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

We have demonstrated that interactions within the conserved serpin breach region play a direct role in the critical step of 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 (Tr...

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Bibliographic Details
Published in:Biochemistry (Easton) 2003-10, Vol.42 (42), p.12260-12272
Main Authors: Blouse, Grant E, Perron, Michel J, Kvassman, Jan-Olov, Yunus, Saadia, Thompson, Jannah H, Betts, Russell L, Lutter, Leonard C, Shore, Joseph D
Format: Article
Language:English
Subjects:
Base Sequence
Circular Dichroism
DNA Primers
Electrophoresis, Polyacrylamide Gel
Kinetics
Models, Molecular
Mutagenesis, Site-Directed
Plasminogen Activator Inhibitor 1 - chemistry
Plasminogen Activator Inhibitor 1 - genetics
Plasminogen Activator Inhibitor 1 - physiology
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Serpins - metabolism
Tryptophan - chemistry
Tryptophan - genetics
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Summary:We have demonstrated that interactions within the conserved serpin breach region play a direct role in the critical step of 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, with phenylalanine altered the kinetics of the reaction mechanism and impeded the ability of PAI-1 to spontaneously become latent without compromising the inherent rate of cleaved loop insertion or partitioning between the final inhibited serpin−proteinase complex and hydrolyzed serpin. Kinetic dissection of the PAI-1 inhibitory mechanism using multiple target proteinases made possible the identification of a single rate-limiting intermediate step coupled to the molecular interactions within the breach region. This step involves the initial insertion of the proximal reactive center loop hinge residue(s) into β-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 retarding the rate-limiting step in tPA reactions in which strong exosite interactions must be overcome for the reaction to proceed.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi034737n