Kinetics of the inhibition of human pancreatic elastase by recombinant eglin c. Influence of elastin.
Recombinant eglin c is a potent reversible inhibitor of human pancreatic elastase. At pH 7.4 and 25 degrees C, kass. = 7.3 x 10(5) M-1.s-1, kdiss. = 2.7 x 10(-4) s-1 and Ki = 3.7 x 10(-10) M. Stopped-flow kinetic indicate that the formation of the stable enzyme-inhibitor complex is not preceded by a fast pre-equilibrium complex or that the latter has a dissociation constant greater than 0.3 microM. The elastase-eglin c complex is much less stable at pH 5.0 and 25 degrees C, where kdiss. = 1.1 x 10(-2) s-1 and Ki = 7.3 x 10(-8) M. At pH 7.4 the activation energy for kass. is 43.9 kJ.mol-1 (10.5 kcal.mol-1). The kass. increases between pH 5.0 and 8.0 and remains essentially constant up to pH 9.0. This pH-dependence could not be described by a simple ionization curve. Both alpha 2-macroglobulin and alpha 1-proteinase inhibitor are able to dissociate the elastase-eglin c complex, as evidenced by measurement of the enzymic activity of alpha 2-macroglobulin-bound elastase or by polyacrylamide-gel electrophoresis of mixtures of alpha 1-proteinase inhibitor and elastase-eglin c complex. The rough estimate of kdiss. obtained with the alpha 2-macroglobulin dissociation experiment (1.6 x 10(-4) s-1) was of the same order of magnitude as the constant measured with the progress curve method. Eglin c strongly inhibits the solubilization of human aorta elastin by human pancreatic elastase. The extent of inhibition is the same whether elastase is added to a suspension of elastin and eglin c or whether elastase is preincubated with elastin for 3 min before addition of eglin c. However, the efficiency of the inhibitor sharply decreases if elastase is reacted with elastin for more prolonged periods.
Faller
B
B
I.N.S.E.R.M. Unité 237, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg, Illkirch, France.
Dirrig
S
S
Rabaud
M
M
Bieth
J G
JG
D004549:Elastin / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors*; D011506:Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D015843:Serpins; D013696:Temperature; D000515:alpha 1-Antitrypsin / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000378:metabolism
D011506:Proteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D015843:Serpins; D000515:alpha 1-Antitrypsin; D000511:alpha-Macroglobulins; C028656:eglin proteinase inhibitors; D004549:Elastin; D010196:Pancreatic Elastase