Pseudomonas aeruginosa Elastase

The elastase produced by Pseudomonas aerughosa is probably responsible for the tissue destruction observed during pulmonary and corneal infections by this pathogen. We have synthesized a new substrate, AbzAla-Gly-Leu-Ala-Nba, for P. aeruginosa elastase. Cleavage of the peptide by elastase at pH 7.2 results in 6to 7-fold increase in fluorescence (h,,, 340 nm; X, 415 nm). A sensitive rate assay utilizing this substrate was developed and used to study inhibitors. Elastase was strongly inhibited competitively by peptides containing the hydroxamic acid (HONHCOCH(CH&H5)CO-AlaGly-NHZ, K, = 44 n~), N-hydroxypeptide (CHO-HOLeuAla-Gly-NHZ, KI = 8 PM), and thiol (HSCH2CH(CHzCsH5)CO-Ala-Gly-NH2, KI = 64 a) functional groups. The hydroxamic acid (HONHCOCH&O-AIaGly-NH2) lacking the benzyl side chain was a 6000-fold poorer inhibitor, indicating that the inhibitors are binding to the primary substrate specificity site (S',) of elastase. The nomenclature used for the individual amino acid residues (Pl, P1, PZ, etc.) of a substrate or inhibitor and the subsites (S1, S I , S'z, etc.) of the enzyme is that of Schechter and Berger (Schechter, I., and Berger, A. (1967) Biochem Biophys. Res. Commun, 27, 157-162). In each case, the inhibitors contain a ligating group to interact with active site zinc atom of the elastase. The affinity resin HONHCOCH(CH2CsHs)COAla-Gly-NH(CH&-agarose was utilized to purify P. aeruginosa elastase. The enzyme was applied at pH 7.2 and could be eluted with a pH 10.0, 0.1 M 'h i s solution safely. Enzymatically inactive material washed straight through the column. Elastase was not inhibited by ClCH2CO-HOLeu-OCH3, a thermolysin inhibitor, but was irreversibly inhibited by the tripeptide analog C1CHzCO-HOLeu-Ala-G1y-NH2. This indicates that P. aeruginosa elastase in common with porcine pancreatic elastase has an extended substrate recognition site. Some of the inhibitors may find use in the clinical treatment of P. aeruginosa infections. The abbreviations used are: Abz, 2-aminobenzoyl; Nba, NHCH2CeH4N02 (4-nitrobenzylamide); HOLeu, N-hydroxyleucine.