title: Effect of zymogen domains and active site occupation on activation of prothrombin by von Willebrand factor-binding protein.
abstract: Prothrombin is conformationally activated by von Willebrand factor-binding protein (vWbp) from Staphylococcus aureus through insertion of the NH(2)-terminal residues of vWbp into the prothrombin catalytic domain. The rate of prothrombin activation by vWbp(1-263) is controlled by a hysteretic kinetic mechanism initiated by substrate binding. The present study evaluates activation of prothrombin by full-length vWbp(1-474) through activity progress curve analysis. Additional interactions from the COOH-terminal half of vWbp(1-474) strengthened the initial binding of vWbp to prothrombin, resulting in higher activity and an ∼100-fold enhancement in affinity. The affinities of vWbp(1-263) or vWbp(1-474) were compared by equilibrium binding to the prothrombin derivatives prethrombin 1, prethrombin 2, thrombin, meizothrombin, and meizothrombin(des-fragment 1) and their corresponding active site-blocked analogs. Loss of fragment 1 in prethrombin 1 enhanced affinity for both vWbp(1-263) and vWbp(1-474), with a 30-45% increase in Gibbs free energy, implicating a regulatory role for fragment 1 in the activation mechanism. Active site labeling of all prothrombin derivatives with D-Phe-Pro-Arg-chloromethyl ketone, analogous to irreversible binding of a substrate, decreased their K(D) values for vWbp into the subnanomolar range, reflecting the dependence of the activating conformational change on substrate binding. The results suggest a role for prothrombin domains in the pathophysiological activation of prothrombin by vWbp, and may reveal a function for autocatalysis of the vWbp·prothrombin complexes during initiation of blood coagulation.
authors.affiliation: Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Heather K
authors.initials: HK
authors.lastname: Kroh
authors.forename: Paul E
authors.initials: PE
authors.lastname: Bock
mesh_terms: D001667:Binding, Competitive; D001777:Blood Coagulation; D002352:Carrier Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D004792:Enzyme Precursors / Q000737:chemistry*; D005337:Fibrin / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D013211:Staphylococcus aureus / Q000378:metabolism*; D013816:Thermodynamics; D013917:Thrombin / Q000737:chemistry; D037521:Virulence Factors / Q000737:chemistry; D014841:von Willebrand Factor / Q000737:chemistry*
chemical_list: D002352:Carrier Proteins; D004792:Enzyme Precursors; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D037521:Virulence Factors; D014841:von Willebrand Factor; C028254:von Willebrand factor receptor; D011516:Prothrombin; D005337:Fibrin; D013917:Thrombin