NMR identification of transient complexes critical to adenylate kinase catalysis
NMR identification of transient complexes critical to adenylate kinase catalysis
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DOI：
10.1021/ja075055g
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发表时间：
2007-11-14
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;Wolf-Watz, Magnus
蛋白质化学中的一个基本问题是，酶的天然能量格局如何使化学反应能够有效催化。腺苷酸激酶是一种催化AMP和ATP可逆转化为两个ADP分子的小单体酶。以往的结构研究表明，底物结合伴随着ATP和AMP结合基序的大限速空间位移。在这份报告中，溶液状态核磁共振方法被用来探索腺苷酸激酶在其自由形式、与其天然底物的复合体以及在紧密结合的抑制剂存在的情况下的天然能量格局。ATP的结合诱导了一种动态平衡，在这种平衡中，ATP结合基序以几乎相等的布居分布在开放和闭合构象中。AMP结合也有类似的情况，它导致AMP结合基序的开放构象和闭合构象之间的平衡。这些与ATP和AMP结合的结构簇代表了在催化过程中短暂存在的络合物。AMP和ATP的同时结合需要迫使两个底物结合基序协同关闭。此外，还发现了先前未知的ATP和AMP结合位点之间的单向能量耦合。基于这些和以前的结果，我们认为腺苷酸激酶属于一组酶，其底物作用于将先前存在的平衡转移到催化活性状态。
A fundamental question in protein chemistry is how the native energy landscape of enzymes enables efficient catalysis of chemical reactions. Adenylate kinase is a small monomeric enzyme that catalyzes the reversible conversion of AMP and ATP into two ADP molecules. Previous structural studies have revealed that substrate binding is accompanied by large rate-limiting spatial displacements of both the ATP and AMP binding motifs. In this report a solution-state NMR approach was used to probe the native energy landscape of adenylate kinase in its free form, in complex with its natural substrates, and in the presence of a tight binding inhibitor. Binding of ATP induces a dynamic equilibrium in which the ATP binding motif populates both the open and the closed conformations with almost equal populations. A similar scenario is observed for AMP binding, which induces an equilibrium between open and closed conformations of the AMP binding motif. These ATP- and AMP-bound structural ensembles represent complexes that exist transiently during catalysis. Simultaneous binding of AMP and ATP is required to force both substrate binding motifs to close cooperatively. In addition, a previously unknown unidirectional energetic coupling between the ATP and AMP binding sites was discovered. On the basis of these and previous results, we propose that adenylate kinase belongs to a group of enzymes whose substrates act to shift pre-existing equilibria toward catalytically active states.