Interconversion of active and inactive conformations of urokinase-type plasminogen activator

The catalytic activity of serine proteases depends on a salt-bridge between the amino group of residue 16 and the side chain of Asp194. The salt-bridge stabilizes the oxyanion hole and the S1 specificity pocket of the protease. Some serine proteases exist in only partially active forms, in which the amino group of residue 16 is exposed to the solvent. Such a partially active state is assumed by a truncated form of the murine urokinase-type plasminogen activator (muPA), consisting of residues 16-243. Here we investigated the allosteric interconversion between partially active states and the fully active state. Both a monoclonal antibody (mU3) and a peptidic inhibitor (mupain-1--16) stabilize the active state. The epitope of mU3 is located in the 37- and 70-loops at a site homologous to exosite I of thrombin. The N-terminus((Ile16)) of muPA((16--243)) was less exposed upon binding of mU3 or mupain-1--16. In contrast, introduction of the mutations F40Y or E137A into muPA((16--243)) increased exposure of the N-terminus((Ile16)) and resulted in large changes in the thermodynamic parameters for mupain-1--16 binding. We conclude that the distorted state of muPA((16--243)) is conformationally ordered upon binding of ligands to the active site and upon binding of mU3 to the 37- and 70-loops. Our study establishes the 37- and 70-loops as a unique site for binding to compounds stabilizing the active state of serine proteases.