New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops

New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops

Author Rehault, S. Google Scholar
Brillard-Bourdet, M. Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Gauthier, F. Google Scholar
Moreau, T. Google Scholar
Institution Univ Tours
Universidade Federal de São Paulo (UNIFESP)
Abstract Cathepsin G has both trypsin- and chymotrypsin-like activity, but studies on its enzymatic properties have been limited by a lack of sensitive synthetic substrates. Cathepsin G activity is physiologically controlled by the fast acting serpin inhibitors alpha(1)-antichymotrypsin and alpha(1)-proteinase inhibitor, in which the reactive site loops are cleaved during interaction with their target enzymes. We therefore synthesized a series of intramolecularly quenched fluorogenic peptides based on the sequence of various serpin loops. Those peptides were assayed as substrates for cathepsin G and other chymotrypsin-like enzymes including chymotrypsin and chymase. Peptide substrates derived from the alpha(1)-antichymotrypsin loop were the most sensitive for cathepsin G; with k(cat)/K-m values of 5-20 mM(-1) s(-1). Substitutions were introduced at positions P-1 and P-2 in alpha(1)-antichymotrypsin-derived substrates to tentatively improve their sensitivity. Replacement of Leu-Leu in ortho-aminobenzoyl (Abz)-Thr-Leu-Leu-Ser-Ala-Leu-Gln-N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) by Pro-Phe in Abz-Thr-Pro-Phe-Ser-Ala-Leu-Gln-EDDnp produced the most sensitive substrate of cathepsin G ever reported. It was cleaved with a specificity constant k(cat)/K-m of 150 mM(-1) s(-1). Analysis by molecular modeling of a peptide substrate bound into the cathepsin G active site revealed that, in addition to the protease S-1 subsite, subsites S-1' and S-2' significantly contribute to the definition of the substrate specificity of cathepsin G.
Language English
Date 1999-05-14
Published in Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 274, n. 20, p. 13810-13817, 1999.
ISSN 0021-9258 (Sherpa/Romeo, impact factor)
Publisher Amer Soc Biochemistry Molecular Biology Inc
Extent 13810-13817
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000080322200014

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