A FLUORESCENT-PROBE STUDY OF PLASMINOGEN-ACTIVATOR INHIBITOR-1 - EVIDENCE FOR REACTIVE CENTER LOOP INSERTION AND ITS ROLE IN THE INHIBITORYMECHANISM

A mutant recombinant plasminogen activator inhibitor 1 (PAI-1) was cre ated (Ser-338 --> Cys) in which cysteine was placed at the P-9 positio n of the reactive center loop. Labeling this mutant with -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 s table. Complex formation with tissue or urokinase type plasminogen act ivator (tPA or uPA), and cleavage between P-3 and P-4 with a catalytic concentration of elastase, all resulted in identical 13-nm blue shift s of the peak fluorescence emission wavelength and 6.2-fold fluorescen ce enhancements. Formation of latent PAI showed the same 13-nm spectra l shift with a 6.7-fold fluorescence emission increase, indicating tha t the NBD probe is in a slightly more hydophobic milieu. These changes can be attributed to insertion of the reactive center loop into the b eta 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 comp lexation was followed using stopped flow fluorimetry. This rate showed a hyperbolic dependence on tPA concentration, with a half-saturation concentration of 0.96 mu M and a maximum rate constant of 3.4 s(-1), T hese results demonstrate experimentally that complexation with proteas es is presumably associated with loop insertion. The identical fluores cence changes obtained with tPA . PAI-1 and uPA . PAI-1 complexes and elastase-cleaved PAI-1 strongly suggest that in the stable protease-PA I-1 complex the reactive center loop is cleaved and inserted into beta sheet A and that this process is central to the inhibition mechanism.