title: A new proteinase 3 substrate with improved selectivity over human neutrophil elastase.
abstract: We report the synthesis and enzymatic studies on a new proteinase 3 intermolecular quenched substrate with enhanced selectivity over neutrophil elastase. Using combinatorial chemistry methods, we were able to synthesize the hexapeptide library with the general formula ABZ-Tyr-Tyr-Abu-X1'-X2'-X3'-Tyr(3-NO2)-NH2 using the mix and split method. The iterative deconvolution of such a library allowed us to obtain the sequence ABZ-Tyr-Tyr-Abu-Asn-Glu-Pro-Tyr(3-NO2)-NH2 with a high specificity constant (kcat/KM=1534×10(3)M(-1)s(-1)) and superior selectivity over neutrophil elastase and other neutrophil-derived serine proteases. Moreover, using the obtained substrate, we were able to detect a picomolar concentration of proteinase 3 (PR3). Incubation of the above-mentioned substrate with neutrophil lysate resulted in a strong fluorescent signal that was significantly reduced in the presence of a PR3 selective inhibitor.
authors.affiliation: Faculty of Chemistry, University of Gdansk, 80-952 Gdansk, Poland.
authors.forename: J
authors.initials: J
authors.lastname: Popow-Stellmaszyk
authors.forename: M
authors.initials: M
authors.lastname: Wysocka
authors.forename: A
authors.initials: A
authors.lastname: Lesner
authors.forename: B
authors.initials: B
authors.lastname: Korkmaz
authors.forename: K
authors.initials: K
authors.lastname: Rolka
mesh_terms: D020650:Combinatorial Chemistry Techniques; D006801:Humans; D019272:Leukocyte Elastase / Q000378:metabolism*; D053146:Myeloblastin / Q000378:metabolism*; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; D019272:Leukocyte Elastase; D053146:Myeloblastin
keywords: Combinatorial chemistry; Intermolecular quenched substrate; Neutrophil serine proteases; Proteinase 3