Rapid formation of an anion-sensitive active site in stoichiometric complexes of streptokinase and human [Glu1]plasminogen.
We have examined the time-dependent appearance of amidolytic activity in equimolar complexes of streptokinase (SK) and human [Glu1]plasminogen (HPg) under various conditions. When stoichiometric levels of the two proteins are incubated and assayed in hypotonic buffers at 4 degrees C, amidolytic activity toward the chromogenic substrate D-Val-Leu-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 C1- 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 HPg. 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 C1- 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 C1- to a much greater extent than is that of SK-HPg'.
Chibber
B A
BA
Radek
J T
JT
Morris
J P
JP
Castellino
F J
FJ
D000838:Anions / Q000378:metabolism*; D001665:Binding Sites; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010958:Plasminogen / Q000378:metabolism*; D013300:Streptokinase / Q000378:metabolism*
D000838:Anions; D046911:Macromolecular Substances; D010958:Plasminogen; D013300:Streptokinase