Streptokinase binds to human plasmin with high affinity, perturbs the plasmin active site, and induces expression of a substrate recognition exosite for plasminogen.
Binding of streptokinase (SK) to plasminogen (Pg) conformationally activates the zymogen and converts both Pg and plasmin (Pm) into specific Pg activators. The interaction of SK with Pm and its relationship to the mechanism of Pg activation were evaluated in equilibrium binding studies with active site-labeled fluorescent Pm derivatives and in kinetic studies of SK-induced changes in the catalytic specificity of Pm. SK bound to fluorescein-labeled and native Pm with dissociation constants of 11 +/- 2 pm and 12 +/- 4 pm, which represented a 1,000-10,000-fold higher affinity than determined for Pg. Stoichiometric binding of SK to native Pm was followed by generation of a two-fragment form of SK cleaved at Lys(59) (SK'), which exhibited an indistinguishable affinity for labeled Pm, while a truncated, SK(55-414) species had a 120-360-fold reduced affinity. Binding of SK to native Pm was accompanied by a >50-fold enhancement in specificity for activation of Pg, which was paralleled by a surprising 2.6-10-fold loss of specificity of Pm for 8 of 11 tripeptide-pNA substrates. Further studies with Pm labeled at the active site with 2-anilinonaphthalene-6-sulfonic acid demonstrated directly that binding of SK to Pm resulted in expression of a new substrate binding exosite for Pg on the SK.Pm complex. It is concluded that SK activates Pg in part by preferential binding to the active zymogen conformation. High affinity binding of SK to Pm enhances Pg substrate specificity principally through emergence of a substrate recognition exosite.
Boxrud
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Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Fay
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Bock
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D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005341:Fibrinolysin / Q000378:metabolism*; D005456:Fluorescent Dyes; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D010958:Plasminogen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013300:Streptokinase / Q000378:metabolism*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D010958:Plasminogen; D013300:Streptokinase; D005341:Fibrinolysin