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首页> 外文期刊>Journal of Molecular Biology >Determination of electrostatic interaction energies and protonation state populations in enzyme active sites
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Determination of electrostatic interaction energies and protonation state populations in enzyme active sites

机译：酶活性位点中静电相互作用能和质子态种群的确定

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The pH-dependence of the NMR chemical shift for titratable groups in proteins often deviate from a standard Henderson-Hasselbalch (HH) titration curve. A non-HH dependence of the chemical shift for a given residue can arise from a single-site, non-HH titrational event for that residue, or if the chemical shift of the group is influenced by additional titrational events occurring in other residues. We show that simultaneous fits of several non-HH NMR titration curves of interacting protein residues to a statistical mechanical model can be used to distinguish between these two cases. From fitting of non-HH titrations, we can extract electrostatic interaction energies between protein residues. Furthermore, by performing simultaneous fits of NMR titration curves and enzymatic pH-activity profiles, we can gain information on the identity and populations of the catalytically competent protonation states in enzymes. We apply the global fitting of titrational events (GloFTE) method to experimental data on five enzyme systems and on a single non-enzyme system, and show that the extracted electrostatic interaction energies and effective dielectric constants for a subset of these systems agree excellently with experimentally determined values as well as with theoretical calculations. In the case of reduced Escherichia coli thioredoxin we use GloFTE analysis to distinguish between two possible interpretations of the NMR titration curves of the active site residues. We also show that for the strongly coupled system of titratable groups in the active site of the Bacillus circulans xylanase (BCX) N35D mutant, GloFTE fits of a single titration curve and an enzymatic pH-activity profile can give a full description of the energetics of the titrational events in the enzyme's active site. Using only the X-ray crystallographic structure of the enzyme and the electrostatic interaction energies extracted from such a GloFTE fit, we can uniquely identify the three catalytic groups in this system. This raises the prospect of completely characterising active site titrational events from a single unassigned NMR titration curve and an enzymatic pH-activity profile. (C) 2007 Elsevier Ltd. All rights reserved.
机译：蛋白质中可滴定基团的NMR化学位移的pH依赖性通常偏离标准的Henderson-Hasselbalch（HH）滴定曲线。给定残基的化学位移与HH的非依赖性可能源自该残基的单点非HH滴定事件，或者该基团的化学位移受其他残基中发生的其他滴定事件影响。我们表明相互作用蛋白残基的几个非HH NMR滴定曲线的同时拟合到统计力学模型可以用来区分这两种情况。通过非HH滴定的拟合，我们可以提取蛋白质残基之间的静电相互作用能。此外，通过同时拟合NMR滴定曲线和酶促pH活性曲线，我们可以获得有关酶催化质子化态的身份和种群的信息。我们将滴定事件的全局拟合（GloFTE）方法应用于五个酶系统和单个非酶系统的实验数据，并表明这些系统的子集的提取的静电相互作用能和有效介电常数与实验非常吻合确定值以及理论计算。对于减少的大肠杆菌硫氧还蛋白，我们使用GloFTE分析来区分活性位点残留的NMR滴定曲线的两种可能解释。我们还显示，对于环化芽孢杆菌木聚糖酶（BCX）N35D突变体活性位点中可滴定基团的强耦合系统，GloFTE拟合单个滴定曲线和酶促pH活性曲线可以全面描述酶活性部位的滴定事件。仅使用酶的X射线晶体学结构和从此类GloFTE拟合中提取的静电相互作用能，我们就可以唯一识别该系统中的三个催化基团。这提出了从单个未分配的NMR滴定曲线和酶促pH活性曲线完全表征活性位点滴定事件的前景。 （C）2007 Elsevier Ltd.保留所有权利。

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