title: 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 alpha1-antichymotrypsin and alpha1-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 alpha1-antichymotrypsin loop were the most sensitive for cathepsin G with kcat/Km values of 5-20 mM-1 s-1. Substitutions were introduced at positions P1 and P2 in alpha1-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 kcat/Km 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 S1 subsite, subsites S1' and S2' significantly contribute to the definition of the substrate specificity of cathepsin G.
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D056649:Cathepsin G; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism*; D002918:Chymotrypsin / Q000378:metabolism; D018360:Crystallography, X-Ray; D004136:Dinitrobenzenes / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011487:Protein Conformation; D012697:Serine Endopeptidases; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism