The Contribution of the Exosite Residues of Plasminogen Activator Inhibitor-1 to Proteinase Inhibition

• Published in: The Journal of biological chemistry Vol. 279; no. 5; pp. 3643 - 3650
• Main Authors: Ibarra, Catherine A, Blouse, Grant E, Christian, Thomas D, Shore, Joseph D
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 30-01-2004
• Subjects: Alanine - chemistry; Amino Acid Sequence; Animals; Binding Sites; Cattle; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Kinetics; Models, Chemical; Molecular Sequence Data; Mutation; Plasminogen Activator Inhibitor 1 - physiology; Protease Inhibitors - pharmacology; Protein Binding; Protein Structure, Secondary; Recombinant Proteins - metabolism; Serpins - metabolism; Spectrometry, Fluorescence; Spectrophotometry; Swine; Thermodynamics; Trypsin - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M310601200

The binding of plasminogen activator inhibitor-1 (PAI-1) to serine proteinases, such as tissue-type plasminogen activator
(tPA) and urokinase-type plasminogen activator (uPA), is mediated by the exosite interactions between the surface-exposed
variable region-1, or 37-loop, of the proteinase and the distal reactive center loop (RCL) of PAI-1. Although the contribution
of such interactions to the inhibitory activity of PAI-1 has been established, the specific mechanistic steps affected by
interactions at the distal RCL remain unknown. We have used protein engineering, stopped-flow fluorimetry, and rapid acid
quenching techniques to elucidate the role of exosite interactions in the neutralization of tPA, uPA, and β-trypsin by PAI-1.
Alanine substitutions at the distal P4′ (Glu-350) and P5′ (Glu-351) residues of PAI-1 reduced the rates of Michaelis complex
formation ( k a ) and overall inhibition ( k app ) with tPA by 13.4- and 4.7-fold, respectively, whereas the rate of loop insertion or final acyl-enzyme formation ( k lim ) increased by 3.3-fold. The effects of double mutations on k a , k lim , and k app were small with uPA and nonexistent with β-trypsin. We provide the first kinetic evidence that the removal of exosite interactions
significantly alters the formation of the noncovalent Michaelis complex, facilitating the release of the primed side of the
distal loop from the active-site pocket of tPA and the subsequent insertion of the cleaved reactive center loop into β-sheet
A. Moreover, mutational analysis indicates that the P5′ residue contributes more to the mechanism of tPA inhibition, notably
by promoting the formation of a final Michaelis complex.