TY - JOUR
T1 - Human α-thrombin inhibition by the highly selective compounds N-ethoxycarbonyl-D-Phe-Pro-α-azaLys p-nitrophenyl ester and N-carbobenzoxy-Pro-α-azaLys p-nitrophenyl ester
T2 - A kinetic, thermodynamic and x-ray crystallographic study
AU - De Simone, Giuseppina
AU - Balliano, Gianni
AU - Milla, Paola
AU - Gallina, Carlo
AU - Giordano, Cesare
AU - Tarricone, Cataldo
AU - Rizzi, Menico
AU - Bolognesi, Martino
AU - Ascenzi, Paolo
PY - 1997/6/20
Y1 - 1997/6/20
N2 - Kinetics, thermodynamics and structural aspects of human α-thrombin (thrombin) inhibition by newly synthesized low molecular weight derivatives of α-azalysine have been investigated. The thrombin catalyzed hydrolysis of N-ethoxycarbonyl-D-Phe-Pro-α-azaLys p-nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) and N-carbobenzoxy-Pro-α-azaLys p-nitrophenyl ester (Cbz-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0°C, and analyzed in parallel with that of N-α-(N,N-dimethylcarbamoyl)-α-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). Decarboxylation following the enzymatic hydrolysis of these p-nitrophenyl esters gave the corresponding 1-peptidyl-2(4-aminobutyl) hydrazines (peptidyl Abh) showing properties of thrombin competitive inhibitors. Therefore, thermodynamics for the reversible binding of D-Phe-Pro-Abh, Cbz-Pro-Abh and Dmc-Abh to thrombin was examined. These results are consistent with the minimum four-step catalytic mechanism for product inhibition of serine proteinases. Eoc-D-Phe-Pro-azaLys-ONp and Eoc-D-Phe-Pro-Abh display a sub-micromolar affinity for thrombin together with a high selectivity versus homologous plasmatic and pancreatic serine proteinases acting on cationic substrates. The three-dimensional structures of the reversible non-covalent thrombin:Eoc-D-Phe-Pro-Abh and thrombin:Cbz-Pro-Abh complexes have been determined by X-ray crystallography at 2.0 Å resolution (R-factor = 0.169 and 0.179, respectively), and analyzed in parallel with that of the thrombin:Dmc-azaLys acyl.enzyme adduct. Both Eoc-D-Phe-Pro-Abh and Cbz-Pro-Abh competitive inhibitors are accommodated in the thrombin active center, spanning the region between the aryl binding site and the S1 primary specificity subsite.
AB - Kinetics, thermodynamics and structural aspects of human α-thrombin (thrombin) inhibition by newly synthesized low molecular weight derivatives of α-azalysine have been investigated. The thrombin catalyzed hydrolysis of N-ethoxycarbonyl-D-Phe-Pro-α-azaLys p-nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) and N-carbobenzoxy-Pro-α-azaLys p-nitrophenyl ester (Cbz-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0°C, and analyzed in parallel with that of N-α-(N,N-dimethylcarbamoyl)-α-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). Decarboxylation following the enzymatic hydrolysis of these p-nitrophenyl esters gave the corresponding 1-peptidyl-2(4-aminobutyl) hydrazines (peptidyl Abh) showing properties of thrombin competitive inhibitors. Therefore, thermodynamics for the reversible binding of D-Phe-Pro-Abh, Cbz-Pro-Abh and Dmc-Abh to thrombin was examined. These results are consistent with the minimum four-step catalytic mechanism for product inhibition of serine proteinases. Eoc-D-Phe-Pro-azaLys-ONp and Eoc-D-Phe-Pro-Abh display a sub-micromolar affinity for thrombin together with a high selectivity versus homologous plasmatic and pancreatic serine proteinases acting on cationic substrates. The three-dimensional structures of the reversible non-covalent thrombin:Eoc-D-Phe-Pro-Abh and thrombin:Cbz-Pro-Abh complexes have been determined by X-ray crystallography at 2.0 Å resolution (R-factor = 0.169 and 0.179, respectively), and analyzed in parallel with that of the thrombin:Dmc-azaLys acyl.enzyme adduct. Both Eoc-D-Phe-Pro-Abh and Cbz-Pro-Abh competitive inhibitors are accommodated in the thrombin active center, spanning the region between the aryl binding site and the S1 primary specificity subsite.
KW - Human α-thrombin
KW - N-carbobenzoxy-Pro-α-azaLys p-nitrophenyl ester
KW - N-ethoxycarbonyl-D-Phe-Pro-α-azaLys p-nitrophenyl ester
KW - Serine proteinase inhibition
KW - X-ray crystal structure
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U2 - 10.1006/jmbi.1997.1037
DO - 10.1006/jmbi.1997.1037
M3 - Article
C2 - 9217260
AN - SCOPUS:0031580204
VL - 269
SP - 558
EP - 569
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 4
ER -