title: Subnanometre enzyme mechanics probed by single-molecule force spectroscopy.
abstract: Enzymes are molecular machines that bind substrates specifically, provide an adequate chemical environment for catalysis and exchange products rapidly, to ensure fast turnover rates. Direct information about the energetics that drive conformational changes is difficult to obtain. We used subnanometre single-molecule force spectroscopy to study the energetic drive of substrate-dependent lid closing in the enzyme adenylate kinase. Here we show that in the presence of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A), closing and opening of both lids is cooperative and tightly coupled to inhibitor binding. Surprisingly, binding of the substrates ADP and ATP exhibits a much smaller energetic drive towards the fully closed state. Instead, we observe a new dominant energetic minimum with both lids half closed. Our results, combining experiment and molecular dynamics simulations, give detailed mechanical insights into how an enzyme can cope with the seemingly contradictory requirements of rapid substrate exchange and tight closing, to ensure efficient catalysis.
authors.affiliation: Physik Department E22, Technische Universität München, James Franck Strasse 1, 85748 Garching, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Pelz
authors.affiliation: Physik Department E22, Technische Universität München, James Franck Strasse 1, 85748 Garching, Germany.
authors.forename: Gabriel
authors.initials: G
authors.lastname: Žoldák
authors.affiliation: Physik Department T38, Technische Universität München, 85748 Garching, Germany.
authors.forename: Fabian
authors.initials: F
authors.lastname: Zeller
authors.affiliation: Physik Department T38, Technische Universität München, 85748 Garching, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Zacharias
authors.affiliation: Physik Department E22, Technische Universität München, James Franck Strasse 1, 85748 Garching, Germany.|Munich Center for Integrated Protein Science, 81377 München, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Rief
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000263:Adenylate Kinase / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002384:Catalysis; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D004798:Enzymes; D056004:Molecular Dynamics Simulation; D036103:Nanotechnology; D052898:Optical Tweezers; D013057:Spectrum Analysis
chemical_list: D001426:Bacterial Proteins; D015226:Dinucleoside Phosphates; D004798:Enzymes; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase