Full Time Course Kinetics of the Streptokinase-Plasminogen Activation Pathway

Abstract

Background:We previously proposed a mechanism for plasminogen (Pg) activation by streptokinase (SK). Results: We tested the mechanism using new discontinuous assays that resolved the full time courses of all the reaction species. Conclusion: Analysis of the results independently validates the proposed SK-Pg activation pathway. Significance: The assays can be applied to SK-Pg activation in other contexts.

Rapid Binding of Plasminogen to Streptokinase in a Catalytic Complex Reveals a Three-step Mechanism

“…Whereas cooperative Lys 414 and other lysine interactions with K4 and K5 and non-LBS interactions with the protease domain contribute to formation of the SK⅐Pm complex, the SK⅐Pg* complex assembly appears to be driven by non-LBS interactions and by SK Lys 414 binding to Pg kringle K4. Consistent with the experimentally determined K m of Ն2 M for substrate Pg binding to the SK⅐Pg* complex and of 270 nM for Pg binding to the SK⅐Pm complex (7) and in the absence of a crystal structure of the SK⅐Pg* complex, we hypothesize that the weaker interaction with Pg in the catalytic complex results in expression of a pro-exosite that binds substrate Pg with lower affinity than the corresponding exosite on the SK⅐Pm complex.…”

“…Group A streptococcal M-like surface proteins bind Pg with high affinity, and the M1 subset lacking Pm-binding motifs binds fibrinogen (Fbg). This allows indirect activation by way of SK⅐Pg*⅐Fbg ternary complex formation (7,77), which proceeds by Fbg binding to the SK⅐Pg* complex rather than SK recruitment on the Pg⅐Fbg complex (7). Group A Streptococcus SK exhibits significant polymorphism (78 -80), and considerable differences exist among SK allelic variants in their efficiency of activating Pg and their recruitment in complexes with Fbg, Fbg fragment D, fibrin, and the plasminogen-binding group A streptococcal M protein (81).…”

Activity Regulation by Fibrinogen and Fibrin of Streptokinase from Streptococcus Pyogenes

“…Thus, Fgn at normal physiological concentrations would appear to be important regulator of the activity of rSK-M1GAS and mechanistic details were investigated by kinetic model building. Fig 2 shows a scheme developed to explain the activity of rSK-M1GAS with Fgn and Pgn, where the core of the model was based on the work of Bock and colleagues using the “trigger and bullet” mechanism [ 13 , 14 , 35 ]. For simplicity, lys-Pgn was studied, to avoid additional reactions involving rSK-M1GAS binding to different conformers of glu-Pgn, or conversion between glu- and lys-Pgn.…”

“…The initial SK•Pgn complex requires no cleavage of zymogen Pgn at Arg 561 -Val 562 , to generate an active site in bound Pgn, but rather the N-terminal Ile residue of SK forms a salt bridge with Asp740 of Pgn to induce an active conformation by a “molecular sexuality” mechanism [ 11 , 12 ]. The active SK•Pgn complex generates Pm (Pm) which results in the formation of the more active SK•Pm complex [ 13 ]. Detailed mechanistic studies have labelled this pathway of Pm generation, through sequential formation of SK•Pgn and SK•Pm complexes, the “trigger and bullet” mechanism [ 14 ].…”

Pathogen activators of plasminogen

“…This approach allows determining the binding and kinetic parameters of both SK•Pg* and SK•Pm formation. This analysis was validated independently by full time courses of Pg activation and three discontinuous chromogenic substrate initial rate assays, orthogonal to the assays described above . The assays use different conditions of quenching with active site‐blocked Pm (FFR‐Pm), 6‐AHA, and α2‐AP to allow quantitation of time courses of Pg depletion, transient formation of conformationally activated SK•Pg*, and formation of Pm and SK•Pm.…”