Mapping of the substrate binding site of human leukocyte chymotrypsin (cathepsin G) using tripeptidyl-p-nitroanilide substrates.
The kinetic constants, KM, kcat and kcat/KM of human leukocyte chymotrypsin (Cathepsin G, E.C.3.4.21.20.) were determined with 33 N-protected and 16 N-unprotected tripeptidyl-p-nitroanilide substrates. The individual contributions of the amino acid side chains of the substrates at P1-P4 subsites to the kinetic parameters were calculated by regression analysis. As far as KM is concerned, the highest contributions yielded the structure of an "optimum" substrate PhCO-Ala-Val-Tyr-pNA. The contribution values permitted us to characterize the S1-S4 binding segment in the enzyme's binding site, interacting with the P1-P4 moieties of the substrate. The enzyme prefers uniformly hydrophobic substituents at the S1-S4 sites. The S1 primary specificity subsite of the enzyme can bind Leu and 2-aminohexanoic acid as well, besides the aromatic amino acids Phe, Tyr, Trp. Data were compared with those obtained when studying pancreatic chymotrypsin with the same substrates.
D000813:Anilides; D001665:Binding Sites; D056649:Cathepsin G; D002403:Cathepsins / Q000097:blood*; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000201:enzymology*; D009574:Nitro Compounds; D009842:Oligopeptides; D011485:Protein Binding; D012697:Serine Endopeptidases; D013379:Substrate Specificity
D000813:Anilides; D009574:Nitro Compounds; D009842:Oligopeptides; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G