Kinetic Mechanism of the Inhibition of Cathepsin G by α1-Antichymotrypsin and α1-Proteinase Inhibitor

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Abstract
Uncontrolled proteolysis due to cathepsin G (cat G) may cause severe pathological disorders. Cat G is inhibited by α1-antichymotrypsin (ACT) and α1-proteinase inhibitor (α1PI), two members of the serpin superfamily of proteins. To see whether these two inhibitors play a physiological proteolysis-preventing function, we have made a detailed kinetic investigation of their reaction with cat G. The kinetics of inhibition of cat G in the presence of inhibitor and substrate evidenced a two-step inhibition mechanism: E + I EI* EI. The cat G/ACT interaction is described by Ki* = 6.2 × 10-8 M and k2 = 2.8 × 10-2 s-1, while the cat G/α1PI association is governed by Ki* = 8.1 × 10-7 M and k2 = 5.5 × 10-2 s-1. The reliability of these kinetic constants was checked using a number of experiments which all gave consistent results: (i) both EI* complexes were found to be enzymatically inactive, (ii) the Ki* values were determined directly using initial velocity experiments of cat G-catalyzed hydrolysis of substrate in the presence of inhibitor, (iii) the second-order rate constants k2/Ki* were measured using second-order inhibition experiments in the absence of substrate, and (iv) the ratio of the two second-order rate constants was determined by measuring the partition of cat G between the two fluorescently labeled serpins. Since the plasma concentrations of ACT and α1PI are much higher than their Ki* values, cat G released from neutrophils will be fully taken up as rapidly forming EI* complexes, that is, 70% with ACT and 30% with α1PI. Both ACT and α1PI are thus physiological cat G inhibitors whose inhibitory potential does not depend on the formation of the stable inhibitory species EI characteristic of serpins. Such an in vivo inhibition mechanism might take place with other serpin/proteinase systems.