Thermal-activated protein mobility and its correlation with catalysis in thermophilic alcohol dehydrogenase
Thermal-activated protein mobility and its correlation with catalysis in thermophilic alcohol dehydrogenase
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DOI：
10.1073/pnas.0403337101
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发表时间：
2004-06-29
影响因子：
11.1
通讯作者：
Klinman, JP
中科院分区：
文献类型：
--
作者：
Liang, ZX;Lee, T;Klinman, JP
采用液相色谱耦合质谱法研究了嗜热乙醇脱氢酶（htADH）的温度依赖性氢-氘（H/D）交换。对蛋白质衍生肽H/D交换模式变化的分析表明，htADH的一些区域在低温下处于刚性构象亚态，蛋白质的协同运动有限。酶在大约30度和45度处经历两次离散的转变，以获得更动态的构象亚态。在WC以上发生转变的5个多肽中，有4个与辅因子直接接触，并且NAD（+）结合亲和力在此温度范围内也发生了变化，这意味着辅因子结合结构域的迁移率发生了变化。相比之下，表现出30度转变的5个肽位于底物结合域。这种转变与氢化物转移的k（cat）活化能的变化相一致，导致k（cat）与五种肽的加权平均汇率常数k（HX(WA)）之间呈线性相关。这些观察结果表明，氢化物转移与htADH中蛋白质迁移率之间存在直接耦合，并且迁移率的增加至少是高温下E-act降低的部分原因。这些数据为蛋白质动力学在控制酶活性位点的氢隧穿中起关键作用的假设提供了支持。
Temperature-dependent hydrogen-deuterium (H/D) exchange of the thermophilic alcohol dehydrogenase (htADH) has been studied by using liquid chromatography-coupled mass spectrometry. Analysis of the changes in H/D exchange patterns for the protein-derived peptides suggests that some regions of htADH are in a rigid conformational substate at reduced temperatures with limited cooperative protein motion. The enzyme undergoes two discrete transitions at approximate to30 and 45degreesC to attain a more dynamic conformational substate. Four of the five peptides exhibiting the transition above WC are in direct contact with the cofactor, and the NAD(+)-binding affinity is also altered in this temperature range, implicating a change in the mobility of the cofactor-binding domain >45degreesC. By contrast, the five peptides exhibiting the transition at 30degreesC reside in the substrate-binding domain. This transition coincides with a change in the activation energy of k(cat) for hydride transfer, leading to a linear correlation between k(cat) and the weighted average exchange rate constant k(HX(WA)) for the five peptides. These observations indicate a direct coupling between hydride transfer and protein mobility in htADH, and that an increased mobility is at least partially responsible for the reduced E-act at high temperature. The data provide support for the hypothesis that protein dynamics play a key role in controlling hydrogen tunneling at enzyme active sites.