Plasminogen activation by single-chain urokinase in functional isolation. A kinetic study

Area of Science:

• Biochemistry
• Enzymology
• Molecular Biology

Background:

• Single-chain urokinase (sc urokinase) is a proenzyme form of urokinase.
• Understanding the kinetics of sc urokinase is crucial for its therapeutic applications.
• Plasminogen activation is a key step in fibrinolysis.

Purpose of the Study:

• To investigate and compare the kinetics of Glu- and Lys-plasminogen activation by sc urokinase and two-chain urokinase (tc urokinase).
• To characterize the activation of sc urokinase to tc urokinase by plasmin.
• To elucidate the distinct properties of sc urokinase compared to conventional serine protease zymogens.

Main Methods:

• Kinetic analysis of plasminogen activation using fluorescence polarization with fluorescein-labeled aprotinin.
• Measurement of plasmin generation in functional isolation.
• Characterization of sc urokinase activation by plasmin using kinetic parameters (Km, kcat).

Main Results:

• sc urokinase exhibited Michaelis-Menten kinetics for plasminogen activation, with lower catalytic efficiency (kcat/Km) than tc urokinase (16-27-fold lower).
• sc urokinase showed minimal activity against low molecular weight peptide substrates, suggesting an inaccessible substrate-binding pocket.
• The activation of sc urokinase to tc urokinase by plasmin was characterized, with kinetic parameters (Km = 3.0 microM, kcat = 105 min-1).

Conclusions:

• sc urokinase possesses distinct enzymatic properties compared to tc urokinase and conventional serine protease zymogens.
• Plasminogen appears to function as both an effector and a substrate for sc urokinase.
• The findings highlight the unique catalytic mechanism and substrate accessibility of sc urokinase.