Zymogen activation: Effect of peptides sequentially related to the bovine β‐trypsin N‐terminus on kazal inhibitor and benzamidine binding to bovine trypsinogen

The activating effect of peptides sequentially related to the Ile 16‐Vall7‐Gly 18 N ‐terminus of bovine β‐trypsin (nemely Ile‐Val‐Gly, Ile‐Val, Ile‐Leu Ile‐Ala, Val‐Val, Leu‐Val, and Val‐Leu) on the thermodynamic parameters for the binding of the porcine pancreatic secretory trypsin inhibitor (Kazal inhibtor) and benzamidine to bovine trypsinogen was investigated at pH 5.5 (Bis tris‐HCI buffer, I=0.1 M ) and T =21 ± 0.5°C. Thermodynamic parameters for Kazal inhibitor and benzamidine association to the binary peptide/zymogen adducts are more favorable than those observed for ligand binding to the proenzyme alone, although never as much as those reported for the formation of bovine β‐trypsin/Kazal inhibitor and bovine β‐trypsin/benzamidine adducts. Analogously, the affinity of activating peptides for the binary proenzume/Kazal inhibitor and binary proenzyme/benzamidine complexes is higher than that observed for peptide binding to free bovine trysinogen. Differences in affinity for ligand binding to free bovine trypsinogen. Differences in affinity for ligand binding to free bovine trypsinogen, to its binary adducts and to bovine β‐trypsin suggest the presence of different activation levels of the proenzyme, none of which structurally coincide with that achieved in bovine β‐trypsin. The existence of different discrete states suggests that the zymogen‐to‐active enzyme transition should not be considered as a two‐state process but as a multistep event.