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Fourteen-member-ring macrolides are antibiotics with a variety of anti-inflammatory activities, and have repeatedly been reported to reduce mucus hypersecretion in conditions such as cystic fibrosis and bronchiectasis. Their structure is characterized by a macrocyclic lactone ring. Because human neutrophil elastase (HNE) plays a crucial role in the vicious circle leading to mucus hypersecretion, and lactones are known to be elastase inhibitors, we hypothesized that macrolides might directly inhibit elastase. To investigate this hypothesis we designed a series of spectrophotometric experiments using a chromogenic substrate with two macrolides, erythromycin (Er) and flurythromycin (FE). We determined the 1st order rate constant (kobs) by inhibition and competitive substrate assays, the latter allowing us to calculate the substrate binding constant or inhibition constant and the acylation rate constant (ka). A proflavine displacement assay was used to determine the deacylation rate constant (kd). Both Er and FE are good HNE inhibitors, showing a high ka and a low kd. Because the number of turnovers per inactivation of Er was ≅ 20-fold higher than that of FE, we supposed that the lower reactivation of HNE-FE was due to the formation of a more stable inactivated enzyme. This hypothesis was confirmed by the hydrazine reactivation of the acyl enzyme. For Er we identified a kd only, whereas for FE, in addition to the kd, an alkylation constant (k2) was calculated, correlated to a fully inactivated enzyme. From our kinetics data, we therefore conclude that Er acts as an alternate substrate HNE inhibitor, whereas FE acts as an inactivator.