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.
Sazonova
I Y
IY
Cardiovascular Biology Laboratory, Harvard School of Public Health and the Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
Houng
A K
AK
Chowdhry
S A
SA
Robinson
B R
BR
Hedstrom
L
L
Reed
G L
GL
D000595:Amino Acid Sequence; D000818:Animals; D000933:Antifibrinolytic Agents / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D003001:Cloning, Molecular; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000378:metabolism; D010960:Plasminogen Activators / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013291:Streptococcus / Q000201:enzymology*; D013300:Streptokinase / Q000737:chemistry* / Q000378:metabolism*
D000933:Antifibrinolytic Agents; D011994:Recombinant Proteins; D010958:Plasminogen; D013300:Streptokinase; D010960:Plasminogen Activators; D005341:Fibrinolysin; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus