New chromogenic substrates of human neutrophil cathepsin G containing non-natural aromatic amino acid residues in position P(1) selected by combinatorial chemistry methods.
Specificity of human cathepsin G was explored using combinatorial chemistry methods. Deconvolution of a tetrapeptide library, where 5-amino-2-nitrobenzoic acid served as a chromophore attached at the C-terminus, yielded the active sequence Phe-Val-Thr-Tyr-Anb(5,2)-NH(2). This sequence was used for a second-generation library with the general formula Ac-Phe-Val-Thr-X-Anb(5,2)-NH(2), where position X was replaced with several amino acids: L-pyridyl- alanine (Pal), 4-nitro-L-phenylalanine (Nif), 4-amino-L- phenylalanine (Amf), 4-carboxy-L-phenylalanine (Cbf), 4-guanidine-L-phenylalanine (Gnf), 4-methyloxycarbonyl- L-phenylalanine (Mcf), 4-cyano-L-phenylalanine (Cyf), Phe, Tyr, Arg and Lys. Specificity ligand parameters, k(cat) and K(M), with human cathepsin G were determined for all chromogenic substrates synthesized. The highest value of the specificity constant (k(cat)/K(M)) was obtained for a substrate with the Gnf residue in position P(1). This peptide was 10 times more active than the second most active substrate which contained the Amf residue. The following order of potency was established: Gnf > > Amf > Tyr = Phe > Arg= Lys > Cyf. Substrate specificity for cathepsin G is greatly enhanced when an aromatic side chain and a strong positive charge are incorporated in residue P(1).
Wysocka
Magdalena
M
Bioorganic Chemistry Department, Faculty of Chemistry, University of Gdańsk, Sobieskiego 18, 80-952 Gdańsk, Poland.
Legowska
Anna
A
Bulak
Elzbieta
E
Jaśkiewicz
Anna
A
Miecznikowska
Hanna
H
Lesner
Adam
A
Rolka
Krzysztf
K
D024322:Amino Acids, Aromatic / Q000138:chemical synthesis* / Q000737:chemistry; D032701:Biomimetics; D056649:Cathepsin G; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism*; D002863:Chromogenic Compounds / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques; D006801:Humans; D008954:Models, Biological; D009504:Neutrophils / Q000201:enzymology*; D019151:Peptide Library; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D024322:Amino Acids, Aromatic; D002863:Chromogenic Compounds; D019151:Peptide Library; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G