The mechanism of a bacterial plasminogen activator intermediate between streptokinase and staphylokinase.

The therapeutic properties of plasminogen activators are dictated by their mechanism of action. Unlike staphylokinase, a single domain protein, streptokinase, a 3-domain (alpha, beta, and gamma) molecule, nonproteolytically activates human (h)-plasminogen and protects plasmin from inactivation by alpha(2)-antiplasmin. Because a streptokinase-like mechanism was hypothesized to require the streptokinase gamma-domain, we examined the mechanism of action of a novel two-domain (alpha,beta) Streptococcus uberis plasminogen activator (SUPA). Under conditions that quench trace plasmin, SUPA nonproteolytically generated an active site in bovine (b)-plasminogen. SUPA also competitively inhibited the inactivation of plasmin by alpha(2)-antiplasmin. Still, the lag phase in active site generation and plasminogen activation by SUPA was at least 5-fold longer than that of streptokinase. Recombinant streptokinase gamma-domain bound to the b-plasminogen.SUPA complex and significantly reduced these lag phases. The SUPA-b.plasmin complex activated b-plasminogen with kinetic parameters comparable to those of streptokinase for h-plasminogen. The SUPA-b.plasmin complex also activated h-plasminogen but with a lower k(cat) (25-fold) and k(cat)/K(m) (7.9-fold) than SK. We conclude that a gamma-domain is not required for a streptokinase-like activation of b-plasminogen. However, the streptokinase gamma-domain enhances the rates of active site formation in b-plasminogen and this enhancing effect may be required for efficient activation of plasminogen from other species.

Leucine 42 in the fibronectin motif of streptokinase plays a critical role in fibrin-independent plasminogen activation.

The NH(2) terminus (residues 1-59) of streptokinase (SK) is a molecular switch that permits fibrin-independent plasminogen activation. Targeted mutations were made in recombinant (r) SK1-59 to identify structural interactions required for this process. Mutagenesis established the functional roles of Phe-37and Glu-39, which were projected to interact with microplasmin in the activator complex. Mutation of Leu-42 (rSK1-59(L42A)), a conserved residue in the SK fibronectin motif that lacks interactions with microplasmin, strongly reduced plasminogen activation (k(cat) decreased 50-fold) but not amidolysis (k(cat) decreased 1.5-fold). Otherwise rSK1-59(L42A) and native rSK1-59 were indistinguishable in several parameters. Both displayed saturable and specific binding to Glu-plasminogen or the remaining SK fragment (rSKDelta59). Similarly rSK1-59 and rSK1-59(L42A) bound simultaneously to two different plasminogen molecules, indicating that both plasminogen binding sites were intact. However, when bound to SKDelta59, rSK1-59(L42A) was less effective than rSK1-59 in restructuring the native conformation of the SK A domain, as detected by conformation-dependent monoclonal antibodies. In the light of previous studies, these data provide evidence that SK1-59 contributes to fibrin-independent plasminogen activation through 1) intermolecular interactions with the plasmin in the activator complex, 2) binding interactions with the plasminogen substrate, and 3) intramolecular interactions that structure the A domain of SK for Pg substrate processing.