Resolution of Michaelis complex, acylation, and conformational change steps in the reactions of the serpin, plasminogen activator inhibitor-1, with tissue plasminogen activator and trypsin

Michaelis complex, acylation, and conformational change steps were resolved in the reactions of the serpin, plasminogen activator inhibitor-1 (PAI-1), with tissue plasminogen activator (tPA) and trypsin 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 fluorescence changes 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 the P9 label perturbing the interactions by 10-60-fold. Rapid kinetic analyses of the labeled PAI-1-inactive enzyme interactions were consistent with a single-step reversible binding process involving no conformational change. Blocking of PAI-1 reactive loop-fi-sheet A interactions through mutation of the P14 Thr --> Arg or annealing a reactive center loop peptide into sheet A did not weaken the binding of the 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 variants previously shown to report acylation and reactive loop-sheet A interactions during the PAI-1-proteinase reaction. Rapid kinetic analyses showed saturation of the reaction rate constant and, in the case of the P1'-labeled PAI-1 reaction, biphasic changes in fluorescence indicative of an intermediate resembling the noncovalent complex on the path to the covalent complex. Indistinguishable K-M and k(lim) values of similar to 20 muM and 80-90 s(-1) for reaction of the two labeled PAI-1s with trypsin suggested that a diffusion-limited association 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 K-M of 1.7 and 0. 1 muM and of k(lim) of 17 and 2.6 s(-1) for tPA reactions with P1' and P9-labeled PAI-Is, respectively, suggested that tPA-PAI-1 I exosite interactions, sensitive to the effects of labeling, promoted a rapid association of PAI-1 and tPA and reversible formation of an acyl-enzyme complex but impeded a rate-limiting burial of the reactive loop leading to trapping of the acyl-enzyme complex. Together, the results suggest a kinetic pathway for formation of the covalent complex between PAI-1 and proteinases involving the initial formation of a Michaelis-type noncovalent complex without significant conformational change, followed by reversible acylation and irreversible reactive loop conformational change steps that trap the proteinase in a covalent complex.