title: Crystallographic structure of a peptidyl keto acid inhibitor and human alpha-thrombin.
abstract: The low molecular weight alpha-keto amide inhibitor CVS-1347, benzyl-SO2-Met(O2)-Pro-Arg(CO)((CONH)CH2)-phenyl, is a slow, tight binding inhibitor of alpha-thrombin amidolytic activity having a Ki = 1.28 x 10(-10) M. A complex between human alpha-thrombin and a hydrolysis product of CVS-1347 has been determined and refined using crystallography. The crystals belong to monoclinic space group C2 with cell dimensions of a = 71.08, b = 72.05 and c = 72.98 A and beta = 100.8 degrees. The structure was solved using isomorphous replacement methods and refined with resolution limits of (8.00-1.76) A to an R-value of 0.162. The Pro-Arg core of the inhibitor binds in the S2 and S1 subsites respectively, as is usually observed for Pro-Arg thrombin inhibitors. The Met(O2) side chain does not make any close contacts with the enzyme but influences the conformation of Glu192; the N-terminal benzylsulfonyl group makes an aromatic-aromatic contact with Trp215 in the hydrophobic part of the active site. The alpha-keto carboxylic acid of the proteolyzed inhibitor binds with the carboxylate group in the oxyanion hole, demonstrating that this region can accommodate an anion in a protease-peptide complex. The alpha-keto carbonyl group interacts closely with the two most important residues in the active site: the carbon atom is within a covalent bond distance of the active site Ser195 O gamma and the carbonyl oxygen is hydrogen bonded to His57.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, Michigan State University, East Lansing 48824-1322, USA.
authors.forename: K
authors.initials: K
authors.lastname: Håkansson
authors.forename: A
authors.initials: A
authors.lastname: Tulinsky
authors.forename: M M
authors.initials: MM
authors.lastname: Abelman
authors.forename: T A
authors.initials: TA
authors.lastname: Miller
authors.forename: G P
authors.initials: GP
authors.lastname: Vlasuk
authors.forename: P W
authors.initials: PW
authors.lastname: Bergum
authors.forename: M S
authors.initials: MS
authors.lastname: Lim-Wilby
authors.forename: T K
authors.initials: TK
authors.lastname: Brunck
mesh_terms: D000595:Amino Acid Sequence; D000991:Antithrombins / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005341:Fibrinolysin / Q000037:antagonists & inhibitors; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D011487:Protein Conformation; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000737:chemistry*
chemical_list: D000991:Antithrombins; C096690:CVS 1347; D007202:Indicators and Reagents; D009842:Oligopeptides; D013917:Thrombin; D005341:Fibrinolysin