Mutation of the Highly Conserved Tryptophan in the Serpin Breach Region Alters the Inhibitory Mechanism of Plasminogen Activator Inhbitor-1

摘要

We have demonstrated that interactions within the conserved serpin breach region play a direct role in the critical step fo the serpin reaction in which the acyl-enzyme intermediate must first be exposed to hydrolyzing water and aqueous deacylation.Substitution of the breach tryptophan in PAI-1(Trp175),a residue found in virtually all known serpins,wiht phenylalanine altered the kinetics of the reaction inherent rate of cleaved loop insertion or partitioning between th final inhibitory mechanism using multiple target molecular interactions within the breach region.This step involves the initial insertion of the proximal reactive center loop hinge residue(s) into beta-sheet A and facilitates translocation of the distal P'-side of the cleaved reactive center loop from the substrate cleft of the proteinase.Substitution of the tryptophan residue raised the kinetic barrier restricting the initial loop insertion event,significantly reatarding the residue raised the kinetic barrier restricting the initial loop insertion eventmsignificantly retarding the rate-limiting step in tPA reactions in which strong exosite interactions must be overcome for the reaction to proceed.