The new fluorogenic substrates of neutrophil proteinase 3 optimized in prime site region.
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.
Wysocka
Magdalena
M
Faculty of Chemistry, University of Gdańsk, 80-952 Gdańsk, Poland.
Lesner
Adam
A
Majkowska
Grazyna
G
Legowska
Anna
A
Guzow
Katarzyna
K
Rolka
Krzysztof
K
Wiczk
Wiesław
W
D000595:Amino Acid Sequence; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D053146:Myeloblastin / Q000378:metabolism*; D009579:Nitrobenzoates / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D009579:Nitrobenzoates; D010455:Peptides; C019251:5-amino-2-nitrobenzoic acid; D053146:Myeloblastin