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

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.