title: 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.
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 A 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 S1 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".
authors.affiliation: Centro Biotecnologie Avanzate IST, Università di Genova, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Nardini
authors.forename: A
authors.initials: A
authors.lastname: Pesce
authors.forename: M
authors.initials: M
authors.lastname: Rizzi
authors.forename: E
authors.initials: E
authors.lastname: Casale
authors.forename: R
authors.initials: R
authors.lastname: Ferraccioli
authors.forename: G
authors.initials: G
authors.lastname: Balliano
authors.forename: P
authors.initials: P
authors.lastname: Milla
authors.forename: P
authors.initials: P
authors.lastname: Ascenzi
authors.forename: M
authors.initials: M
authors.lastname: Bolognesi
mesh_terms: D000818:Animals; D000991:Antithrombins / Q000378:metabolism / Q000494:pharmacology*; D001372:Aza Compounds / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects; D002417:Cattle; D002863:Chromogenic Compounds / Q000378:metabolism / Q000494:pharmacology; D018360:Crystallography, X-Ray; D006629:Hirudins / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D000991:Antithrombins; D001372:Aza Compounds; D002863:Chromogenic Compounds; D006629:Hirudins; D010446:Peptide Fragments; D015842:Serine Proteinase Inhibitors; C062510:hirugen; C075756:N(alpha)(N,N-dimethylcarbamoyl)-alpha-azalysine 4-nitrophenyl ester; D012697:Serine Endopeptidases; D014357:Trypsin; D013917:Thrombin; D008239:Lysine