Kinetics of the inhibition of human leukocyte elastase by elafin, a 6-kilodalton elastase-specific inhibitor from human skin.

We have investigated the kinetics of inhibition of human leukocyte elastase by elafin, a small protein originally isolated from human skin. A single inhibitor molecule was found to bind to a single site on the protease, blocking the reactive serine at the enzyme's catalytic center. Association of the enzyme with the inhibitor proceeds via a single bimolecular process, with a second-order rate constant of 3.6 x 10(6) M-1 s-1 at pH 8.0 and 25 degrees C. Dissociation of the enzyme-inhibitor complex regenerates fully active enzyme with a first-order rate constant of 6.0 x 10(-4) s-1. The species of elafin which is released from the complex simultaneously with the enzyme was estimated to be at least 99.8% active, with association and dissociation kinetics identical to preparations of the inhibitor which had never been exposed to the enzyme. Ki, the equilibrium dissociation constant of the enzyme-inhibitor complex, decreases from 6.7 x 10(-9) to 2.0 x 10(-10) M as the pH is increased from 5.4 to 9.0. The effect of pH on the association rate constant reveals that the reaction rate is dependent on the concentration of the unprotonated form of a group with pKa of 6.8, which we have assigned to the histidine which forms part of the catalytic triad in the enzyme's active site. On the basis of these findings, we conclude that elafin is a potent, substrate-like, but fully reversible inhibitor of human leukocyte elastase.