1. Department of Biochemistry and Howard Hughes Medical Institute,
2. Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA
3. Institute of Physics, Martin Luther-University Halle-Wittenberg, D-06120 Halle, Germany
4. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA
5. Laboratoire de Chimie Biophysique, ISIS, Université Louis Pasteur, F-67000 Strasbourg, France
6. Present addresses: University of Umeå, Department of Chemistry, SE-90187 Umeå, Sweden (M.W.-W.); Departments of Molecular and Cellular Physiology and Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA (T.F.); Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Maryland 21201, USA (E.P.); Department of Biochemistry and the Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588, USA (M.A.W.); University at Lübeck, Institute of Physics, 23538 Lübeck, Germany (C.G.H.).

Correspondence to: Christian G. Hübner^3,6Dorothee Kern¹ Correspondence and requests for materials should be addressed to D.K. (Email: dkern@brandeis.edu) or C.G.H. (Email: huebner@physik.uni-luebeck.de).

Abstract

The mechanisms by which enzymes achieve extraordinary rate acceleration and specificity have long been of key interest in biochemistry. It is generally recognized that substrate binding coupled to conformational changes of the substrate–enzyme complex aligns the reactive groups in an optimal environment for efficient chemistry. Although chemical mechanisms have been elucidated for many enzymes, the question of how enzymes achieve the catalytically competent state has only recently become approachable by experiment and computation. Here we show crystallographic evidence for conformational substates along the trajectory towards the catalytically competent 'closed' state in the ligand-free form of the enzyme adenylate kinase. Molecular dynamics simulations indicate that these partially closed conformations are sampled in nanoseconds, whereas nuclear magnetic resonance and single-molecule fluorescence resonance energy transfer reveal rare sampling of a fully closed conformation occurring on the microsecond-to-millisecond timescale. Thus, the larger-scale motions in substrate-free adenylate kinase are not random, but preferentially follow the pathways that create the configuration capable of proficient chemistry. Such preferred directionality, encoded in the fold, may contribute to catalysis in many enzymes.

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