Modulation of a Pre-existing Conformational Equilibrium Tunes Adenylate Kinase Activity
Modulation of a Pre-existing Conformational Equilibrium Tunes Adenylate Kinase Activity
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DOI：
10.1021/ja3032482
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发表时间：
2012-10-10
影响因子：
15
通讯作者：
Wolf-Watz, Magnus
中科院分区：
文献类型：
--
作者：
Aden, Jorgen;Verma, Abhinav;Wolf-Watz, Magnus
在酶促反应循环中，结构可塑性通常是不同微观步骤所必需的。来自大肠杆菌的腺苷酸激酶（AK（eco））在溶液中占据两种主要构象;开放（非活性）和封闭（活性）状态，并且总体周转率与活性构象的寿命成反比。因此，结构可塑性与AK（eco）中的酶促周转密切相关。在这里，我们探测开放到封闭的构象平衡的情况下，结合基板与NMR光谱和分子动力学模拟。在没有底物的情况下的构象平衡，反过来，营业额可以调制与突变和渗透压驱动的扰动。去除ATP和AMP结合亚结构域之间的一个氢键导致群体向开放构象转变，并导致k（cat）增加。向AK（eco）中添加渗透剂TMAO导致群体向闭合构象转变并且k（cat）显著降低。米氏常数（K-M）与k（cat）的变化成比例，这是由于闭合构象的群体对底物结合亲和力的影响。因此，k（cat）和K-M是相互依赖的，并且在AK（eco）的情况下，调制k（cat）的任何扰动都与K-M中的比例响应相反映。因此，我们的结果表明，预先存在的构象平衡的平衡常数直接影响酶催化。从进化的角度来看，我们的研究结果表明，对于AK（ECO），存在足够的灵活性，以获得与所施加的细胞选择压力相称的特异性常数（k（cat）/K-M）。
Structural plasticity is often required for distinct microscopic steps during enzymatic reaction cycles. Adenylate kinase from Escherichia coli (AK(eco)) populates two major conformations in solution; the open (inactive) and closed (active) state, and the overall turnover rate is inversely proportional to the lifetime of the active conformation. Therefore, structural plasticity is intimately coupled to enzymatic turnover in AK(eco). Here, we probe the open to closed conformational equilibrium in the absence of bound substrate with NMR spectroscopy and molecular dynamics simulations. The conformational equilibrium in absence of substrate and, in turn, the turnover number can be modulated with mutational- and osmolyte-driven perturbations. Removal of one hydrogen bond between the ATP and AMP binding subdomains results in a population shift toward the open conformation and a resulting increase of k(cat). Addition of the osmolyte TMAO to AK(eco) results in population shift toward the closed conformation and a significant reduction of k(cat). The Michaelis constants (K-M) scale with the change in k(cat), which follows from the influence of the population of the closed conformation for substrate binding affinity. Hence, k(cat) and K-M are mutually dependent, and in the case of AK(eco), any perturbation that modulates k(cat) is mirrored with a proportional response in K-M. Thus, our results demonstrate that the equilibrium constant of a pre-existing conformational equilibrium directly affects enzymatic catalysis. From an evolutionary perspective, our findings suggest that, for AK(eco), there exists ample flexibility to obtain a specificity constant (k(cat)/K-M) that commensurate with the exerted cellular selective pressure.