Enzymatic properties of single‐chain and two‐chain forms of a Lys158→ Glu158 mutant of urokinase–type plasminogen activator

Abstract

Recombinant single‐chain urokinase‐type plasminogen activator (rscu‐PA), in which the plasmin‐sensitive peptide bond Lys158‐Ile159 is destroyed by site‐specific mutagenesis of LYS158 to Glu (rscu‐PA‐Glu158), is quantitatively converted to two‐chain urokinase‐type plasminogen activator (rtcu‐PA‐Glu158) by treatment with endoproteinase Glu‐C (Staphylococcus aureus V8 proteinase). The catalytic efficiency (k2/Km) of rscu‐PA‐Glu158 for the activation of plasminogen is 20 times lower (0.0001 μM−1 s−l) than that of rscu‐PA (0.002 μM−1 s−1). In contrast, rtcu‐PA‐Glu158 has very similar properties to rtcu‐PA obtained by digestion of rscu‐PA with plasmin, including binding to benzamidine‐Sepharose, catalytic efficiency for the activation of plasminogen (0.035 μM−1 s−1 versus 0.046 μM−1 s−1) and fibrinolytic activity in an in vitro plasma clot lysis system. It is concluded that the amino acid in position 158 is a main determinant of the functional properties of single‐chain urokinase‐type plasminogen activator but not of the two‐chain form. Copyright © 1988, Wiley Blackwell. All rights reserved