A Hybrid QM/MM Simulation Study of Intramolecular Proton Transfer in the Pyridoxal 5′-Phosphate in the Active Site of Transaminase: Influence of Active Site Interaction on Proton Transfer
A Hybrid QM/MM Simulation Study of Intramolecular Proton Transfer in the Pyridoxal 5′-Phosphate in the Active Site of Transaminase: Influence of Active Site Interaction on Proton Transfer
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DOI：
10.1021/jp506196m
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发表时间：
2014-09-25
影响因子：
3.3
通讯作者：
Chandra, Amalendu
中科院分区：
文献类型：
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作者：
Banik, Sindrila Dutta;Chandra, Amalendu
吡哆醛 5'-磷酸 (PLP) 席夫碱是一种多功能辅助因子，表现出互变异构平衡，涉及 N-质子化两性离子酮烯胺互变异构体和 O-质子化共价烯醇胺互变异构体之间的分子内质子转移。据推测，为了实现催化活性，PLP 必须呈两性离子酮烯胺互变异构形式。然而，PLP依赖性酶活性位点席夫碱互变平衡平衡的确切位置尚不清楚。在目前的工作中，我们利用量子力学和分子力学相结合的模拟研究了天冬氨酸转氨酶（AspAT）活性位点中PLP天冬氨酸（PLP-Asp）席夫碱的外部醛亚胺态的互变平衡。本研究的主要重点是分析控制各种 PLP 依赖性酶活性位点互变异构平衡的因素。结果表明，在气相和水相以及 AspAT 的活性位点中，酮烯胺互变异构体比烯醇胺互变异构体更优选。目前的模拟表明，与烯醇胺互变异构体相比，AspAT 的活性位点更适合酮烯胺互变异构体。有趣的是，Tyr225 在酮烯胺互变异构体中充当酚氧的质子供体，而在共价烯醇胺互变异构体中，它充当酚氧的质子受体。最后，元动力学研究证实了这一结果。计算出的自由能垒约为7.5 kcal/mol。对三种不同 PLP 依赖性酶（天冬氨酸转氨酶、多巴脱羧酶和丙氨酸消旋酶）的活性位点残基创建的微环境进行了比较分析，以了解互变异构平衡的控制因素。分析表明，活性位点残基和 PLP 酚氧之间的分子间氢键使互变异构平衡向 N-质子化酮烯胺互变异构形式移动。
Pyridoxal 5'-phosphate (PLP) Schiff base, a versatile cofactor, exhibits a tautomeric equilibrium that involves an intramolecular proton transfer between the N-protonated zwitterionic ketoenamine tautomer and the O-protonated covalent enolimine tautomer. It has been postulated that for the catalytic activity, the PLP has to be in the zwitterionic ketoenamine tautomeric form. However, the exact position of the tautomeric equilibrium of Schiff base in the active site of PLP-dependent enzyme is not known yet. In the present work, we investigated the tautomeric equilibrium for the external aldimine state of PLP aspartate (PLP-Asp) Schiff base in the active site of aspartate aminotransferase (AspAT) using combined quantum mechanical and molecular mechanical simulations. The main focus of the present study is to analyze the factors that control the tautomeric equilibrium in the active sites of various PLP-dependent enzymes. The results show that the ketoenamine tautomer is more preferred than the enolimine tautomer both in the gas and aqueous phases as well as in the active site of AspAT. Current simulations show that the active site of AspAT is more suitable for the ketoenamine tautomer compared to the enolimine tautomer. Interestingly, the Tyr225 acts as a proton donor to the phenolic oxygen in the ketoenamine tautomer, while in the covalent enolimine tautomer, it acts as a proton acceptor to the phenolic oxygen. Finally, the metadynamics study confirms this result. The calculated free energy barrier is about 7.5 kcal/mol. A comparative analysis of the microenvironment created by the active site residues of three different PLP-dependent enzymes (aspartate aminotransferase, Dopa decarboxylase, and Ala-racemase) has been carried out to understand the controlling factor(s) of the tautomeric equilibrium. The analysis shows that the intermolecular hydrogen bonding between active site residues and the phenolic oxygen of PLP shifts the tautomeric equilibrium toward the N-protonated ketoenamine tautomeric form.