Abstract

p-Guanidinobenzoate-trypsinogen is transformed into a trypsin-like conformation upon binding of Ile-Val as evidenced by specific changes in its circular dichroism spectrum. By means of this signal 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 have been determined at different Ca 2+ concentrations. Ile-Val and Ile-Val-Gly exhibit the strongest binding affinity of all peptides investigated. Replacement of the first isoleucine or of the second valine residue by other amino acids considerably reduces the peptide affinity. Discussion of these is based on the known spatial arrangement of the Ile16-Val17-Gly18 N-terminus and of the Ile-Val dipeptide in the Ile16 cleft (crystal structures of bovine trypsin and of the trypsinogen-PTI ‡ ‡ Abbreviations used: PTI, pancreatic trypsin inhibitor, Trasylol R (Bayer AG); pGB, p-guanidinobenzoate; NPGB, p-nitrophenole- p′-guanidinobenzoate; Tg, trypsinogen. The amino acid sequence numbers refer to the chymotrypsinogen enumeration (Hartley & Shotton, 1971); residues of PTI and soybean trypsin inhibitor are indicated by (I). -Ile-Val complex; Bode et al., 1978). The free energies of binding of the first and of the second peptide residue are almost additive indicating independency between both subsites. The third residue, glycine, does not significantly contribute to binding. The peptide analogues of various trypsinogen N-termini exhibit no measurable affinity for the Ile 16 cleft. The equilibrium constant for the binding of PTI to trypsinogen and the affinity of Ile-Val for the resulting binary complex have been determined in the presence and absence of Ca 2+, using the competitive PTI-binding to α-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 (+43 kJ mol −1, 20 °C; stabilization of the “activation domain”; Fehlhammer et al., 1977), due to the binding of the trypsin N-terminus (—55 kJ mol −1) and of the peptide analogues (e.g. Ile-Val; −28 kJ mol −1) into the preformed Ile 16 cleft, and due to the specific burying of the covalently linked pGB group in the fixed specificity pocket (— 39 kJ mol −1). This pocket is co-operatively linked with the Ile 16 cleft according to a free-energy change coupling of —43 kJ mol −1.

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