| Abstract
| - Michaelis complex, acylation, and conformational change steps were resolved in the reactionsof the serpin, plasminogen activator inhibitor-1 (PAI-1), with tissue plasminogen activator (tPA) andtrypsin by comparing the reactions of active and Ser 195-inactivated enzymes with site-specific fluorescent-labeled PAI-1 derivatives that report these events. Anhydrotrypsin or S195A tPA-induced fluorescencechanges in P1‘-Cys and P9-Cys PAI-1 variants labeled with the fluorophore, NBD, indicative of a substrate-like interaction of the serpin reactive loop with the proteinase active-site, with the P1‘ label but not theP9 label perturbing the interactions by 10−60-fold. Rapid kinetic analyses of the labeled PAI-1-inactiveenzyme interactions were consistent with a single-step reversible binding process involving noconformational change. Blocking of PAI-1 reactive loop-β-sheet A interactions through mutation of theP14 Thr → Arg or annealing a reactive center loop peptide into sheet A did not weaken the binding ofthe inactive enzymes, suggesting that loop-sheet interactions were unlikely to be induced by the binding.Only active trypsin and tPA induced the characteristic fluorescence changes in the labeled PAI-1 variantspreviously shown to report acylation and reactive loop-sheet A interactions during the PAI-1-proteinasereaction. Rapid kinetic analyses showed saturation of the reaction rate constant and, in the case of theP1‘-labeled PAI-1 reaction, biphasic changes in fluorescence indicative of an intermediate resembling thenoncovalent complex on the path to the covalent complex. Indistinguishable KM and klim values of ∼20μM and 80−90 s-1 for reaction of the two labeled PAI-1s with trypsin suggested that a diffusion-limitedassociation of PAI-1 and trypsin and rate-limiting acylation step, insensitive to the effects of labeling,controlled covalent complex formation. By contrast, differing values of KM of 1.7 and 0.1 μM and of klimof 17 and 2.6 s-1 for tPA reactions with P1‘ and P9-labeled PAI-1s, respectively, suggested that tPA-PAI-1 exosite interactions, sensitive to the effects of labeling, promoted a rapid association of PAI-1 andtPA and reversible formation of an acyl−enzyme complex but impeded a rate-limiting burial of the reactiveloop leading to trapping of the acyl−enzyme complex. Together, the results suggest a kinetic pathwayfor formation of the covalent complex between PAI-1 and proteinases involving the initial formation ofa Michaelis-type noncovalent complex without significant conformational change, followed by reversibleacylation and irreversible reactive loop conformational change steps that trap the proteinase in a covalentcomplex.
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