Inhibition of Serine Proteinases by P-Carbethoxyphenyl Esters of ϵ-Guanidino- And ϵ-Amino Caproic Acid: Thermodynamic and Molecular Modeling Study

Abstract

The inhibitory effect of the clinically used p-carbethoxyphenyl ester of ϵ-guanidino-caproic acid metha-nesulphonate (ϵ-GCA-CEP) on the catalytic properties of human LYS⁷⁷-plasmin (EC 3.4.21.7), bovine factor Xa (EC 3.4.21.6), bovine α-thrombin (EC 3.4.21.5), ancrod (EC 3.4.21.28), crotalase (EC 3.4.21.30), bovine β-trypsin (EC 3.4.21.4), porcine pancreatic β-kallikrein-B (EC 3.4.21.39, human urinary kallikrein (EC 3.4.21.35) and the M_r 54,000 species of human urokinase (EC 3.4.21.31) was investigated (between pH 2.0 and 8.5, I = 0.1 M;T = 21 ϵ 0.5ϵC), and analyzed in parallel with that of the homologous derivative p-carbethoxyphenyl ϵ-amino-caproate hydro chloride (ϵ-ACA-CEP). On lowering the pH from 5.5 to 3.0, values of the apparent dissociation inhibition constant (K_i) for ϵ-GCA. CEP and ϵ-ACA-CEP interaction with the serine proteinases considered increase, reflecting the acidic pK-shift upon inhibitor binding of a single ionizing group. Over the whole pH range explored, (i) ϵ-GCA-CEP interacts with bovine factor Xa and bovine α-thrombin with an higher affinity than that observed for ϵ-ACA-CEP binding; (ii) both inhibitors associate to bovine β-trypsin with the same affinity; and (iii) ϵ-ACA-CEP inhibits human Lys⁷⁷-plasmin and the M_r 54,000 species of human urokinase with an higher affinity than that reported for ϵ-GCA-CEP association, thus reflecting the known enzyme primary specificity properties. However, the affinity of ϵ-ACA-CEP for ancrod, crotalase, porcine pancreatic β-kallikrein-B and human urinary kallikrein, all of which preferably bind arginyl rather than lysyl side chains at the primary position of substrates and/or inhibitors, is paradoxically higher than that displayed by ϵ-GCA-CEP. By considering the amino acid sequences, the X-ray three-dimensional structures and/or the computer-generated molecular models of serine proteinase: inhibitor adducts, the observed binding behaviour of ϵ-GCA-CEP and ϵ-ACA-CEP to the enzymes considered has been related to the inferred stereochemistry of proteinase: inhibitor contact region(s).

Key Words: :

• serine proteinase inhibitors. serine proteinase
• inhibitor adduct formation (thermodynamics of) serine proteinase
• inhibitor adducts (molecular modeling of)