Structure-Function Analysis of the Streptokinase Amino Terminus (Residues 1-59)

Streptokinase (SK) binds to plasminogen (Pg) to form a complex that converts substrate Pg to plasmin. Residues 1-59 of SK regulate its capacity to induce an active site in bound Pg by a nonproteolytic mechanism and to activate substrate Pg in a fibrin-independent manner. We analyzed 24 SK mutants to better define the functional properties of SK-(1-59). Mutations within the ^αβ₁ strand (residues 17-26) of SK completely prevented non-proteolytic active site induction in bound Pg and rendered SK incapable of protecting plasmin from inhibition by α ₂-antiplasmin. However, when fibrin-bound, the activities of ^αβ₁ strand mutants were similar to that of wild-type (WT) SK and resistant to α₂-antiplasmin. Mutation of Ile¹ of SK also prevented nonproteolytic active site induction in bound Pg. However, unlike ^αβ₁ strand mutants, the functional defect of Ile¹ mutants was not relieved by fibrin, and complexes of Ile¹ mutants and plasmin were resistant to α₂-antiplasmin. Plasmin enhanced the activities of ^αβ₁ strand and Ile¹ mutants, suggesting that SK-plasmin complexes activated mutant SK·Pg complexes by hydrolyzing the Pg Arg⁵⁶¹-Val⁵⁶² bond. Mutational analysis of Glu³⁹ of SK suggested that a salt bridge between Glu ³⁹ and Arg⁷¹⁹ of Pg is important, but not essential, for nonproteolytic active site induction in Pg. Deleting residues 1-59 rendered SK dependent on plasmin and fibrin to generate plasminogen activator (PA) activity. However, the PA activity of SK-(60-414) in the presence of fibrin was markedly reduced compared with WT SK. Despite its reduced PA activity, the fibrinolytic potency of SK-(60-414) was greater than that of WT SK at higher (but not lower) SK concentrations due to its capacity to deplete plasma Pg. These studies define mechanisms by which the SK α domain regulates rapid active site induction in bound Pg, contributes to the resistance of the SK-plasmin complex to α₂-antiplasmin, and controls fibrin-independent Pg activation.