title: Critical role of W60d in thrombin allostery.
abstract: Mutation of residue W60d of thrombin, located 17 A from the Na+ binding site, suppresses Na+ binding and the functional differences between the slow and fast forms. The molecular basis for the long-range effect of this mutation is provided by a conspicuous network of water molecules which connects the Na+ binding environment to the specificity sites S1 and S2 of the enzyme. The mutation appears to stabilize thrombin in a hybrid conformation that is overall similar to the slow form, but with the fibrinogen recognition site functioning as in the fast form. It also affects the switch in specificity from fibrinogen to protein C linked to the release Na+ and the fast-->slow conversion. Under physiological conditions of pH, temperature and NaCl concentration, the W60dS mutant behaves as an anticoagulant. It has a reduced activity toward fibrinogen by 22-fold, while the reduction of protein C activation in the presence of saturating concentrations of thrombomodulin is less than 2-fold. Even more remarkable is the cleavage of fibrin I monomer leading to release of fibrinopeptide B, which is reduced by more than 130-fold. This property is reminiscent of the snake venom ancrod, which only releases fibrinopeptide A, and adds substantially to the anticoagulant potency of the W60dS mutant. In fact, the clotting time in the presence of this mutant is prolonged more than 40-fold compared to the wild-type.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: E R
authors.initials: ER
authors.lastname: Guinto
authors.forename: E
authors.initials: E
authors.lastname: Di Cera
mesh_terms: D000494:Allosteric Regulation; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D012964:Sodium; D013917:Thrombin