1AFE

HUMAN ALPHA-THROMBIN INHIBITION BY CBZ-PRO-AZALYS-ONP

Summary for 1AFE

• Entry DOI: 10.2210/pdb1afe/pdb
• Descriptor: ALPHA-THROMBIN (SMALL SUBUNIT), ALPHA-THROMBIN (LARGE SUBUNIT), HIRUGEN, ... (6 entities in total)
• Functional Keywords: complex (serine protease-inhibitor), blood coagulation, n-carbobenzoxy-pro-alfa-azalys-p-nitrophenylester, serine proteinase inhibition, glycosylated protein, hydrolase-hydrolase inhibitor complex, hydrolase/hydrolase inhibitor
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 3
• Total formula weight: 35795.77

Authors

De Simone, G.,Balliano, G.,Milla, P.,Gallina, C.,Giordano, C.,Tarricone, C.,Rizzi, M.,Bolognesi, M.,Ascenzi, P. (deposition date: 1997-03-06, release date: 1997-12-03, Last modification date: 2024-10-23)

Primary citation

De Simone, G.,Balliano, G.,Milla, P.,Gallina, C.,Giordano, C.,Tarricone, C.,Rizzi, M.,Bolognesi, M.,Ascenzi, P.
Human alpha-thrombin inhibition by the highly selective compounds N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester: a kinetic, thermodynamic and X-ray crystallographic study.
J.Mol.Biol., 269:558-569, 1997

PubMed Abstract: Kinetics, thermodynamics and structural aspects of human alpha-thrombin (thrombin) inhibition by newly synthesized low molecular weight derivatives of alpha-azalysine have been investigated. The thrombin catalyzed hydrolysis of N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester (Cbz-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C, and analyzed in parallel with that of N-alpha-(N,N-dimethylcarbamoyl)-alpha-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 A 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.

PubMed: 9217260
DOI: 10.1006/jmbi.1997.1037

Experimental method

X-RAY DIFFRACTION (2 Å)