Abstract
Binding of the fibrinolytic proteinase plasmin (Pm) to streptokinase (SK) in a tight stoichiometric complex transforms Pm into a potent proteolytic activator of plasminogen. SK binding to the catalytic domain of Pm, with a dissociation constant of 12 pm, is assisted by SK Lys(414) binding to a Pm kringle, which accounts for a 11-20-fold affinity decrease when Pm lysine binding sites are blocked by 6-aminohexanoic acid (6-AHA) or benzamidine. The pathway of SK.Pm catalytic complex formation was characterized by stopped-flow kinetics of SK and the Lys(414) deletion mutant (SKDeltaK414) binding to Pm labeled at the active site with 5-fluorescein ([5F]FFR-Pm) and the reverse reactions by competitive displacement of [5F]FFR-Pm with active site-blocked Pm. The rate constants for the biexponential fluorescence quenching caused by SK and SKDeltaK414 binding to [5F]FFR-Pm were saturable as a function of SK concentration, reporting encounter complex affinities of 62-110 nm in the absence of lysine analogs and 4900-6500 and 1430-2200 nm in the presence of 6-AHA and benzamidine, respectively. The encounter complex with SKDeltaK414 was approximately 10-fold weaker in the absence of lysine analogs but indistinguishable from that of native SK in the presence of 6-AHA and benzamidine. The studies delineate for the first time the sequence of molecular events in the formation of the SK.Pm catalytic complex and its regulation by kringle ligands. Analysis of the forward and reverse reactions supports a binding mechanism in which SK Lys(414) binding to a Pm kringle accompanies near-diffusion-limited encounter complex formation followed by two slower, tightening conformational changes.
Highlights
Drug and streptococcal pathogenicity factor, streptokinase (SK) activates Pg to Pm through a unique mechanism [1, 2]
The present study describes the first rapid-reaction kinetics investigation of active site fluorescently labeled Pm binding to SK, characterizes the binding and conformational intermediates on the pathway, and identifies lysine binding sites (LBS)-dependent steps
Expression of the data as ⌬F/Fo showed that the reactions started at zero, indicating no change associated with encounter complex formation
Summary
Protein Purification and Characterization—Human [Glu]Pg carbohydrate form 2 ([Glu]Pg2) was purified from plasma by published procedures [38, 39]. Measurements were corrected for background (Յ10%) by subtraction of blanks lacking [5F]FFR-Pm. Simultaneous nonlinear least-squares fitting with SCIENTIST software was performed of the [5F]FFR-Pm titrations with SK or SK⌬K414 in the absence and presence of fixed FFR-Pm concentrations by the cubic equation for tight competitive binding of a single ligand (SK or SK⌬K414) to labeled ([5F]FFR-Pm) and non-labeled acceptor (FFR-Pm) (49 –51). The cubic equation is an exact solution for competitive binding of one ligand to two acceptors, typically one fluorescently labeled that reports the interaction and the non-labeled competitor, under conditions where the assumption cannot be made that free and total ligand concentrations are approximately equal (49 –51) This analysis gave the dissociation constant (KC) and stoichiometric factor (m) for competitive binding of SK or SK⌬K414 to FFR-Pm as well as the maximum fluorescence intensity change (⌬Fmax/Fo), the dissociation constant (KD), and the stoichiometric factor (n) for binding of SK or SK⌬K414 to [5F]FFR-Pm (49 –51)
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