Inhibition of neutrophil cathepsin G by oxidized mucus proteinase inhibitor. Effect of heparin

Oxidation of mucus proteinase inhibitor (MPI) transforms Met(73), the P-1' residue of its active center into methionine sulfoxide and lowers its affin ity for neutrophil elastase [Boudier, C., and Bieth, J. G. (1994) Biochem. J. 303, 61-68]. Here, we show that the oxidized inhibitor has also a decrea sed affinity for neutrophil cathepsin G and pancreatic chymotrypsin. The K- i of the oxidized MPI-cathepsin G complex (1.2 mu M) is probably too high t o be compatible with significant inhibition of cathepsin G in inflammatory lung secretions. Stopped-flow kinetics shows that, within the inhibitor con centration range used, the mechanism of inhibition of cathepsin G and chymo trypsin by oxidized MPI is consistent with a one-step reaction, E + I (kdis s)reversible arrow(kass) EI, whereas the inhibition of elastase takes place in two steps, E + I reversible arrow(Ki*) EI* (k-2)reversible arrow(k2) EI . Heparin, which accelerates the inhibition of the three proteinases by nat ive MPI, also favors their interaction with oxidized MPI. Flow calorimetry shows that heparin binds oxidized MPI with K-d, Delta H degrees, and Delta S degrees values close to those reported for native MPI. In the presence of heparin, oxidized MPI inhibits cathepsin G via a two-step reaction charact erized by K-i*= 0.22 mu M, k(2) = 0.1 s(-1), k(-2) = 0.023 s(-1), and K-i = 42 nM. Under these conditions, in vivo inhibition of cathepsin G is again possible. Heparin also improves the inhibition of chymotrypsin and elastase by oxidized MPI by increasing their k(ass) or k(2)/K-i* and decreasing the ir K-i. Our data suggest that oxidation of MPI during chronic bronchitis ma y lead to cathepsin G-mediated lung tissue degradation and that heparin may be a useful adjuvant of MPI-based therapy of acute lung inflammation in cy stic fibrosis.