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Kinetic mechanism of the inhibition of cathepsin G by alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor.

Abstract：

:Uncontrolled proteolysis due to cathepsin G (cat G) may cause severe pathological disorders. Cat G is inhibited by alpha 1-antichymotrypsin (ACT) and alpha 1-proteinase inhibitor (alpha 1PI), 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 x 10(-)8 M and k2 = 2.8 x 10(-)2 s-1, while the cat G/alpha 1PI association is governed by Ki = 8.1 x 10(-)7 M and k2 = 5.5 x 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 alpha 1PI 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 alpha 1PI. Both ACT and alpha 1PI 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.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Duranton J,Adam C,Bieth JG

doi

10.1021/bi980223q

subject

Has Abstract

pub_date

1998-08-11 00:00:00

pages

11239-45

issue

eissn

0006-2960

issn

1520-4995

pii

bi980223q

journal_volume

pub_type

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Kinetic mechanism of the inhibition of cathepsin G by alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor.