Kinetics for the hydrolysis of the chromogenic active site titrant N-alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp) catalyzed by bovine beta-trypsin, bovine alpha-thrombin, human alpha-thrombin, human Lys77-plasmin, human urinary kallikrein, the M(r) 33,000 and M(r) 54,000 species of human urokinase, as well as by porcine pancreatic beta-kallikrein-A and B have been obtained between pH 6.0 and 8.0, at 21.0 degrees C. Moreover, the three dimensional structure of the human alpha-thrombin-(hirugen). Dmc-azaLys acyl . enzyme complex has been analyzed and refined by X-ray crystallography at 2.0 Angstrom resolution (R-factor = 0.168). As observed for bovine beta-trypsin, the acylating inhibitor molecule is covalently bound to the Ser195 catalytic residue, filling the human alpha-thrombin S-1 primary specificity subsite with its lysyl side-group. However, the carbonyl group of the scissile human alpha-thrombin . Dmc-azalys acyl bond does not occupy properly the oxyanion binding hole. At variance from the bovine beta-trypsin . Dmc-azaLys acyl . enzyme structure, a second, not covalently bound, inhibitor molecule, partly shielded by the 60-insertion loop of human alpha-thrombin, is contacting the enzyme ''aryl-binding site''.

Human alpha-thrombin inhibition by the active site titrant N-alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester: A comparative kinetic and X-ray crystallographic study

Ferraccioli R;
1996

Abstract

Kinetics for the hydrolysis of the chromogenic active site titrant N-alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp) catalyzed by bovine beta-trypsin, bovine alpha-thrombin, human alpha-thrombin, human Lys77-plasmin, human urinary kallikrein, the M(r) 33,000 and M(r) 54,000 species of human urokinase, as well as by porcine pancreatic beta-kallikrein-A and B have been obtained between pH 6.0 and 8.0, at 21.0 degrees C. Moreover, the three dimensional structure of the human alpha-thrombin-(hirugen). Dmc-azaLys acyl . enzyme complex has been analyzed and refined by X-ray crystallography at 2.0 Angstrom resolution (R-factor = 0.168). As observed for bovine beta-trypsin, the acylating inhibitor molecule is covalently bound to the Ser195 catalytic residue, filling the human alpha-thrombin S-1 primary specificity subsite with its lysyl side-group. However, the carbonyl group of the scissile human alpha-thrombin . Dmc-azalys acyl bond does not occupy properly the oxyanion binding hole. At variance from the bovine beta-trypsin . Dmc-azaLys acyl . enzyme structure, a second, not covalently bound, inhibitor molecule, partly shielded by the 60-insertion loop of human alpha-thrombin, is contacting the enzyme ''aryl-binding site''.
1996
Istituto di Scienze e Tecnologie Molecolari - ISTM - Sede Milano
File in questo prodotto:
File Dimensione Formato  
prod_203381-doc_48716.pdf

solo utenti autorizzati

Descrizione: Human-Thrombin Inhibition by the Active Site Titrant N-(N,N-dimethylcarbamoyl)-azalys-p-nitrophenyl ester: A Kinetic and X-ray Crystallographic Study
Dimensione 676.73 kB
Formato Adobe PDF
676.73 kB Adobe PDF   Visualizza/Apri   Richiedi una copia

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.14243/180120
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact