title: Full time course kinetics of the streptokinase-plasminogen activation pathway.
abstract: Our previously hypothesized mechanism for the pathway of plasminogen (Pg) activation by streptokinase (SK) was tested by the use of full time course kinetics. Three discontinuous chromogenic substrate initial rate assays were developed with different quenching conditions that enabled quantitation of the time courses of Pg depletion, plasmin (Pm) formation, transient formation of the conformationally activated SK·Pg* catalytic complex intermediate, formation of the SK·Pm catalytic complex, and the free concentrations of Pg, Pm, and SK. Analysis of full time courses of Pg activation by five concentrations of SK along with activity-based titrations of SK·Pg* and SK·Pm formation yielded rate and dissociation constants within 2-fold of those determined previously by continuous measurement of parabolic chromogenic substrate hydrolysis and fluorescence-based equilibrium binding. The results obtained with orthogonal assays provide independent support for a mechanism in which the conformationally activated SK·Pg* complex catalyzes an initial cycle of Pg proteolytic conversion to Pm that acts as a trigger. Higher affinity binding of the formed Pm to SK outcompetes Pg binding, terminating the trigger cycle and initiating the bullet catalytic cycle by the SK·Pm complex that converts the residual Pg into Pm. The new assays can be adapted to quantitate SK-Pg activation in the context of SK- or Pg-directed inhibitors, effectors, and SK allelic variants. To support this, we show for the first time with an assay specific for SK·Pg* that fibrinogen forms a ternary SK·Pg*·fibrinogen complex, which assembles with 200-fold enhanced SK·Pg* affinity, signaled by a perturbation of the SK·Pg* active site.
authors: From the Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2561.
authors: Miranda
authors: M
authors: Nolan
authors: Samantha D
authors: SD
authors: Bouldin
authors: Paul E
authors: PE
authors: Bock
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D002863:Chromogenic Compounds; D004789:Enzyme Activation; D005341:Fibrinolysin / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D010958:Plasminogen / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013291:Streptococcus / Q000201:enzymology; D013300:Streptokinase / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D002863:Chromogenic Compounds; D046912:Multiprotein Complexes; D010958:Plasminogen; D013300:Streptokinase; D005341:Fibrinolysin
keywords: Enzyme Kinetics; Fibrinolysis; Full Time Course Kinetics; Plasmin; Plasminogen; Serine Protease; Streptokinase; Zymogen Activation
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