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, and the resulting complexes initiate proteolytic activity of host plasminogen which facilitates bacterial colonization of the host organism. We have investigated the kinetic mechanism of the plasminogen activation mediated by a novel two-domain (alpha, beta) streptokinase isolated from Streptococcus uberis (Sk(U)) with specificity toward bovine plasminogen. The interaction between Sk(U) and plasminogen occurred in two steps: (1) rapid association of 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 parameters: K(m) < or = 1.5 microM and k(cat) = 0.55 s(-)(1). The ability of proteolytic fragments of Sk(U) to activate plasminogen was investigated. 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 plasminogen activation in complex with plasmin, but not with plasminogen, and thereby exhibited functional similarity to the staphylokinase. The fusion protein 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) the N-terminal alpha-domain, including a native N-terminus, was necessary for "virgin" activation of the associated plasminogen in the Sk(U)-PgA complex and (2) the C-terminal beta-domain of Sk(U) is important for recognition of the substrate in the Sk(U)-PgA complex.
Johnsen
L B
LB
Protein Chemistry Laboratory and Laboratory of Gene Expression, Department of Molecular and Structural Biology, University of Aarhus, Denmark.
Rasmussen
L K
LK
Petersen
T E
TE
Etzerodt
M
M
Fedosov
S N
SN
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D015951:Factor Xa / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008970:Molecular Weight; D010958:Plasminogen / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013291:Streptococcus / Q000201:enzymology; D013300:Streptokinase / Q000737:chemistry* / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D010958:Plasminogen; D013300:Streptokinase; D015951:Factor Xa