Demonstration of a two-step reaction mechanism for the inhibition of heparin-bound neutrophil elastase by alpha 1-proteinase inhibitor.
Heparin decreases the rate of inhibition of neutrophil elastase by alpha 1-proteinase inhibitor as a result of its strong binding to the enzyme. Here, we used the slow-binding kinetic approach to decide whether the enzyme-inhibitor interaction proceeds via a two-step mechanism and to identify the step that is affected by heparin. The inhibition kinetics was assessed under pseudo-first-order conditions using conventional or stopped-flow spectrophotometry. In the absence of heparin, the pseudo-first-order rate constant of inhibition increased linearly with the inhibitor concentration indicating that within the experimental concentration range (< or = 6 microM) the enzyme-inhibitor association conforms either to a simple bimolecular reaction (E+I kass-->EI with kass = 10(7) M-1 s-1) or to a two-step reaction (E+I Ki*<==>EI* k2-->EI with Ki* > 0.4 microM and k2 > 4 s-1). In the presence of heparin, the rate constant of inhibition varied hyperbolically with the inhibitor concentration, indicating that the inhibition is a two-step process with Ki* = 80 nM and K2 = 0.15 s-1. Thus, heparin has two opposite effects on the elastase + alpha 1-proteinase inhibitor interaction: it favors the association by decreasing Ki* but impairs it by decreasing k2. This rationalizes the previously demonstrated rate-depressing effect of the sulfated polymer. Heparin does not significantly alter the stability of the irreversible elastase-alpha 1-proteinase inhibitor complex.
Faller
B
B
Laboratoire d'Enzymologie, INSERM Unité 237, Université Louis Pasteur de Strasbourg, Illkirch, France.
Cadène
M
M
Bieth
J G
JG
D000595:Amino Acid Sequence; D017022:Flow Injection Analysis; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000201:enzymology*; D010196:Pancreatic Elastase / Q000378:metabolism*; D013053:Spectrophotometry; D000515:alpha 1-Antitrypsin / Q000378:metabolism*
D046911:Macromolecular Substances; D000515:alpha 1-Antitrypsin; D006493:Heparin; D010196:Pancreatic Elastase