Inhibitory Mechanism of Serpins:  Loop Insertion Forces Acylation of Plasminogen Activator by Plasminogen Activator Inhibitor-1

• Published in: Biochemistry (Easton) 1998-11, Vol.37 (44), p.15491-15502
• Main Authors: Kvassman, Jan-Olov, Verhamme, Ingrid, Shore, Joseph D
• Format: Article
• Language: English
• Subjects: Acylation; Animals; Binding Sites; Catalysis; Cattle; Electrophoresis, Polyacrylamide Gel; Humans; Kinetics; Plasminogen Activator Inhibitor 1 - chemistry; Plasminogen Activator Inhibitor 1 - metabolism; Plasminogen Activator Inhibitor 1 - pharmacology; Plasminogen Activators - antagonists & inhibitors; Plasminogen Activators - chemistry; Plasminogen Activators - metabolism; Protein Structure, Secondary; Serine - metabolism; Substrate Specificity; Tissue Plasminogen Activator - metabolism; Trypsin - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9814787

Serpin inhibitors are believed to form an acyl enzyme intermediate with their target proteinases which is stabilized through insertion of the enzyme-linked part of the reactive center loop (RCL) as strand 4 in β-sheet A of the inhibitor. To test critically the role and timing of these steps in the reaction of the plasminogen activator inhibitor PAI-1, we blocked the vacant position 4 in β-sheet A of this serpin with an octapeptide. The peptide-blocked PAI-1 was a substrate for both tissue-type plasminogen activator (tPA) and trypsin and was hydrolyzed at the scissile bond. The reactivity of the peptide-blocked substrate PAI-1 was compared to that of the unmodified inhibitor by rapid acid quenching as well as photometric techniques. With trypsin as target, the limiting rate constants for enzyme acylation were essentially the same with inhibitor and substrate PAI-1 (21−23 s-1), as were also the associated apparent second-order rate constants (2.8−2.9 μM-1 s-1). With tPA, inhibitor and substrate PAI-1 reacted identically to form a tightly bound Michaelis complex (K d ≈ K m ≈ 20 nM). The limiting rate constant for acylation of tPA, however, was 57 times faster with inhibitor PAI-1 (3.3 s-1) than with the substrate form (0.059 s-1), resulting in a 5-fold difference in the corresponding second-order rate constants (13 vs 2.5 μM-1 s-1). We attribute the ability of tPA to discriminate between the two PAI-1 forms to exosite bonds that cannot occur with trypsin. The exosite bonds retain specifically the distal part of the PAI-1 RCL in the substrate pocket, which favors a reversal of the acylation step. Acylation of tPA becomes effective only by separating the products of the acylation step. With substrate PAI-1, this depends on passive displacement of bonds, whereas with inhibitor PAI-1, separation is accomplished by loop insertion that pulls tPA from its docking site on PAI-1, resulting in faster acylation than with substrate PAI-1.