Inhibition of bovine β-trypsin, human α-thrombin and porcine pancreatic β-kallikrein-B by benzamidine and its bis-, tris- and tetra-derivatives: Thermodynamic and molecular modeling study

The inhibitory effect of bis-, tris- and tetra-benzamidine derivatives (DAPP, TAPB and TAPP, respectively) on the catalytic properties of bovine β-trypsin (β-trypsin), human α-thrombin (α-thrombin) and porcine pancreatic β-kallikrein-B (β-kallikrein-B) was investigated (between pH 2.0 and 7.0, I= 0.1 M; T= 37.0 ± 0.5°C), and analyzed in parallel with that of benzamidine, commonly taken as a molecular inhibitor model of serine proteinases. Over the whole pH range explored, benzamidine, DAPP, TAPB and TAPP, show the same value of the association inhibition constant (K(i) M^-1) for β-trypsin; at variance, the affinity of DAPP, TAPB and TAPP for α-thrombin and β-kallikrein-B is higher than that found for benzamidine association around neutrality, but tends to converge in the acidic pH limb. On lowering the pH from 5.5 to 3.0, the decrease in affinity for benzamidine binding to β-trypsin, α-thrombin and β-kallikrein-B as well as for DAPP, TAPB and TAPP association to β-trypsin reflects the acidic-pK shift, upon inhibitor binding, of a single ionizing group. Over the same pH range, values of K(i) for DAPP, TAPB and TAPP binding to α-thrombin and β-kallikrein-B appear to be modulated by the acidic-pK shift, upon inhibitor association, of two equivalent proton-binding residues. Considering the X-ray three dimensional structures and the computer-generated molecular models of the serine proteinase inhibitor complexes, the observed binding behaviour of benzamidine, DAPP, TAPB and TAPP to β-trypsin, α-thrombin and β-kallikrein-B has been related to the inferred stereochemistry of the enzyme: inhibitor contact region(s).