Rapid formation of an anion-sensitive active site in stoichiometric complexes of streptokinase and human [Glu1]plasminogen.

Abstract

We have examined the time-dependent appearance of amidolytic activity in equimolar complexes of streptokinase (SK) and human [Glul]plasminogen (HPg) under various conditions. When stoichiometric levels of the two proteins are incubated and assayed in hypotonic buffers at 4°C, amidolytic activity toward the chromogenic substrate D-ValLeu-Lys-p-nitroanilide (S-2251), within the resulting complex, appears with an observed first-order rate constant of 1.03 ± 0.06/min. On the other hand, when the assay for amidolytic activity is conducted at a Cl-concentration of 0.15 M, this same activity develops with an observed first-order rate constant of 0.13 ± 0.01/min. Under all conditions of assay of importance to the mechanism proposed, the only molecular components present are SK and H]Pg. The rate of appearance of an enzyme species displaying amidolytic activity is dependent on the anion in its assay; a much slower rate constant is obtained with Cl than with AcO-. These observations are consistent with the formation, within the complex, of an early anion-sensitive active site (SK-HPg*) that is converted to a form (SK-HPg') that is much less sensitive to the presence of anions. During the time period of this process, no conversion of plasminogen to plasmin occurs within the complex. Steadystate kinetic properties of SK-HPg* and SK-HPg' have been measured toward the substrate S-2251. Consistent with the mechanism suggested above, the amidolytic activity of SK-HPg* is inhibited by Cl to a much greater extent than is that of SK-HPg'.Fibrinolytic activity in mammalian plasma is primarily expressed through the action of plasmin, a serine protease generated consequent to activation of its plasma protein precursor, plasminogen. This activation occurs as a result of cleavage of a single peptide bond, ArgSWq-Vall6l, in the zymogen (1) and is mediated by a variety of proteins, such as urokinase (2), streptokinase (SK) (3), and tissue activators (4). Of these, the bacterial protein SK is unique in that it does not appear to be a protease, since a synthetic substrate, or a protein substrate, for SK, other than plasminogen, has not as yet been identified.Current understanding of the mechanism whereby SK activates human plasminogen (HPg) is based on studies that correlate appearance of enzymic activity with the molecular species of proteins present in equimolar complexes of HPg and SK (for a comprehensive review of this area, see ref. 5). From such work, it has been concluded that the major activators of HPg are equimolar complexes of SK and HPg (6), in which an active site has developed in the plasminogen moiety of the complex (7), and a similar complex of SK and human plasmin (HPm), which uses the active site present in HPm for its activator activity (8). This latter complex forms by intramolecular (9, 10) peptide bond cleavage of HPg, within the SK-HPg complex, and/or by stoichiometric interaction of SK with previously formed HPm.Recent work from our laboratory has shown that at very early reaction times, at least two types o...

Resolution of Conformational Activation in the Kinetic Mechanism of Plasminogen Activation by Streptokinase

“…SK binding to Pg and conformational expression of the Pg catalytic site in the SK⅐Pg* complex occurred rapidly and without rate-controlling intermediates on the seconds time scale. The slow (minutes) isomerization of the SK⅐Pg* complex reported in previous studies (22,23) with [Glu]Pg at low temperature and/or low chloride concentrations was not observed in our studies. This does not mean that this conformational change does not occur but likely represents differences in the experimental conditions, where this event apparently occurs rapidly under our conditions.…”

“…These results parallel the effects of 6-AHA on SK binding and conformational activation and indicate that the fast phase of SK binding to [Lys]Pg is linked to interactions with lysine-binding sites on Pg. One of these fast reactions may correspond to the isomerization of the SK⅐Pg* complex at low chloride concentrations described previously (22). The results indicate that further rapid-reaction kinetic studies should enable resolution of the individual molecular events on the pathway of conformational Pg activation by SK.…”

“…Early studies (14,16,17) demonstrated that interaction of SK with Pg produced the activated Pg catalytic site in the SK⅐Pg* complex. Subsequent kinetic studies indicated that Pg activation involved an initially formed SK⅐Pg* activation complex and an isomerized form of the complex (22,23). The isomerization was time-, chloride ion-, and fibrinogen-dependent, and the active complexes interconverted slowly under certain experimental conditions.…”

The Lack of Binding of VEK-30, an Internal Peptide from the Group A Streptococcal M-like Protein, PAM, to Murine Plasminogen Is due to Two Amino Acid Replacements in the Plasminogen Kringle-2 Domain

“…For more quantitative estimates, the absorbance at 405 nm, which measures both the activation rate of the tested hPg and also the cleavage of the substrate, S2251, was plotted against time squared (Fig. 4, C and D) (30), and the slopes compared with each other under identical initial concentrations of all components. The data show that the activity generated in the WT⅐hPg complex with SK is accelerated 14-, 305-, and 360-fold by 1.…”

Roles for chloride ion and fibrinogen in the activation of [Glu1]plasminogen in human plasma.

“…This hypothesis, while explaining some phenomena seen during the classic hypercoagulable state known as disseminated intravascular coagulation, may not encompass all molecular events occurring during thrombotic episodes and use of TPA as a thrombolytic agent. Roles for Cl -and fibrinogen have been implicated in human plasminogen ([Glu1]Pg) activation in purified systems using streptokinase (2,3), urokinase (4,5), and TPA (6) as activators. These data show that Cl-, which exists in plasma at 90 mM, could have a significant inhibitory effect on [Glu']Pg activation and that the influence of fibrinogen on the initial rate of [Glu']Pg activation could be physiologically important during enhanced fibrinolysis in vivo.…”