DEMONSTRATION OF A 2-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 alpha1-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 (less-than-or-equal-to 6 muM) the enzyme-inhibitor association conforms either to a simple bimolecular reaction (E + I --> k(ass) EI with k(ass) = 10(7) M-1 s-1) or to a two-step reaction (E + I half arrow right over half arrow left K(i)* EI* --> k2 EI with K(i)* > 0.4 muM 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 K(i)* = 80 nM and K2 = 0.15 s-1. Thus, heparin has two opposite effects on the elastase + alpha1-proteinase inhibitor interaction: it favors the association by decreasing K(i)* 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-alpha1-proteinase inhibitor complex.