A fluorescent probe study of plasminogen activator inhibitor-1. Evidence for reactive center loop insertion and its role in the inhibitory mechanism.
A mutant recombinant plasminogen activator inhibitor 1 (PAI-1) was created (Ser-338-->Cys) in which cysteine was placed at the P9 position of the reactive center loop. Labeling this mutant with N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) provided a molecule with a fluorescent probe at that position. The NBD-labeled mutant was almost as reactive as wild type but was considerably more stable. Complex formation with tissue or urokinase type plasminogen activator (tPA or uPA), and cleavage between P3 and P4 with a catalytic concentration of elastase, all resulted in identical 13-nm blue shifts of the peak fluorescence emission wavelength and 6.2-fold fluorescence enhancements. Formation of latent PAI showed the same 13-nm spectral shift with a 6.7-fold fluorescence emission increase, indicating that the NBD probe is in a slightly more hydrophobic milieu. These changes can be attributed to insertion of the reactive center loop into the beta sheet A of the inhibitor in a manner that exposes the NBD probe to a more hydrophobic milieu. The rate of loop insertion due to tPA complexation was followed using stopped flow fluorimetry. This rate showed a hyperbolic dependence on tPA concentration, with a half-saturation concentration of 0.96 microM and a maximum rate constant of 3.4 s-1. These results demonstrate experimentally that complexation with proteases is presumably associated with loop insertion. The identical fluorescence changes obtained with tPa.PAI-1 and uPA.PAI-1 complexes and elastase-cleaved PAI-1 strongly suggest that in the stable protease-PAI-1 complex the reactive center loop is cleaved and inserted into beta sheet A and that this process is central to the inhibition mechanism.
Shore
J D
JD
Division of Biochemical Research, Henry Ford Hospital, Detroit, Michigan 48202-2689.
Day
D E
DE
Francis-Chmura
A M
AM
Verhamme
I
I
Kvassman
J
J
Lawrence
D A
DA
Ginsburg
D
D
D009327:4-Chloro-7-nitrobenzofurazan; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D005456:Fluorescent Dyes; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010069:Oxadiazoles; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D012694:Serine; D013050:Spectrometry, Fluorescence; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors
D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D010069:Oxadiazoles; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D012694:Serine; C035573:4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; D009327:4-Chloro-7-nitrobenzofurazan; D003545:Cysteine