Crystallographic structure of a peptidyl keto acid inhibitor and human α-thrombin Original Research Article

The low molecular weight α-keto amide inhibitor CVS-1347, benzyl-SO2-Met(O2)-Pro-Arg(CO)((CONH)CH2)-ph4enyl, is a slow, tight binding inhibitor of α-thrombin amidolytic activity having a Ki = 1.28 × 10−10 M. A complex between human α-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 Å and β = 100.8°. The structure was solved using isomorphous replacement methods and refined with resolution limits of (8.00–1.76) Å 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 α-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 α-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 Oγ and the carbonyl oxygen is hydrogen bonded to His57. These residues thus act as a Lewis acid and base in catalysis with respect to the substrate carbonyl carbon and peptide nitrogen atoms of the scissile bond of substrate.