title: NMR identification of transient complexes critical to adenylate kinase catalysis.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Umeå, SE-901 87 Umeå, Sweden.
authors.forename: Jörgen
authors.initials: J
authors.lastname: Adén
authors.forename: Magnus
authors.initials: M
authors.lastname: Wolf-Watz
mesh_terms: D000244:Adenosine Diphosphate / Q000138:chemical synthesis* / Q000737:chemistry; D000249:Adenosine Monophosphate / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000263:Adenylate Kinase / Q000737:chemistry*; D002384:Catalysis; D009682:Magnetic Resonance Spectroscopy / Q000379:methods* / Q000592:standards; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012015:Reference Standards
chemical_list: D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase