Article | Published:

Intrinsic motions along an enzymatic reaction trajectory

Nature volume 450, pages 838844 (06 December 2007) | Download Citation

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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Acknowledgements

We thank V. Orekhov at the Swedish NMR Centre for 800 MHz NMR spectrometer time, L. Kay for providing pulse programs, D. Korzhnev for sharing software for NMR relaxation data analysis and J. Hohlbein for Monte Carlo simulation software. We are grateful to K. O. Stetter for providing DNA isolated from A. aeolicus and the Advanced Biomedical Computing Center for CPU hours. This work was supported by NIH grants to D.K. and K.A.H.-W., a DOE grant to D.K., a fellowship from the American Heart Association to M.L., a Volkswagen Foundation grant to C.G.H. and M.O., and the Studienstiftung des Deutschen Volkes to M.O. The research at Harvard was supported in part by a grant from NIH to M.K.

Author Contributions K.A.H.-W., V.T., M.L. and M.O. contributed equally to this work. V.T. solved the X-ray structures with assistance from T.F., E.P., M.A.W. and G.A.P. NMR experiments were performed by K.A.H.-W., V.T. and M.W.-W., with assistance from D.K. Computation was carried out by M.L., with assistance and supervision from M.K. and D.K. FRET experiments were designed and performed by M.O. and K.A.H.-W., with assistance and supervision from C.G.H. and D.K. K.A.H.-W. and D.K. wrote the manuscript, and D.K. supervised all aspects of this work.

Author information

Author notes

    • Magnus Wolf-Watz
    • , Tim Fenn
    • , Ed Pozharski
    • , Mark A. Wilson
    •  & Christian G. Hübner

    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.).

Affiliations

  1. Department of Biochemistry and Howard Hughes Medical Institute,

    • Katherine A. Henzler-Wildman
    • , Vu Thai
    • , Ming Lei
    • , Magnus Wolf-Watz
    •  & Dorothee Kern
  2. Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA

    • Tim Fenn
    • , Ed Pozharski
    • , Mark A. Wilson
    •  & Gregory A. Petsko
  3. Institute of Physics, Martin Luther-University Halle-Wittenberg, D-06120 Halle, Germany

    • Maria Ott
    •  & Christian G. Hübner
  4. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Martin Karplus
  5. Laboratoire de Chimie Biophysique, ISIS, Université Louis Pasteur, F-67000 Strasbourg, France

    • Martin Karplus

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Correspondence to Christian G. Hübner or Dorothee Kern.

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