Human α-Thrombin Inhibition by the Active Site Titrant Nα-(N,N-dimethylcarbamoyl)-α-azalysine p-nitrophenyl ester: A Comparative Kinetic and X-ray Crystallographic Study

doi:10.1006/jmbi.1996.0292

Journal of Molecular Biology

Volume 258, Issue 5, 24 May 1996, Pages 851-859

https://doi.org/10.1006/jmbi.1996.0292 Get rights and content

Abstract

Kinetics for the hydrolysis of the chromogenic active site titrantN^α-(N,N-dimethylcarbamoyl)-α-azalysinep-nitrophenyl ester (Dmc-azaLys-ONp) catalyzed by bovine β-trypsin, bovine α-thrombin, human α-thrombin, human Lys77-plasmin, human urinary kallikrein, theM_r33,000 andM_r54,000 species of human urokinase, as well as by porcine pancreatic β-kallikrein-A and B have been obtained between pH 6.0 and 8.0, at 21.0 °C. Moreover, the three dimensional structure of the human α-thrombin-(hirugen)·Dmc-azaLys acyl·enzyme complex has been ana lyzed and refined by X-ray crystallography at 2.0 Å resolution (R-factor=0.168). As observed for bovine β-trypsin, the acylating inhibitor molecule is covalently bound to the Ser195 catalytic residue, filling the human α-thrombin S₁primary specificity subsite with its lysyl side-group. However, the carbonyl group of the scissile human α-thrombin·Dmc- azaLys acyl bond does not occupy properly the oxyanion binding hole. At variance from the bovine β-trypsin·Dmc-azaLys acyl·enzyme structure, a second, not covalently bound, inhibitor molecule, partly shielded by the 60-insertion loop of human α-thrombin, is contacting the enzyme “aryl-binding site”.

References (0)

Cited by (16)

Cross-enzyme inhibition by gabexate mesylate: Formulation and reactivity study
1998, Journal of Pharmaceutical Sciences
Gabexate mesylate (GM; commercialized under the brand name FOY) is a nonantigenic synthetic inhibitor of plasmatic and pancreatic serine proteinases that is used therapeutically in the treatment of pancreatitis and disseminated intravascular coagulation and as a regional anticoagulant for hemodialysis. The inhibitory effect of GM on nitric oxide synthase as well as serine proteinases and swine kidney copper amine oxidase, all acting on cationic substrates, has been investigated. On the basis of the available X-ray crystal structures of the enzymes considered, the possible binding mode(s) of GM has(have) been analyzed. The enzyme cross-inhibition by GM suggests that the use of this drug should be under careful control. With the aim to improve the scarce plasma stability of GM, the positively charged drug has been complexed to the surface of preformed anionic liposomes. The liposome-complexed GM half-life increases about five-fold, indicating the protective effect of liposomes on GM degradation. Moreover, the GM complexation with liposomes does not alter its inhibitory activity on NOS–I and porcine pancreatic trypsin.
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: A kinetic, thermodynamic and x-ray crystallographic study
1997, Journal of Molecular Biology
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.
N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-α-azalysine p-nitrophenyl ester: A novel, high selective and optimal chromogenic active site titrant for human and bovine α-, β- and γ-thrombin
1996, Biochemical and Biophysical Research Communications
The serine proteinase catalyzed hydrolysis ofN-ethoxycarbonyl-D-phenylalanyl-L-prolyl-α-azalysinep-nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0°C. The results are consistent with the minimum three-step catalytic mechanism. The acylation step is rate limiting for human (Lys77 species) and porcine plasmin, and for bovine β-trypsin, the deacylation rate being limiting, on the other hand,for human and bovine α-, β- and γ-thrombin. Moreover, theM_r33,000 species of human urokinase and the neuraminidase-treated porcine pancreatic β-kallikrein-B do not catalyze the hydrolysis of the tripeptide. According to the specificity properties of the serine proteinases considered, Eoc-D-Phe-Pro-azaLys-ONp shows the characteristics of a novel, high selective and optimal chromogenic active site titrant for human and bovine α-, β- and γ-thrombin.
Novel Inhibitors and Activity-Based Probes Targeting Trypsin-Like Serine Proteases
2022, Frontiers in Chemistry
Crystal structure of a biosynthetic sulfo-hirudin complexed to thrombin
2007, Journal of the American Chemical Society
Thrombin inhibition by the highly selective 'reversible suicide substrate' N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-α-azalysine p-nitrophenyl ester
2005, Protein and Peptide Letters

View all citing articles on Scopus

^f1: Corresponding author.

View full text

Journal of Molecular Biology

Regular articleHuman α-Thrombin Inhibition by the Active Site Titrant Nα-(N,N-dimethylcarbamoyl)-α-azalysine p-nitrophenyl ester: A Comparative Kinetic and X-ray Crystallographic Study

Abstract

Regular article
Human α-Thrombin Inhibition by the Active Site Titrant N^α-(N,N-dimethylcarbamoyl)-α-azalysine p-nitrophenyl ester: A Comparative Kinetic and X-ray Crystallographic Study