Active centre studies on bovine pancreatic chymotrypsin with tripeptidyl-p-nitroanilide substrates.
The kinetic behaviour of bovine pancreatic chymotrypsin was studied with 22 N-protected and 17 N-unprotected tripeptidyl-p-nitroanilide substrates. The contribution of the individual side chains to the kinetic parameters were calculated by regression analysis. At subsite P1 (notation of Schechter and Berger, 1967, Biochem. Biophys. Res. Commun. 27, 157) Tyr seems to be better than Phe and Trp, concerning kcat values. At P2 subsite the best KM values were obtained with Gly and Ser, whereas the hydrophobicity of P2 subsite appears to be necessary for efficient catalytic activity. At P3 mainly polar amino acids, both with D and L configuration, were tested. They improve the solubility of substrates in aqueous medium, as well as the kinetic parameters. Suc(OMe) and Suc protecting groups at P4 increase significantly the catalytic activity compared to the aromatic ones. The obtained data were compared to the known substrate binding site of bovine pancreatic chymotrypsin.
D000813:Anilides; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D007700:Kinetics; D009574:Nitro Compounds; D009842:Oligopeptides; D010179:Pancreas / Q000201:enzymology*; D011485:Protein Binding; D013379:Substrate Specificity
D000813:Anilides; D009574:Nitro Compounds; D009842:Oligopeptides; D002918:Chymotrypsin