KINETIC AND CRYSTALLOGRAPHIC STUDIES OF THROMBIN WITH AC-(D)PHE-PRO-BOROARG-OH AND ITS LYSINE, AMIDINE, HOMOLYSINE, AND ORNITHINE ANALOGS

The X-ray crystallographic structure of Ac-(D)Phe-Pro-boroArg-OH [DuP714, K-i = 0.04 nM; Kettner, C., Mersinger, L., and Knabb, R. (1990) J. Biol. Chem. 265, 18289] complexed with human alpha-thrombin shows the boron atom covalently bonded to the side-chain oxygen of the active site serine, Ser195. The boron adopts a nearly tetrahedral geometry, and the boronic acid forms a set of interactions with the protein that mimic the tetrahedral transition state of serine proteases. Contributions of the arginine side chain to inhibitor affinity were examined by synthesis of the ornithine, lysine, homolysine, and amidine analogs of DuP714. The basic groups interact with backbone carbonyl groups, water molecules, and an aspartic acid side chain (Asp189) located in the thrombin S1 specificity pocket. The variation in inhibition constant by 3 orders of magnitude appears to reflect differences in the energetics of interactions made with thrombin and differences in ligand flexibility in solution. Kinetic and crystallographic data are reported for the following thrombin inhibitors: DuP714 (space group C2, a = 70.8 Angstrom, b = 72.3 Angstrom, c = 72.6 Angstrom, beta = 100.6 degrees, crystallographic R-factor = 0.204 to 1.95 Angstrom resolution); Ac-(D)Phe-Pro-boroLys-OH (K-i = 0.24 nM, C2, a = 70.3 Angstrom, b = 71.9 Angstrom, c = 71.9 Angstrom, beta = 100.9 degrees, R-factor = 0.201 to 2.35 Angstrom resolution); Ac-(D)Phe-Pro-boro-homoLys-OH (K-i = 8.1 nM, C2, a = 70.3 Angstrom, b = 71.9 Angstrom, c = 71.9 Angstrom, beta = 100.9 degrees, R-factor = 0.212 to 2.4 Angstrom resolution); Ac-(D)Phe-Pro-boroOrn-OH (K-i = 79 nM, C2, a = 70.4 Angstrom, b = 72.2 Angstrom, c = 72.2 Angstrom, beta = 100.1 degrees, R-factor = 0.195 to 2.25 Angstrom resolution); and Ac-(o)Phe-Pro-boro-n-butylamidinoGly-OH (K-i = 0.29 nM, C2, a = 70.8 Angstrom, b = 72.4 Angstrom, c = 72.2 Angstrom, beta = 100.3 degrees, R-factor = 0.197 to 2.25 Angstrom resolution).