Resolution of conformational activation in the kinetic mechanism of plasminogen activation by streptokinase

Abstract

Streptokinase (SK) activates plasminogen (Pg) by specific binding and nonproteolytic expression of the Pg catalytic site, initiating Pg proteolysis to form the fibrinolytic proteinase, plasmin (Pm). The SK-induced conformational activation mechanism was investigated in quantitative kinetic and equilibrium binding studies. Progress curves of Pg activation by SK monitored by chromogenic substrate hydrolysis were parabolic, with initial rates (v1) that indicated no transient species and subsequent rate increases (v2). The v1 dependence on SK concentration for [Glu]Pg and [Lys]Pg was hyperbolic with dissociation constants corresponding to those determined in fluorescence-based binding studies for the native Pg species, identifying v 1 as rapid SK binding and conformational activation. Comparison of [Glu]Pg and [Lys]Pg activation showed an ∼12-fold higher affinity of SK for [Lys]Pg that was lysine-binding site dependent and no such dependence for [Glu]Pg. Stopped-flow kinetics of SK binding to fluorescently labeled Pg demonstrated at least two fast steps in the conformational activation pathway. Characterization of the specificity of the conformationally activated SK·[Lys]Pg* complex for tripeptide-p-nitroanilide substrates demonstrated 5-18- and 10-130-fold reduced specificity (kcat/K m) compared with SK·Pm and Pm, respectively, with differences in Km and kcat dependent on the P1 residue. The results support a kinetic mechanism in which SK binding and reversible conformational activation occur in a rapid equilibrium, multistep process.