Urea-Dependent Adenylate Kinase Activation following Redistribution of Structural States
Urea-Dependent Adenylate Kinase Activation following Redistribution of Structural States
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DOI：
10.1016/j.bpj.2016.08.028
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发表时间：
2016-10-04
影响因子：
3.4
通讯作者：
Wolf-Watz, Magnus
中科院分区：
文献类型：
--
作者：
Rogne, Per;Wolf-Watz, Magnus
蛋白质在功能上通常依赖于构象变化，从而使它们能够在没有底物或结合配偶体的情况下对稀疏的结构状态进行采样。这种结构微观状态的分布由它们的相对稳定性决定，并且它们相互转化的动力学由相关激活势垒的大小决定。在这里，我们通过监测其酶活性对尿素添加的响应变化，探索了选定酶腺苷酸激酶 (Adk) 的结构、稳定性和功能之间的相互作用。为此，我们使用了 P-31 NMR 测定，该测定对于异质样品成分（例如尿素的存在）很有用。研究发现，Adk 在低尿素浓度下被激活，而较高尿素浓度则展开，从而使该酶失活。通过化学位移的定量分析，发现尿素重新分布了预先存在的结构微观状态，以牺牲底物结合的闭合状态为代价来稳定底物结合的开放状态。 Adk 受到底物结合结构域缓慢打开的速率限制，并且结构状态的尿素依赖性重新分布与结构域打开速率常数增加导致活性增加的模型一致。此外，我们还检测到催化自由能和底物（ATP）结合自由能之间存在很强的相关性，这也与Adk的催化模型一致。从一般角度来看，对于所涉及的状态之间溶剂可及表面积存在相当大差异的情况，尿素似乎可用于将折叠蛋白质的构象平衡调节为更扩展的状态。这种效应补充了渗透剂的作用，例如三甲胺 N-氧化物，有利于更紧凑的蛋白质状态。
Proteins are often functionally dependent on conformational changes that allow them to sample structural states that are sparsely populated in the absence of a substrate or binding partner. The distribution of such structural microstates is governed by their relative stability, and the kinetics of their interconversion is governed by the magnitude of associated activation barriers. Here, we have explored the interplay among structure, stability, and function of a selected enzyme, adenylate kinase (Adk), by monitoring changes in its enzymatic activity in response to additions of urea. For this purpose we used a P-31 NMR assay that was found useful for heterogeneous sample compositions such as presence of urea. It was found that Adk is activated at low urea concentrations whereas higher urea concentrations unfolds and thereby deactivates the enzyme. From a quantitative analysis of chemical shifts, it was found that urea redistributes preexisting structural microstates, stabilizing a substrate-bound open state at the expense of a substrate-bound closed state. Adk is rate-limited by slow opening of substrate binding domains and the urea-dependent redistribution of structural states is consistent with a model where the increased activity results from an increased rate-constant for domain opening. In addition, we also detected a strong correlation between the catalytic free energy and free energy of substrate (ATP) binding, which is also consistent with the catalytic model for Adk. From a general perspective, it appears that urea can be used to modulate conformational equilibria of folded proteins toward more expanded states for cases where a sizeable difference in solvent-accessible surface area exists between the states involved. This effect complements the action of osmolytes, such as trimethylamine N-oxide, that favor more compact protein states.