Coupling of Conformational and Proteolytic Activation in the Kinetic Mechanism of Plasminogen Activation by Streptokinase
Journal of Biological Chemistry
Binding of streptokinase (SK) to plasminogen (Pg) induces conformational activation of the zymogen and initiates its proteolytic conversion to plasmin (Pm). The mechanism of coupling between conformational activation and Pm formation was investigated in kinetic studies. Parabolic time courses of Pg activation by SK monitored by chromogenic substrate hydrolysis had initial rates (v 1 ) representing conformational activation and subsequent rates of activity increase (v 2 ) corresponding to the
... e of Pm generation determined by a specific discontinuous assay. The v 2 dependence on SK concentration for [Lys]Pg showed a maximum rate at a Pg to SK ratio of ϳ2:1, with inhibition at high SK concentrations. [Glu]Pg and [Lys]Pg activation showed similar kinetic behavior but much slower activation of [Glu]Pg, due to an ϳ12-fold lower affinity for SK and an ϳ20-fold lower k cat /K m . Blocking lysine-binding sites on Pg inhibited SK⅐Pg* cleavage of [Lys]Pg to a rate comparable with that of [Glu]Pg, whereas [Glu] Pg activation was not significantly affected. The results support a kinetic mechanism in which SK activates Pg conformationally by rapid equilibrium formation of the SK⅐Pg* complex, followed by intermolecular cleavage of Pg to Pm by SK⅐Pg* and subsequent cleavage of Pg by SK⅐Pm. A unified model of SK-induced Pg activation suggests that generation of initial Pm by SK⅐Pg* acts as a self-limiting triggering mechanism to initiate production of one SK equivalent of SK⅐Pm, which then converts the remaining free Pg to Pm.