Rapid-reaction kinetic characterization of the pathway of streptokinase-plasmin catalytic complex formation.
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.
Verhamme
Ingrid M
IM
Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Bock
Paul E
PE
D015119:Aminocaproic Acid / Q000737:chemistry; D001550:Benzamidines / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D005341:Fibrinolysin / Q000737:chemistry*; D017353:Gene Deletion; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013300:Streptokinase / Q000737:chemistry*
D001550:Benzamidines; D008024:Ligands; D013300:Streptokinase; D005341:Fibrinolysin; D008239:Lysine; C032157:benzamidine; D015119:Aminocaproic Acid