Identification of a Long-range Protein Network That Modulates Active Site Dynamics in Extremophilic Alcohol Dehydrogenases
Identification of a Long-range Protein Network That Modulates Active Site Dynamics in Extremophilic Alcohol Dehydrogenases
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DOI：
10.1074/jbc.m113.453951
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发表时间：
2013-05-17
影响因子：
4.8
通讯作者：
Klinman, Judith P.
中科院分区：
文献类型：
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作者：
Nagel, Zachary D.;Cun, Shujian;Klinman, Judith P.
来自嗜热脂肪芽孢杆菌的四聚体嗜热醇脱氢酶（ht-ADH）在活性位点（Trp-87）的芳香族侧链处发生突变。ht-W87 A突变导致野生型酶在30 ° C下观察到的Arabius断裂的丧失和归因于活性四聚体形式的不稳定的冷不稳定性的增加。动力学同位素效应（KIE）在实验温度范围内几乎不依赖于温度，并且与野生型酶在30 ℃以上测得的幅度相似。这表明ht-W87 A在低于30 ℃的野生型酶中不发生硬化。ht-ADH的热不稳定嗜冷同源物ps-A25 Y中二聚体-二聚体界面处的突变导致更耐热的酶和低温下速率决定步骤的变化。ht-ADH的相互突变ht-Y25 A导致与W87 A相似的动力学行为。总的来说，结果表明，在活性位点的灵活性是密切相关的亚基相互作用20埃。先前报道的ht-ADH的凸Arrhenius曲线（Kohen，A.，坎尼奥河Bartolucci，S.，和Klinman，J. P.（1999）Nature 399，496 - 499）被认为至少部分是由亚基相互作用的变化引起的，所述亚基相互作用在30 ℃以下使底物结合结构域刚性化，并阻碍酶对催化相关构象景观进行取样的能力。这些结果暗示了在原核生物乙醇脱氢酶中控制C-H激活的进化上保守的长距离动态通信网络。
A tetrameric thermophilic alcohol dehydrogenase from Bacillus stearothermophilus (ht-ADH) has been mutated at an aromatic side chain in the active site (Trp-87). The ht-W87A mutation results in a loss of the Arrhenius break seen at 30 degrees C for the wild-type enzyme and an increase in cold lability that is attributed to destabilization of the active tetrameric form. Kinetic isotope effects (KIEs) are nearly temperature-independent over the experimental temperature range, and similar in magnitude to those measured above 30 degrees C for the wild-type enzyme. This suggests that the rigidification in the wild-type enzyme below 30 degrees C does not occur for ht-W87A. A mutation at the dimer-dimer interface in a thermolabile psychrophilic homologue of ht-ADH, ps-A25Y, leads to a more thermostable enzyme and a change in the rate-determining step at low temperature. The reciprocal mutation in ht-ADH, ht-Y25A, results in kinetic behavior similar to that of W87A. Collectively, the results indicate that flexibility at the active site is intimately connected to a subunit interaction 20 angstrom away. The convex Arrhenius curves previously reported for ht-ADH (Kohen, A., Cannio, R., Bartolucci, S., and Klinman, J. P. (1999) Nature 399, 496499) are proposed to arise, at least in part, from a change in subunit interactions that rigidifies the substrate-binding domain below 30 degrees C, and impedes the ability of the enzyme to sample the catalytically relevant conformational landscape. These results implicate an evolutionarily conserved, long-range network of dynamical communication that controls C-H activation in the prokaryotic alcohol dehydrogenases.