PDBsum entry: 1ae8

Hydrolase/hydrolase inhibitor

PDB id: 1ae8

Contents

Protein chains

36 a.a. *

253 a.a. *

Ligands

ASP-PHE-GLU-GLU-ILE-PRO-GLU-GLU-TYS-LEU

NAG

AZL

Waters ×159

* Residue conservation analysis

PDB id:

1ae8

Name:

Hydrolase/hydrolase inhibitor

Title:

Human alpha-thrombin inhibition by eoc-d-phe-pro-azalys-onp

Structure:

Alpha-thrombin (small subunit). Chain: l. Alpha-thrombin (large subunit). Chain: h. Hirugen. Chain: i. Synonym: n-acetylhirudin 53 - 64 with sulfato-tyr 63

Source:

Homo sapiens. Human. Organism_taxid: 9606. Organ: blood. Tissue: plasma. Hirudo medicinalis. Medicinal leech. Organism_taxid: 6421

Biol. unit:

Hexamer (from PQS)

Resolution:

2.00Å R-factor: 0.169

Authors:

G.De Simone,G.Balliano,P.Milla,C.Gallina,C.Giordano,C.Tarric M.Rizzi,M.Bolognesi,P.Ascenzi

Key ref:

G.De Simone et al. (1997). 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. PubMed id: 9217260 DOI: 10.1006/jmbi.1997.1037

Date:

06-Mar-97 Release date: 03-Dec-97

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin

Seq: 622 a.a.

Struc: 36 a.a.

Protein chain

P00734  (THRB_HUMAN) -  Prothrombin

Seq: 622 a.a.

Struc: 253 a.a.

Enzyme reactions

• Enzyme class: Chains L, H: E.C.3.4.21.5 - Thrombin.
• Reaction: Preferential cleavage: Arg-|-Gly; activates fibrinogen to fibrin and releases fibrinopeptide A and B.

 Gene Ontology (GO) functional annotation 

• Cellular component: extracellular region (1 term)
• Biological process: blood coagulation (2 terms)
• Biochemical function: catalytic activity (3 terms)

DOI no: 10.1006/jmbi.1997.1037

J Mol Biol 269:558-569 (1997)

PubMed id: 9217260

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.

G.De Simone, G.Balliano, P.Milla, C.Gallina, C.Giordano, C.Tarricone, M.Rizzi, M.Bolognesi, P.Ascenzi.

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.

Selected figure(s)

Figure 3.
Figure 3. A, Stereo view of the active site region in the thrombin:Eoc- Image -Phe-Pro-Abh complex. In all panels, residues of the catalytic triad, from the specificity S[1] subsite, from the 60-insertion loop, and from the aryl binding site have been labeled. The inhibitor molecules are identified with the I label. B, Binding mode of Cbz-Pro-Abh to the thrombin active site, in the same orientation adopted in A. Omit electron density, relative to the inhibitor molecule is shown overlaid. The unfilled electron density lobe on the left-hand side of Pro P2 accounts for the alternative conformation of the Cbz group (not shown in the picture). For further details see the text.

Figure 5.
Figure 5. Chemical structures of Eoc- Image -Phe-Pro-azaLys-ONp (A), Cbz-Pro-azaLys-ONp (B), Dmc-azaLys-ONp (C), Eoc-D-Phe-Pro-Abh (D), Cbz-Pro-Abh (E) and Dmc-Abh (F). The N, O and C atoms of amino acid derivatives, relevant for the discussion, have been labeled.

The above figures are reprinted by permission from Elsevier: J Mol Biol (1997, 269, 558-569) copyright 1997.

Figures were selected by an automated process.