Local protein dynamics and catalysis: Detection of segmental motion associated with rate-limiting product release by a glutathione transferase
Local protein dynamics and catalysis: Detection of segmental motion associated with rate-limiting product release by a glutathione transferase
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DOI：
10.1021/bi026776p
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发表时间：
2002-12-24
期刊：
影响因子：
2.9
通讯作者：
Armstrong, RN
中科院分区：
文献类型：
--
作者：
Codreanu, SG;Ladner, JE;Armstrong, RN
谷胱甘肽转移酶rGSTM1-1催化谷胱甘肽(GSH)与1-氯-2，4-二硝基苯的加成反应，该反应的化学步骤比物理步骤快60倍。Y115的羟基位于活性中心通道中，控制着产物从活性中心的输出。Y115F突变体酶的k(CAT)(72 S(-1))是天然酶(20 S(-1))的3.6倍。结晶学观察和来自酰胺质子交换动力学的证据与Y115F突变体节段运动程度的局部增加相一致，这与产物释放速度的提高相结合。与Y115的羟基有关的氢键相互作用的丧失反映在主链位置的细微变化，构成活性位点通道的结构元素的B因子增加，以及最戏剧性的是，突变位点附近明确定义的电子密度的丧失。在这些区域，酰胺质子交换的动力学也被提高了3到12倍，为影响产物释放的局部蛋白质动力学的变化提供了直接的、定量的证据。增强的产物释放率被认为是由于蛋白质构象平衡群体的微小移动而导致的，该平衡群体允许产物从活性部位流出。
Glutathione transferase rGSTM1-1 catalyzes the addition of glutathione (GSH) to 1-chloro-2,4-dinitrobenzene, a reaction in which the chemical step is 60-fold faster than the physical step of product release. The hydroxyl group of Y115, located in the active site access channel, controls the egress of product from the active site. The Y115F mutant enzyme has a k(cat) (72 s(-1)) that is 3.6-fold larger than that of the native enzyme (20 s(-1)). Crystallographic observations and evidence from amide proton exchange kinetics are consistent with localized increases in the degree of segmental motion of the Y115F mutant that are coupled to the enhanced rate of product release. The loss of hydrogen bonding interactions involving the hydroxyl group of Y115 is reflected in subtle alterations in the backbone position, an increase in B-factors for structural elements that comprise the channel to the active site, and, most dramatically, a loss of well-defined electron density near the site of mutation. The kinetics of amide proton exchange are also enhanced by a factor between 3 and 12 in these regions, providing direct, quantitative evidence for changes in local protein dynamics affecting product release. The enhanced product release rate is proposed to derive from a small shift in the equilibrium population of protein conformers that permit egress of the product from the active site.