The new fluorogenic substrates of neutrophil proteinase 3 optimized in prime site region.

Magdalena Wysocka, Adam Lesner, Grazyna Majkowska, Anna Legowska, Katarzyna Guzow, Krzysztof Rolka, Wiesław Wiczk

Index: Anal. Biochem. 399(2) , 196-201, (2010)

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Abstract

Previously selected by the combinatorial chemistry approach, potent fluorogenic substrate of proteinase 3 was used as the starting structure to design new substrates. The general formula of the synthesized peptides is as follows: ABZ-Tyr-Tyr-Abu-ANB-X-NH(2), where ANB (5-amino-2-nitrobenzoic acid) served as a chromophore and an acceptor of fluorescence, ABZ (aminobenzoic acid) is a donor of fluorescence in these fluorescence resonance energy transfer (FRET) peptides, and X is a proteinogenic amino acid (except Cys). The introduced modifications influenced substrate activity of the synthesized peptides. The highest value of specificity constant for proteinase 3 was obtained for the single peptide with Gln in the discussed position (k(cat)/K(M) = 275,000 M(-1) s(-1)), which was nearly twice as active as the reference compound (lacking a substituent in the X position). In addition, more efficient energy transfer was observed, due mainly to the bathochromic effect for the introduced modification. This approach opens a new possibility to design potent and highly specific substrates of proteinase 3 and other proteinases optimized in the prime site region.Copyright 2010 Elsevier Inc. All rights reserved.