Open AccessCharacterization of the Interaction Between Plasminogen and Staphylokinase
Author(s) -
Lijnen H. Roger,
Cock Frans,
Hoef Berthe,
Schlott Bernhard,
Collen Désiré
Publication year - 1994
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1994.tb20005.x
Subject(s) - staphylokinase , plasmin , streptokinase , chemistry , binding site , recombinant dna , kringle domain , lysine , dissociation constant , stereochemistry , active site , enzyme , microbiology and biotechnology , biochemistry , biology , amino acid , receptor , myocardial infarction , gene , psychology , psychiatry
Binding parameters [association ( k a ) and dissociation ( k d ) rate constants, and affinity constants ( K a = k a / k d )] for the interaction between recombinant staphylokinase (SakSTAR) and plasmin(ogen) were determined by real‐time biospecific interaction analysis. The K a value for binding of SakSTAR to native human Glu‐plasminogen was 0.93×10 8 M ‐1 as compared to 2.0×10 8 M ‐1 and 1.6×10 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 K a , values of 4.0×10 8 M ‐1 and 8.4×10 8 M‐ ‐1 , respectively, whereas active‐site blocked LMM‐plasmin (a plasmin derivative lacking kringles 1– 4) and the plasmin B‐chain bound with K a values of 1.0×10 8 M ‐1 and 0.49×10 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 ( K a values of 1.2×10 5 M ‐1 and 2.9×10 5 M ‐1 , respectively). The binding of these plasminogen derivatives to streptokinase occurred with similar relative K a values. The K a 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 K a 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 Lysplasminogen 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.