title: Identifying sequential substrate binding at the single-molecule level by enzyme mechanical stabilization.
abstract: Enzyme-substrate binding is a dynamic process intimately coupled to protein structural changes, which in turn changes the unfolding energy landscape. By the use of single-molecule force spectroscopy (SMFS), we characterize the open-to-closed conformational transition experienced by the hyperthermophilic adenine diphosphate (ADP)-dependent glucokinase from Thermococcus litoralis triggered by the sequential binding of substrates. In the absence of substrates, the mechanical unfolding of TlGK shows an intermediate 1, which is stabilized in the presence of Mg·ADP(-), the first substrate to bind to the enzyme. However, in the presence of this substrate, an additional unfolding event is observed, intermediate 1*. Finally, in the presence of both substrates, the unfolding force of intermediates 1 and 1* increases as a consequence of the domain closure. These results show that SMFS can be used as a powerful experimental tool to investigate binding mechanisms of different enzymes with more than one ligand, expanding the repertoire of protocols traditionally used in enzymology.
authors.affiliation: †Department of Biological Sciences, Columbia University, Northwest Corner Building, 550 West 120 Street, New York, New York 10027, United States.|‡Laboratorio de Bioquímica y Biología Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago, Chile.
authors.forename: Jaime Andrés
authors.initials: JA
authors.lastname: Rivas-Pardo
authors.affiliation: †Department of Biological Sciences, Columbia University, Northwest Corner Building, 550 West 120 Street, New York, New York 10027, United States.
authors.forename: Jorge
authors.initials: J
authors.lastname: Alegre-Cebollada
authors.affiliation: ‡Laboratorio de Bioquímica y Biología Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago, Chile.
authors.forename: César A
authors.initials: CA
authors.lastname: Ramírez-Sarmiento
authors.affiliation: †Department of Biological Sciences, Columbia University, Northwest Corner Building, 550 West 120 Street, New York, New York 10027, United States.
authors.forename: Julio M
authors.initials: JM
authors.lastname: Fernandez
authors.affiliation: ‡Laboratorio de Bioquímica y Biología Molecular, Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago, Chile.
authors.forename: Victoria
authors.initials: V
authors.lastname: Guixé
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005941:Glucokinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D055595:Mechanical Phenomena; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D058767:Protein Unfolding; D019712:Thermococcus / Q000201:enzymology
chemical_list: D004791:Enzyme Inhibitors; D000244:Adenosine Diphosphate; D005941:Glucokinase
keywords: force−extension; mechanical clamp; mechanical intermediate; single-molecule force spectroscopy; substrate stabilization
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