Origin of the Activity Drop with the E50D Variant of Catalytic Antibody 34E4 for Kemp Elimination
Origin of the Activity Drop with the E50D Variant of Catalytic Antibody 34E4 for Kemp Elimination
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DOI：
10.1021/jp8076084
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发表时间：
2009-01-15
影响因子：
3.3
通讯作者：
Jorgensen, William L.
中科院分区：
文献类型：
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作者：
Alexandrova, Anastassia N.;Jorgensen, William L.
在酶中，多种结构效应协同作用导致高的催化活性，而单独这些效应可能很小。人工酶的设计需要理解并有能力操纵这种微妙的效果。催化5-硝基苯并恶唑Kemp消除的34134催化抗体及其Glu50Asp （E50D）变体是本研究的主题。仅去除一个亚甲基就可使催化Kemp消除的速率降低约30倍。在这里，我们的目的是了解催化性能的这种差异。利用QM/MM蒙特卡罗模拟和自由能摄动理论，阐明了34E4和E50D突变体催化Kemp消除的机理。在这两种蛋白质中，反应都遵循一个单一的、协调一致的机制。突变体激活势垒升高2.4 kcal/mol，速率降低62倍，与实验结果吻合较好。在整个反应过程中监测活性位点残基的位置和功能。结果表明，在34E4的E50D变体中，与碱的接触较松、与asn58的接触较短、与Trp91在反应过渡态的pi堆积较差以及不同的溶剂化模式都是导致反应速率降低的原因。
In enzymes, multiple structural effects cooperatively lead to the high catalytic activity, while individually these effects can be small. The design of artificial enzymes requires the understanding and ability to manipulate such subtle effects. The 34134 catalytic antibody, catalyzing Kemp elimination of 5-nitrobenzisoxazole, and its Glu50Asp (E50D) variant are the subject of the present investigation. This removal of only a methylene group yields an approximately 30-fold reduction in the rate for the catalyzed Kemp elimination. Here, the aim is to understand this difference in the catalytic performance. The mechanism of Kemp elimination catalyzed by 34E4 and the E50D mutant is elucidated using QM/MM Monte Carlo simulations and free energy perturbation theory. In both proteins, the reaction is shown to follow a single-step, concerted mechanism. In the mutant, the activation barrier rises by 2.4 kcal/mol, which corresponds to a 62-fold rate deceleration, which is in good agreement with the experimental data. The positions and functionality of the residues in the active site are monitored throughout the reaction. It is concluded that the looser contact with the base, shorter base-Asn58 contact, less favorable pi-stacking with Trp91 in the transition state of the reaction, and different solvation pattern all contribute to the reduction of the reaction rate in the E50D variant of 34E4.