A Concerted Structural Transition in the Plasminogen Activator Inhibitor-1 Mechanism of Inhibition

Abstract

The inhibition mechanism of serpins requires a change in structure to entrap the target proteinase as a stable acyl-enzyme complex. Although it has generally been assumed that reactive center loop insertion and associated conformational change proceeds in a concerted manner, this has not been demonstrated directly. Through the substitution of tryptophan with 7-azatryptophan and an analysis of transient reaction kinetics, we have described the formation of an inhibited serpin-proteinase complex as a single concerted transition of the serpin structure. Replacement of the four tryptophans of plasminogen activator inhibitor type-1 (PAI-1) with the spectrally unique analogue 7-azatryptophan permitted observations of conformational changes in the serpin but not those of the proteinase. Formation of covalent acyl-enzyme complexes, but not noncovalent Michaelis complexes, with tissue-type plasminogen activator (t-PA) or urokinase (u-PA) resulted in rapid decreases of fluorescence coinciding with insertion of the reactive center loop and expansion of beta-sheet A. Insertion of an octapeptide consisting of the P14-P7 residues of the reactive center loop into beta-sheet A produced the same conformational change in serpin structure measured by 7-azatryptophan fluorescence, suggesting that introduction of the proximal loop residues induces the structural rearrangement of the serpin molecule. The atom specific modification of the tryptophan indole rings through analogue substitution produced a proteinase specific effect on function. The reduced inhibitory activity of PAI-1 against t-PA but not u-PA suggested that the mechanism of loop insertion is sensitive to the intramolecular interactions of one or more tryptophan residues.

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

“…The fluorescence change accompanying the reaction of NBD-labeled PAI-1 variants with at least a 5-fold molar excess of each proteinase was monitored at 480 nm, using an emission filter (Oriel 51300) with a cut-off below 515 nm (21,22,27). As found previously, all stopped-flow traces fit best to a double exponential function, yielding pseudo-first-order rate constants k obs1 and k obs2 (27).…”

“…Mutations were validated by DNA sequencing of the entire PAI-1 gene. All mutant and wild-type constructs were expressed in the E. coli strain BL21(DE3)pLysS and purified as previously described (22,26). Labeling of single Cys-containing PAI-1 variants (i.e.…”

“…The concentrations of recombinant PAI-1 variants were measured at 280 nm, using an absorption coefficient of 0.93 ml mg Ϫ1 cm Ϫ1 and an M r of 43,000 (26,28). Activity of each labeled PAI-1 variant was assessed by formation of a covalent complex with a 2-fold molar excess of proteinase and nonreducing SDS-PAGE analysis (22,29).…”

“…stoichiometry of inhibition) were determined as described (22). Following incubation for 20 min in HEPES buffer containing 0.1% polyethylene glycol 8000, residual enzyme activity was determined by diluting an aliquot of the reaction into a 100 M solution of appropriate chromogenic substrate (Spectrozyme tPA for tPA, Spectrozyme UK for uPA, and S-2222 for ␤-trypsin) and monitoring the linear increase in absorbance at 405 nm for 1 min.…”

“…The progress curves for inhibition were monitored at 405 nm and fitted to a single exponential function with a linear component to obtain the pseudo-first-order rate constant, k obs . The second-order rate constant, k app , was calculated for each proteinase-PAI-1 pair as described (22), using the following K m values: 77.9, 40.0, and 38.1 M for Spectrozyme tPA , Spectrozyme UK , and S-2222, respectively (23).…”