Measurement of the kinetic parameters mediating protease-serpin inhibition.

Serine protease inhibition by proteins of the serpin family is a unique and complex process involving physical, chemical, and conformational changes. After encounter with the reactive site of inhibitor, the protease is conformationally trapped as a covalent complex resembling the acyl-protease intermediate of catalysis. The stability of the trap is not permanent and may vary for different proteases. In addition, the trapping mechanism is not 100% efficient and a fraction of the serpin may be consumed like a substrate before inactivation is complete. Characterization of protease-serpin inhibition therefore requires the measurement of three parameters: the apparent second order rate constant of inhibition (k(inh)), the stoichiometry of inhibition (SI), and the rate of complex breakdown (k(brkdn)). The basic kinetic methods to establish these parameters are described.

Mast cell chymase modifies cell-matrix interactions and inhibits mitogen-induced proliferation of human airway smooth muscle cells.

The hallmarks of chronic, severe asthma include prominent airway inflammation and airway smooth muscle (ASM) hypertrophy and hyperplasia. One of the factors that contribute to the injury and repair process within the airway is activation of proteases and turnover of extracellular matrix components. Mast cells, which are present in increased numbers in the asthmatic airway, are a rich source of the neutral protease chymase, which can degrade several basement membrane components. Recent data suggest that proteases also play a critical role in regulating the expression of CD44, the primary receptor for the matrix glycosaminoglycan hyaluronan. In this study we investigated the effects of chymase treatment on human ASM cell function. We found that chymase degraded the smooth muscle cell pericellular matrix. This was accompanied by an increased release of fibronectin and soluble CD44, but not soluble ICAM-1 or soluble hyaluronan, into the conditioned medium. In addition, chymase inhibited T cell adhesion to ASM and dramatically reduced epidermal growth factor-induced smooth muscle cell proliferation. These data suggest that the local release of mast cell chymase may have profound effects on ASM cell function and airway remodeling.

The effects of reactive site location on the inhibitory properties of the serpin alpha(1)-antichymotrypsin.

The large size of the serpin reactive site loop (RSL) suggests that the role of the RSL in protease inhibition is more complex than that of presenting the reactive site (P1 residue) to the protease. This study examines the effect on inhibition of relocating the reactive site (Leu-358) of the serpin alpha(1)-antichymotrypsin either one residue closer (P2) or further (P1') from the base of the RSL (Glu-342). alpha(1)-Antichymotrypsin variants were produced by mutation within the P4-P2' region; the sequence ITLLSA was changed to ITLSSA to relocate the reactive site to P2 (Leu-357) and to ITITLS to relocate it to P1' (Leu-359). Inhibition of the chymotrypsin-like proteases human chymase and chymotrypsin and the non-target protease human neutrophil elastase (HNE) were analyzed. The P2 variant inhibited chymase and chymotrypsin but not HNE. Relative to P1, interaction at P2 was characterized by greater complex stability, lower inhibition rate constants, and increased stoichiometry of inhibition values. In contrast, the P1' variant inhibited HNE (stoichiometry of inhibition = 4) but not chymase or chymotrypsin. However, inhibition of HNE was by interaction with Ile-357, the P2 residue. The P1' site was recognized by all proteases as a cleavage site. Covalent-complexes resistant to SDS-PAGE were observed in all inhibitory reactions, consistent with the trapping of the protease as a serpin-acyl protease complex. The complete loss in inhibitory activity associated with lengthening the Glu-342-reactive site distance by a single residue and the enhanced stability of complexes associated with shortening this distance by a single residue are compatible with the distorted-protease model of inhibition requiring full insertion of the RSL into the body of the serpin and translocation of the linked protease to the pole opposite from that of encounter.

Heterogeneity in serpin-protease complexes as demonstrated by differences in the mechanism of complex breakdown.

Serpins trap their target proteases in the form of an acyl-enzyme complex. The trap is kinetic, however, and thus serpin-protease complexes ultimately break down, releasing a cleaved inactive serpin and an active protease. The rates of this deacylation process vary greatly depending on the serpin-protease pair with half-lives ranging from minutes to months. The reasons for the diversity in breakdown rates are not clearly understood. In the current study, pH and solvent isotope effects were utilized to probe the mechanism of breakdown for an extremely stable complex and several unstable complexes. Two different patterns for the pH dependence of k(bkdn), the first-order rate constant of breakdown, were found. The stable complex, which breaks down at neutral pH with a half-life of approximately 2 weeks, exhibited a pH-k(bkdn) profile consistent with solvent-hydroxide ion mediated ester hydrolysis. There was no evidence for the participation of the catalytic machinery in the breakdown of this complex, suggesting extensive distortion of the active site. The unstable complexes, which break down with half-lives ranging from minutes to hours, exhibited a bell-shaped pH profile for k(bkdn), typical of the pH-rate profiles of free serine proteases. In the low to neutral pH range k(bkdn) increased with increasing pH in a manner characteristic of His57-mediated catalysis. In the alkaline pH range a decrease in k(bkdn) was observed, consistent with the titration of the Ile16-Asp194 salt bridge (chymotrypsinogen numbering). The alkaline pH dependence was not exhibited in pH-rate profiles of free or substrate-bound HNE, indicating that the salt bridge was significantly destabilized in the complexed protease. These results indicate that breakdown is catalytically mediated in the unstable complexes although, most likely, the protease is not in its native conformation and the catalytic machinery functions inefficiently. However, a mechanism in which breakdown is determined by the equilibrium between distorted and undistorted forms of the complexed protease cannot be completely dismissed. Overall, the results of this study suggest that the protease structure in unstable complexes is distorted to a lesser extent than in stable complexes.