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Interaction between serine (pro)enzymes, and kazal and kunitz inhibitors

1983; Elsevier BV; Volume: 165; Issue: 3 Linguagem: Inglês

10.1016/s0022-2836(83)80219-8

1089-8638

Eraldo Antonini, Paolo Ascenzi, Martino Bolognesi, Giuseppina Gatti, Mario Guarneri, Enea Menegatti,

Complement system in diseases

Equilibrium constants (Kd) for PSTI (porcine pancreatic secretory trypsin inhibitor: Kazal inhibitor) and BPTI (bovine basic pancreatic trypsin inhibitor: Kunitz inhibitor) binding to bovine β-trypsin, bovine trypsinogen, bovine α-chymotrypsin, human urinary kallikrein and porcine pancreatic kallikrein have been obtained at pH 8. Kd values have been determined by measuring the loss of the enzymatic activity and the spectral changes accompanying the formation of the PSTI: (pro)enzyme and BPTI: (pro)enzyme adducts. PSTI is characterized by systematically lower affinities than BPTI and does not inhibit either kallikrein up to inhibitor concentrations of 3 mm. As in the case of BPTI, the affinity of PSTI for bovine trypsinogen increases in the presence of 0·02 m-H-Ile-Val-OH, which mimics, in the proenzyme, the N-terminal segment of the activated bovine β-trypsin. BPTI: bovine trypsinogen: H-Ile-Val-OH and PSTI: bovine trypsinogen: H-Ile-Val-OH complexes show absorption spectra similar to those of BPTI: bovine β-trypsin and PSTI: bovine β-trypsin adducts. The pH dependence of the equilibrium constants for PSTI and BPTI binding to bovine β-trypsin, bovine trypsinogen and bovine α-chymotrypsin is roughly the same for both the inhibitors with the following scale of apparent affinities: β-trypsin ≫ α-chymotrypsin > trypsinogen. The simplest model that describes the experimental data, between pH 5 and 9·5, takes into account a single ionizable group with a pKUNL of approximately 7·2 in the free (pro)enzymes, which appears to decrease by about two units upon PSTI and BTPI binding. The most probable ionizing group to account for such a behaviour is His57, present at the active site of (pro)enzymes. Comparison of the three-dimensional structures of PSTI and BPTI adducts shows that the respective reactive sites, despite structural identity around the scissile peptide bond, differ in their geometry of subsites further away from it. In particular, the wider reactive site polypeptide loop of PSTI as compared to that of BPTI may be responsible, at least in part, for the different binding behaviour of the two inhibitors.