The transition of bovine trypsinogen to a trypsin like state upon strong ligand binding part 2 the binding of the pancreatic trypsin inhibitor and of iso leucine valine and of sequentially related peptides to trypsinogen and p guanidino benzoate trypsinogen
Bode, W.
Journal of Molecular Biology 127(4-5): 357-374
1979
ISSN/ISBN: 0022-2836 Accession: 006781565
p-Guanidinobenzoate(pGB)-trypsinogen is transformed into a tryspin-like conformation upon binding of Ile-Val as evidenced by specific changes in its circular dichroism spectrum. The changes were used to determine the association constants for the binding of a variety of peptides sequentially analogous to either the bovine trypsin N-terminus or to the N-terminal activation peptide sequences of several trypsinogens at different Ca2+ concentrations. Ile-Val and Ile-Val-Gly exhibit the strongest binding affinity of all peptides investigated. Replacement of the 1st isoleucine or of the 2nd valine residue by other amino acids considerably reduces the peptide affinity. Discussion is based on the known spatial arrangement of the Ile16-Vall7-Gly18 N-terminus and of the Ile-Val dipeptide in the Ile16 cleft (crystal structures of bovine trypsin and of the trypsinogen-PTI (pancreatic trypsin inhibitor)-Ile-Val complex; Bode et al., 1978). The free energies of binding of the 1st and of the 2nd peptide residue are almost additive indicating independency between the subsites. The 3rd residue, glycine, does not significantly contribute to binding. The peptide analogs of various trypsinogen N-termini exhibit no measurable affinity for the Ile16 cleft. The equilibrium constant for the binding of PTI to trypsinogen and the affinity of Ile-Val for the resulting binary complex were determined in the presence and absence of Ca2+, using the competitive PTI-binding to .alpha.-chymotrypsin. These competition experiments allow the estimation of the standard free-energy changes due to the conformational transition of trypsinogen into a trypsin-like state due to the binding of the trypsin N-terminus and of the peptide analogs into the preformed Ile16 cleft, and due to the specific burying of the covalently linked pGB group in the fixed specificity pocket. This pocket is cooperatively linked with the Ile16 cleft according to a free-energy change coupling of -43 kJ mol-1.