title: Characterization of the interaction between plasminogen and staphylokinase.
abstract: Binding parameters [association (ka) and dissociation (kd) rate constants, and affinity constants (Ka = ka/kd)] for the interaction between recombinant staphylokinase (SakSTAR) and plasmin(ogen) were determined by real-time biospecific interaction analysis. The Ka value for binding of SakSTAR to native human Glu-plasminogen was 0.93 x 10(8) M-1 as compared to 2.0 x 10(8) M-1 and 1.6 x 10(8) M-1, respectively, for the binding to [S741A]recombinant plasminogen or Lys-[S741A]recombinant plasminogen (intact or proteolytically degraded plasminogen with the active site Ser741 replaced by alanine). Binding of SakSTAR to active plasmin or to active-site blocked plasmin occurred with Ka values of 4.0 x 10(8) M-1 and 8.4 x 10(8) M-1, respectively, whereas active-site blocked LMM-plasmin (a plasmin derivative lacking kringles 1-4) and the plasmin B-chain bound with Ka values of 1.0 x 10(8) M-1 and 0.49 x 10(8) M-1, respectively. Lysine-binding site I (a plasminogen derivative consisting of kringles 1-3) and lysine-binding site II (a plasminogen derivative consisting of kringle 4) bound with much lower affinity (Ka values of 1.2 x 10(5) M-1 and 2.9 x 10(5) M-1, respectively). The binding of these plasminogen derivatives to streptokinase occurred with similar relative Ka values. The Ka values for binding of the plasmin-SakSTAR complex to streptokinase and binding of the plasmin-streptokinase complex to SakSTAR, were, respectively, 44-fold and 30-fold lower than the values for free plasmin. The Ka for binding of plasminogen to the inactive mutants [M26R]Sak42D or [M26A]Sak42D (site-specific mutagenesis of Met26 to arginine or alanine) were 10-20-fold lower than that of native staphylokinase. These results indicate that: (a) the affinity of staphylokinase for Glu-plasminogen and Lys-plasminogen is comparable; (b) the active site in the plasmin molecule is not required for binding; (c) kringle structures 1-4 of plasminogen do not contribute significantly to plasminogen binding of staphylokinase; (d) Met26 in staphylokinase is important for its high-affinity binding to plasminogen; (e) the binding sites on plasmin for staphylokinase and streptokinase overlap at least partially.
authors.affiliation: Center for Molecular and Vascular Biology, University of Leuven, Belgium.
authors.forename: H R
authors.initials: HR
authors.lastname: Lijnen
authors.forename: F
authors.initials: F
authors.lastname: De Cock
authors.forename: B
authors.initials: B
authors.lastname: Van Hoef
authors.forename: B
authors.initials: B
authors.lastname: Schlott
authors.forename: D
authors.initials: D
authors.lastname: Collen
mesh_terms: D000327:Adsorption; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D009097:Multienzyme Complexes; D010958:Plasminogen / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D010958:Plasminogen; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus