Kinetics of plasmin activation of single chain urinary-type plasminogen activator (scu-PA) and demonstration of a high affinity interaction between scu-PA and plasminogen.

The activation kinetics of single chain urinary-type plasminogen activator (scu-PA) by plasmin have been studied in detail. Nonstandard Michaelis-Menten kinetics were observed. To explain our results, we propose a model in which plasmin can exist in two conformations of lower activity (kcat/Km = 1.4 x 10(6) M-1 s-1) or higher activity (kcat/Km = 16.7 x 10(6) M-1 s-1) depending on whether a lysine binding site is occupied or free, respectively. These kinetic studies demonstrate that scu-PA interacts at this binding site (KD approximately 30 nM) and so is able to act as both a substrate and effector in this reaction. Binding was also demonstrated between scu-PA and Glu- or Lys-plasminogen at a high affinity site (KD approximately 65 nM), sensitive to the presence of lysine analogs. This suggests that scu-PA may be almost completely bound to plasminogen in plasma under normal physiological conditions and provides a possible explanation for the fibrin specificity of this activator, as discussed.