Inhibition of Serine Proteinases by Tetra-p-Amidinophenoxy-neo-Pentane: Thermodynamic and Molecular Modeling Study

Abstract The inhibitory effect of the aromatic tetra-benzamidine derivative tetra-p-amidinophenoxy-neo-pentane (TAPP) on the catalytic properties of β-trypsin (EC 3.4.21.4), α-thrombin (EC 3.4.21.5), factor Xa (EC 3.4.21.6), Lys77-plasmin (EC 3.4.21.7) and β-kallikrein-B (EC 3.4.21.35) was investigated (between pH 2 and 8, I = 0.1 M; T = 37 ± 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, TAPP and benzamidine show the same values of the dissociation inhibition constant (Ki) for β-trypsin; at variance with the affinity of TAPP for α-thrombin, factor Xa, Lys77-plasmin and β-kallikrein-B which 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, values of Ki for TAPP binding to β-trypsin as well as for benzamidine association to all the enzymes investigated decreased thus reflecting the pK-shift, upon inhibitor binding, of a single ionizing group. Over the same pH range, values of Ki for TAPP binding to α-thrombin, factor Xa, Lys77- plasmin and β-kallikrein-B may be described as depending on the pK-shift, upon inhibitor association, of two equivalent proton-binding amino acid residues. Considering the X-ray three-dimensional structures and the computer-generated molecular models of serine proteinases:TAPP and :benza-midine adducts, the observed binding behaviour of TAPP and benzamidine to the enzymes considered has been related to the inferred stereochemistry of proteinase: inhibitor contact region(s).