title: Kinetic dissection of the pre-existing conformational equilibrium in the trypsin fold.
abstract: Structural biology has recently documented the conformational plasticity of the trypsin fold for both the protease and zymogen in terms of a pre-existing equilibrium between closed (E*) and open (E) forms of the active site region. How such plasticity is manifested in solution and affects ligand recognition by the protease and zymogen is poorly understood in quantitative terms. Here we dissect the E*-E equilibrium with stopped-flow kinetics in the presence of excess ligand or macromolecule. Using the clotting protease thrombin and its zymogen precursor prethrombin-2 as relevant models we resolve the relative distribution of the E* and E forms and the underlying kinetic rates for their interconversion. In the case of thrombin, the E* and E forms are distributed in a 1:4 ratio and interconvert on a time scale of 45 ms. In the case of prethrombin-2, the equilibrium is shifted strongly (10:1 ratio) in favor of the closed E* form and unfolds over a faster time scale of 4.5 ms. The distribution of E* and E forms observed for thrombin and prethrombin-2 indicates that zymogen activation is linked to a significant shift in the pre-existing equilibrium between closed and open conformations that facilitates ligand binding to the active site. These findings broaden our mechanistic understanding of how conformational transitions control ligand recognition by thrombin and its zymogen precursor prethrombin-2 and have direct relevance to other members of the trypsin fold.
authors.affiliation: From the Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104.
authors.forename: Austin D
authors.initials: AD
authors.lastname: Vogt
authors.affiliation: From the Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104.
authors.forename: Pradipta
authors.initials: P
authors.lastname: Chakraborty
authors.affiliation: From the Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104 enrico@slu.edu.
authors.forename: Enrico
authors.initials: E
authors.lastname: Di Cera
mesh_terms: D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000737:chemistry* / Q000235:genetics; D014357:Trypsin
chemical_list: D011516:Prothrombin; C012007:prethrombins; D014357:Trypsin
keywords: allostery; kinetics; protease; rapid kinetics; structure-function; thrombin; trypsin; zymogen