Kinetic and structural characterization of a two-domain streptokinase: Dissection of domain functionality

The mammalian protease plasminogen can be activated by bacterial activators , the three-domain (alpha, beta, gamma) streptokinases and the one-domain ( alpha) staphylokinases. These activators act as plasmin(ogen) cofactors, an d the resulting complexes initiate proteolytic activity of host plasminogen which facilitates bacterial colonization of the host organism. We have inv estigated the kinetic mechanism of the plasminogen activation mediated by a novel two-domain (alpha, beta) streptokinase isolated from Streptococcus u beris (Sk(U)) with specificity toward bovine plasminogen. The interaction b etween Sk(U) and plasminogen occurred in two steps: (1) rapid association o f the proteins and (2) slow transition to the active complex Sk(U)-PgA, The complex Sk(U)-PgA converted plasminogen to plasmin with the following para meters: K-m less than or equal to 1.5 mu M and k(cat) = 0.55 s(-1). The abi lity of proteolytic fragments of Sk(U) to activate plasminogen was investig ated. Only two C-terminal segments (97-261 and 123-261), which both contain the beta-domain (126-261), were shown to be active. They initiated plasmin ogen activation in complex with plasmin, but not with plasminogen, and ther eby exhibited functional similarity to the staphylokinase. The fusion prote in His(6)-Sk(U) (i.e., Sk(U) with a small N-terminal tag) acted exclusively in complex with plasmin as well. These observations demonstrate that (1) t he N-terminal alpha-domain, including a native N-terminus, was necessary fo r "virgin" activation of the associated plasminogen in the Sk(U)-PgA comple x and (2) the C-terminal beta-domain of Sk(U) is important for recognition of the substrate in the Sk(U)-PgA complex.