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

• Published in: Biochemistry (Easton) Vol. 40; no. 39; pp. 11742 - 11756
• Main Authors: Olson, Steven T, Swanson, Richard, Day, Duane, Verhamme, Ingrid, Kvassman, Jan, Shore, Joseph D
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02.10.2001
• Subjects: Acylation; Electrophoresis, Polyacrylamide Gel; Humans; Kinetics; Plasminogen Activator Inhibitor 1 - chemistry; Plasminogen Activator Inhibitor 1 - genetics; Plasminogen Activator Inhibitor 1 - metabolism; Protein Conformation; Serpins - chemistry; Serpins - metabolism; Spectrometry, Fluorescence; Tissue Plasminogen Activator - chemistry; Tissue Plasminogen Activator - metabolism; Trypsin - chemistry; Trypsin - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0107290

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-β-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 ∼20 μM 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 μM and of k lim of 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 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.