NMR Identification of Transient Complexes Critical to Adenylate Kinase Catalysis

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• 作者：Jö ; rgen Å ; dé ; n ; Magnus Wolf-Watz
• 刊名：Journal of the American Chemical Society
• 出版年：2007
• 出版时间：November 14, 2007
• 年：2007
• 卷：129
• 期：45
• 页码：14003 - 14012
• 全文大小：501K
• 年卷期：v.129,no.45(November 14, 2007)
• ISSN：1520-5126

文摘

A fundamental question in protein chemistry is how the native energy landscape of enzymesenables efficient catalysis of chemical reactions. Adenylate kinase is a small monomeric enzyme thatcatalyzes the reversible conversion of AMP and ATP into two ADP molecules. Previous structural studieshave revealed that substrate binding is accompanied by large rate-limiting spatial displacements of boththe ATP and AMP binding motifs. In this report a solution-state NMR approach was used to probe thenative energy landscape of adenylate kinase in its free form, in complex with its natural substrates, and inthe presence of a tight binding inhibitor. Binding of ATP induces a dynamic equilibrium in which the ATPbinding motif populates both the open and the closed conformations with almost equal populations. A similarscenario is observed for AMP binding, which induces an equilibrium between open and closed conformationsof the AMP binding motif. These ATP- and AMP-bound structural ensembles represent complexes thatexist transiently during catalysis. Simultaneous binding of AMP and ATP is required to force both substratebinding motifs to close cooperatively. In addition, a previously unknown unidirectional energetic couplingbetween the ATP and AMP binding sites was discovered. On the basis of these and previous results, wepropose that adenylate kinase belongs to a group of enzymes whose substrates act to shift pre-existingequilibria toward catalytically active states.