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 activate s the zymogen and converts both Pg and plasmin (Pm) into specific Pg activa tors. The interaction of SI( 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-induce d changes in the catalytic specificity of Pm. SK bound to fluorescein-label ed 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 o f a two-fragment form of SK cleaved at Lys(59) (SK'), which exhibited an in distinguishable affinity for labeled Pm, while a truncated, SK55-414 specie s had a 120-360-fold reduced affinity. Binding of SK to native Pm was accom panied 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 ac tive site with 2-anilinonaphthalene-6-sulfonic acid demonstrated directly t hat binding of SK to Pm resulted in expression of a new substrate binding e xosite 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 affi nity binding of SK to Pm enhances Pg substrate specificity principally thro ugh emergence of a substrate recognition exosite.