Rapid-reaction Kinetic Characterization of the Pathway of Streptokinase-Plasmin Catalytic Complex Formation

Full Time Course Kinetics of the Streptokinase-Plasminogen Activation Pathway

“…Comparison of the effects of kringle ligands and a SK Lys 414 deletion mutant identified the encounter complex as the primary source of the LBS dependence of SK⅐Pm catalytic complex formation, whereas the two conformational changes are less affected. The first step in the binding pathway requires on-rate constants of Ն1 nM Ϫ1 s Ϫ1 , indicating near-diffusion controlled encounter complex formation (45). Remarkably, the fitted rate constant for SK⅐Pm formation determined here was 1.2 nM Ϫ1 s Ϫ1 (Table 1).…”

“…Rapid reaction kinetics of SK binding to active site-labeled Pm demonstrate a three-step mechanism of rapid encounter complex formation followed by two sequential affinity-enhancing conformational changes (45). Comparison of the effects of kringle ligands and a SK Lys 414 deletion mutant identified the encounter complex as the primary source of the LBS dependence of SK⅐Pm catalytic complex formation, whereas the two conformational changes are less affected.…”

“…Although this might affect the distribution of Pm Free and SK⅐Pm measured in Assay 1, it does not affect the concentration of Pm Total . Previous studies show that the off-rate constant for displacement of Pm by FFR-Pm is ϳ0.0008 s Ϫ1 , slow enough for the dissociation of Pm to be negligible in the 2-min quench time (45). In any case, only Pm Total appears in the conservation equations used to calculate SK Free , Pg Free , and Pm Free .…”

Plasminogen Substrate Recognition by the Streptokinase-Plasminogen Catalytic Complex Is Facilitated by Arg253, Lys256, and Lys257 in the Streptokinase β-Domain and Kringle 5 of the Substrate

“…Interaction of the COOH-terminal Lys 414 residue of SK with a Pg/Pm kringle domain is responsible for the LBS-dependent enhancement of the affinity of SK⅐[Lys]Pg* and SK⅐Pm catalytic complex formation (22). Recent rapid reaction kinetic studies of the SK⅐Pm binding pathway demonstrated that interaction of Lys 414 with a Pm kringle enhances formation of an initial rapid equilibrium SK⅐Pm encounter complex, succeeded by two sequential, tightening conformational changes, to achieve an overall dissociation constant of ϳ12 pM (26). The Pg/Pm kringle domain responsible for the enhancement of SK⅐Pg* and SK⅐Pm complex formation is not known.…”

Rapid Binding of Plasminogen to Streptokinase in a Catalytic Complex Reveals a Three-step Mechanism

“…Here we explore for the first time the steps on the pathway of SK binding to Pg and identify critical differences with Pm binding (59) that are the basis for the ϳ4,000-fold lower affinity of [5F]FFR-[Lys]Pg for SK (27). Stopped-flow kinetics of SK binding to labeled [Lys]Pg and [Glu]Pg defined the forward reactions of complex stabilization.…”