Inhibition of human α β and γthrombin by mono-, bis-, tris- and tetra-benzamidine structures: Thermodynamic study

The inhibitory effect of mono-, bis-. tris- and tetra-benzamidine structures (benzamidine, DAPP, TAPB and TAPP, respectively) on the catalytic properties of human α β and γthrombin (α β and γthrombin, respectively) was investigated (between pH 2.0 and 7.0, I == 0.1 M; T == 37.0 ± 0.5°C). The affinity of DAPP, TAPB and TAPP for α and βthrombin is higher than that found for benzamidine association around neutrality, converging in the acidic pH limb; in constrast, benzamidine, DAPP, TAPB and TAPP show the same value of the association inhibition constant (K₁; M^-1) for γthrombin over the whole pH range explored. On lowering the pH from 5.5 to 3.0, the decrease in affinity for benzamidine binding to α β and γthrombin, as well as for DAPP, TAPB and TAPP association to γthrombin reflects the acidic-pK shift, upon inhibitor binding of a single ionizing group. On the other hand, values of K_i for DAPP, TAPB and TAPP binding to α and βthrombin appear to be modulated by the acidic-pK shift, upon inhibitor association, of two equivalent proton-binding residues over the same pH range. By considering molecular models of the serine proteinase: inhibitor complexes, the observed binding behaviour of benzamidine, DAPP, TAPB and TAPP to α β and γthrombin has been related to the inferred stereochemistry of the enzyme: inhibitor contact region(s).