of a type 11 dehydroquinase (DHQase) from Streptomyces coelicolor was investigated using molecular dynamics simulation and density functional theory (DFT) calculations. DHQase catalyzes the elimination of a water molecule from dehydroquinate (DHQ), a key step in the biosynthesis of aromatic amino acids in bacteria, fungi, and plants. In the DFT calculations, 10 models, containing up to 230 atoms, were used to investigate different proposals for the reaction mechanism, suggested on the basis of crystal structures and kinetic data. Probing the flexibility of the active site, molecular dynamics simulation reveals that deprotonated Tyr28 can act as the base that catalyzes the first reaction step, the proton abstraction of the pro-S proton at C2 of DHQ, and formation of the enolate intermediate. The computed barrier for the first transition state (TS1), 13-15 kcal/mol, is only slightly affected by the active site model used and is in good agreement with the corresponding experimental barrier of 13.4 kcal/mol for the rate-determining step. The previously proposed enol form of the intermediate is found to be significantly higher in energy than the enolate form and is thus thermodynamically not competitive. In the second and final reaction step, protonation of the hydroxyl group at C1 by His106 followed by water elimination, there is a substantial buildup of dipole moment due to the net transfer of a proton from His106 to Tyr28. A barrier for the second transition state (TS2) that fits well with the corresponding experimental barrier could only be found if the buildup of dipole moment is at least partly compensated during the second reaction step. We speculate that this could be facilitated by regeneration of the Tyr28 anion or by proton transfer to the vicinity of His106 before TS2 is reached. A revised mechanism for type 11 DHQase is discussed in light of the results of the present calculations.