title: Characterizing Active Site Conformational Heterogeneity along the Trajectory of an Enzymatic Phosphoryl Transfer Reaction.
abstract: States along the phosphoryl transfer reaction catalyzed by the nucleoside monophosphate kinase UmpK were captured and changes in the conformational heterogeneity of conserved active site arginine side-chains were quantified by NMR spin-relaxation methods. In addition to apo and ligand-bound UmpK, a transition state analog (TSA) complex was utilized to evaluate the extent to which active site conformational entropy contributes to the transition state free energy. The catalytically essential arginine side-chain guanidino groups were found to be remarkably rigid in the TSA complex, indicating that the enzyme has evolved to restrict the conformational freedom along its reaction path over the energy landscape, which in turn allows the phosphoryl transfer to occur selectively by avoiding side reactions.
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120, Heidelberg, Germany.
authors.forename: Cathleen
authors.initials: C
authors.lastname: Zeymer
authors.affiliation: Division of Biosciences, Institute of Structural and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK.
authors.forename: Nicolas D
authors.initials: ND
authors.lastname: Werbeck
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120, Heidelberg, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Zimmermann
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120, Heidelberg, Germany. Jochen.Reinstein@mpimf-heidelberg.mpg.de.
authors.forename: Jochen
authors.initials: J
authors.lastname: Reinstein
authors.affiliation: Division of Biosciences, Institute of Structural and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK. d.hansen@ucl.ac.uk.
authors.forename: D Flemming
authors.identifier: 0000-0003-0891-220X
authors.initials: DF
authors.lastname: Hansen
mesh_terms: D020134:Catalytic Domain; D004023:Dictyostelium / Q000201:enzymology; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry / Q000378:metabolism*; D011789:Quantum Theory; D013816:Thermodynamics
chemical_list: D008024:Ligands; D009703:Nucleoside-Phosphate Kinase
keywords: NMR spectroscopy; active-site dynamics; arginine; conformational entropy; nucleoside monophosphate kinase