A plausible identification of the secondary binding site in trypsin and trypsinogen

1. The determination of the binding of 4,4'-diazoamino-bis-benzamidine (DABB) to alpha-trypsin by equilibrium measurements in columns indicated a stoichiometry of 2 mol ligand/mol enzyme. One molecule of ligand is bound to the active center, as shown by competitive experiments and Ki. The second molecule binds to the secondary binding site, with Ki2 = 0.63 mM at pH 8.0, 25 degrees C. 2. Bovine pancreatic trypsin inhibitor (BPTI) prevented binding of DABB to both sites, indicating that they are topographically close and within the interface of the trypsin-BPTI complex. 3. On the basis of data from the literature regarding the tertiary structure of the trypsin-BPTI complex, we concluded that the secondary binding site of trypsin is plausibly identified as the same site in trypsin that binds the Arg-17 residue of BPTI, i.e., Tyr-39 and Tyr-151 in bovine trypsin. This site would then correspond to subsite S'2 on the enzyme surface.