The oxidative inactivation of plasminogen activator inhibitor type 1 results from a conformational change in the molecule and does not require the involvement of the P1' methionine.
Plasminogen activator inhibitor 1 (PAI-1) is sensitive to oxidative inactivation, and it has been suggested that specific oxidation of a methionine residue, Met347, situated in the P1' position of the reactive center may be the cause of the inactivation. To test this hypothesis we have purified and biochemically characterized mutant proteins of PAI-1 in which Met347 and either of two other methionines, Met266 or Met354, has been replaced with oxidation-resistant valine residues. The mutant proteins were found to be equally sensitive to oxidation as wild-type PAI-1, suggesting that a specific oxidation of the P1' Met347 is not responsible for the inactivation. When PAI-1 was oxidized, circular dichroism analysis revealed a rapid conformational change that correlated to the loss of inhibitory activity. The oxidation sensitivity of PAI-1 was enhanced dramatically in the presence of 0.001% sodium dodecyl sulfate, and the circular dichroism spectrum was significantly different from that of untreated PAI-1, suggesting that the increased sensitivity to oxidation may be caused by a conformational change in the inhibitor molecule. Taken together, our data suggest that the oxidative inactivation of PAI-1 is not caused by the specific oxidation of the P1' methionine but results from a conformational change in the protein structure.
Strandberg
L
L
Department of Applied Cell and Molecular Biology, Chemistry University of Umeå, Sweden.
Lawrence
D A
DA
Johansson
L B
LB
Ny
T
T
D001483:Base Sequence; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D066298:In Vitro Techniques; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010084:Oxidation-Reduction; D015849:Plasminogen Inactivators / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013329:Structure-Activity Relationship; D013388:Succinimides / Q000737:chemistry
D009841:Oligonucleotides; D015849:Plasminogen Inactivators; D011994:Recombinant Proteins; D013388:Succinimides; C015752:N-chlorosuccinimide; D012967:Sodium Dodecyl Sulfate; D008715:Methionine; D006861:Hydrogen Peroxide