Rapid Binding of Plasminogen to Streptokinase in a Catalytic Complex Reveals a 3-Step Mechanism [Molecular Biophysics]

August 19th, 2014 by Verhamme, I. M., Bock, P. E.

Rapid kinetics demonstrate a 3-step pathway of streptokinase (SK) binding to plasminogen (Pg), the zymogen of plasmin (Pm). Formation of a fluorescently silent encounter complex is followed by two conformational tightening steps reported by fluorescence quenches. Forward reactions were defined by time courses of biphasic quenching during complex formation between SK or its COOH-terminal Lys414 deletion mutant (SKΔK414) and active site-labeled [Lys]Pg ([5F]FFR-[Lys]Pg), and by the SK dependences of the quench rates. Active site-blocked Pm rapidly displaced [5F]FFR-[Lys]Pg from the complex. The encounter and final SK·[5F]FFR-[Lys]Pg complexes were weakened similarly by SK Lys414 deletion and blocking of lysine-binding sites (LBS) on Pg kringles with 6-aminohexanoic acid (6-AHA) or benzamidine. Forward and reverse rates for both tightening steps were unaffected by 6-AHA, whereas benzamidine released constraints on the first conformational tightening. This indicated that binding of SK Lys414 to Pg kringle 4 plays a role in recognition of Pg by SK. The substantially lower affinity of the final SK·Pg complex compared to SK·Pm is characterized by a ~25-fold weaker encounter complex, and ~40-fold faster off-rates for the second conformational step. The results suggest that effective Pg encounter requires SK Lys414 engagement and significant non-LBS interactions with the protease domain, whereas Pm binding additionally requires contributions of other lysines. This difference may be responsible for the lower affinity of the SK·Pg complex, and the expression of a weaker "pro"-exosite for binding of a second Pg in the substrate mode compared to SK·Pm.