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Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor
1Laboratoire d'Enzymologie, INSERM Unité 237, Université Louis Pasteur de Strasbourg, Illkirch, France.
Abstract:
N-chlorosuccinimide oxidizes one of the methionine residues of mucus proteinase inhibitor with a second-order rate constant of 1.5 M-1.s-1. Cyanogen bromide cleavage and NH2-terminal sequencing show that the modified residue is methionine-73, the P'1 component of the inhibitor's active centre. Oxidation of the inhibitor decreases its neutrophil elastase inhibitory capacity but does not fully abolish it. The kinetic parameters describing the elastase-oxidized inhibitor interaction are: association rate constant kass. = 2.6 x 10(5) M-1.s-1, dissociation rate constant kdiss. = 2.9 x 10(-3) s-1 and equilibrium dissociation constant Ki = 1.1 x 10(-8) M. Comparison with the native inhibitor indicates that oxidation decreases kass. by a factor of 18.8 and increases kdiss. by a factor of 6.4, and therefore leads to a 120-fold increase in Ki. Yet, the oxidized inhibitor may still act as a potent elastase inhibitor in the upper respiratory tract where its concentration is 500-fold higher than Ki, i.e. where the elastase inhibition is pseudo-irreversible. Experiments in vitro with fibrous human lung elastin, the most important natural substrate of elastase, support this view: 1.35 microM elastase is fully inhibited by 5-6 microM oxidized inhibitor whether the enzyme-inhibitor complex is formed in the presence or absence of elastin and whether elastase is pre-adsorbed on elastin or not.
Insights
Oxidation of mucus proteinase inhibitor at methionine-73 reduces its effectiveness against neutrophil elastase. However, the modified inhibitor remains potent in the upper respiratory tract due to high local concentrations.
Area of Science:
- Biochemistry
- Enzymology
- Protease Inhibition
Background:
- Mucus proteinase inhibitor (MPI) is crucial for regulating neutrophil elastase (NE) activity.
- Oxidative stress can modify proteins, potentially altering their function.
Purpose of the Study:
- To investigate the effect of N-chlorosuccinimide (NCS) oxidation on MPI's structure and function.
- To determine the kinetic parameters of the interaction between oxidized MPI and NE.
Main Methods:
- Chemical modification of MPI using NCS.
- Cyanogen bromide cleavage and NH2-terminal sequencing to identify the oxidation site.
- Kinetic analysis of MPI-NE interaction using purified components and in vitro assays with human lung elastin.
Main Results:
- NCS selectively oxidized methionine-73 (M73) in MPI, located at the active site.
- Oxidation significantly decreased the association rate constant (kass) and increased the dissociation rate constant (kdiss) for NE, resulting in a 120-fold increase in the equilibrium dissociation constant (Ki).
- Despite reduced potency, oxidized MPI effectively inhibited NE in vitro, even in the presence of elastin, suggesting pseudo-irreversible inhibition at physiological concentrations.
Conclusions:
- Oxidation of M73 in MPI impairs its direct binding kinetics with NE.
- Oxidized MPI retains significant elastase inhibitory capacity, particularly in the upper respiratory tract where local concentrations are high.
- The findings highlight the potential role of modified MPI in modulating protease activity in vivo under oxidative conditions.