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.
Highlights
We previously proposed a mechanism for plasminogen (Pg) activation by streptokinase (SK)
The previous studies were based on continuous steady-state kinetics in the presence of a chromogenic substrate and on competitive equilibrium binding experiments with the native proteins using active sitefluorescein-labeled Pg/Pm analogs as binding probes
The bimolecular rate constants for Pm generation by the SK1⁄7Pg* and SK1⁄7Pm catalytic complexes previously determined were 0.5 Ϯ 0.05 and 1.16 Ϯ 0.04 MϪ1 sϪ1, respectively, whereas here they were 1.17 Ϯ 0.17 and 2.4 Ϯ 0.4 MϪ1 sϪ1, respectively. The reason for this relatively small difference is not known, but it may be due to differences in the assumptions made here for the discontinuous assays and those in the previous studies from analysis of continuous parabolic assays in the presence of chromogenic substrate
Summary
We previously proposed a mechanism for plasminogen (Pg) activation by streptokinase (SK). 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 SK1⁄7Pg* catalytic complex intermediate, formation of the SK1⁄7Pm catalytic complex, and the free concentrations of Pg, Pm, and SK. The results obtained with orthogonal assays provide independent support for a mechanism in which the conformationally activated SK1⁄7Pg* 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 SK1⁄7Pm 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. We show for the first time with an assay specific for SK1⁄7Pg* that fibrinogen forms a ternary SK1⁄7Pg*1⁄7fibrinogen complex, which assembles with 200-fold enhanced SK1⁄7Pg* affinity, signaled by a perturbation of the SK1⁄7Pg* active site
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