Partitioning of Serpin-Proteinase Reactions between Stable Inhibition and Substrate Cleavage Is Regulated by the Rate of Serpin Reactive Center Loop Insertion into β-Sheet A

Abstract

The serpin family of serine proteinase inhibitors is a mechanistically unique class of naturally occurring proteinase inhibitors that trap target enzymes as stable covalent acyl-enzyme complexes. This mechanism appears to require both cleavage of the serpin reactive center loop (RCL) by the proteinase and a significant conformational change in the serpin structure involving rapid insertion of the RCL into the center of an existing ␤-sheet, serpin ␤-sheet A. The present study demonstrates that partitioning between inhibitor and substrate modes of reaction can be altered by varying either the rates of RCL insertion or deacylation using a library of serpin RCL mutants substituted in the critical P 14 hinge residue and three different proteinases. We further correlate the changes in partitioning with the actual rates of RCL insertion for several of the variants upon reaction with the different proteinases as determined by fluorescence spectroscopy of specific RCL-labeled inhibitor mutants. These data demonstrate that the serpin mechanism follows a branched pathway, and that the formation of a stable inhibited complex is dependent upon both the rate of the RCL conformational change and the rate of enzyme deacylation.The serpins are a gene family that encode a wide variety of proteins, including many of the proteinase inhibitors in plasma, as well as other non-inhibitory proteins such as steroid binding globulins and ovalbumin (1). The serpins are thought to share a common tertiary structure (2) and to have evolved from a common ancestor (3). Current understanding of serpin structure is based largely on seminal x-ray crystallographic studies by Loebermann and co-workers (4) on one member of the family, ␣ 1 -antitrypsin, also called ␣ 1 -proteinase inhibitor. An interesting feature of this structure was that the two residues normally comprising the reactive center (Met-Ser) peptide bond were found on opposite ends of the molecule, separated by almost 70 Å. Based on this structure, these authors proposed a model structure for native serpins, where the reactive center is part of an exposed loop, referred to as the reactive center loop (RCL) 1 (4). Upon cleavage, the RCL integrates into the center of the dominant serpin structural feature, ␤-sheet A, becoming strand four of this six-stranded ␤-sheet. This transformation is accompanied by a large increase in thermal stability, presumably due to reorganization of the five-stranded ␤-sheet A to a six-stranded anti-parallel form (5-8). More recent crystallographic structures of several native serpins with intact reactive center loops have confirmed this hypothesis, demonstrating that the overall native serpin structure is very similar to cleaved ␣ 1 -antitrypsin, but that the reactive center loop is exposed above the plane of the molecule (9 -13).The structural versatility of serpins appears to be essential for their inhibitory activity. Serpins are thought to act as "suicide inhibitors" that react only once with a target proteinase, forming an SDS-stable complex. A "bait" am...

Residues in the Human Corticosteroid-binding Globulin Reactive Center Loop That Influence Steroid Binding before and after Elastase Cleavage

“…Thus, the rate of RCL insertion will determine the efficiency of the inhibitory pathway, and it is not surprising that a non-charged, small hydrophobic P14 residue plays an important role in initiating the RCL registration, as its side chain is the first to pack within the hydrophobic core of the cleaved serpin (6). The importance of P14 in affecting the rate of the loop insertion was first demonstrated by a variant of AAT that contains a large, charged arginine residue in place of the small, neutral threonine at P14 (26) and was confirmed in studies of plasminogen activator inhibitor-1 variants in which the P14 threonine was replaced by a battery of different residues (27). In the latter study the largest changes in the stoichiometry of inhibition and the rate constants for RCL insertion were caused by substitutions with charged amino acids, of which arginine caused the greatest effect (27).…”

Mechanistic characterization and crystal structure of a small molecule inactivator bound to plasminogen activator inhibitor-1

“…Like other serpins, PAI-1 has a solvent-exposed, reactive center loop (RCL) that contains an amino acid sequence that confers protease target specificity. The serpin inhibitory mechanism is a multistep process of coordinated conformational changes that are necessary to trap a target protease (18). The first step is the formation of a noncovalent Michaelis complex, followed by the initial steps of a typical serine protease catalytic attack leading to the covalent acyl-enzyme complex (19).…”

Lysine 114 of Antithrombin Is of Crucial Importance for the Affinity and Kinetics of Heparin Pentasaccharide Binding

“…The proteinase is still attached to this segment by an acyl bond and is therefore transported to the opposite pole of the protein. In this location, the proteinase is squeezed against the main body of the inhibitor and is inactivated by the resulting distortion of a large part of its structure, including the active site (3)(4)(5)(6)(7).…”