This paper presents the crystal structure of porcine pancreatic
carboxypeptidase B (pp-CpB) in complex with a variety of
thiol-based inhibitors that were developed as antagonists of activated
thrombin-activatable fibrinolysis inhibitor (TAFIa). Recent studies have indicated that a selective inhibitor of TAFIa could enhance the efficacy of existing
thrombolytic agents for the treatment of acute
myocardial infarction, one of the most prevalent forms of
heart attacks. Unfortunately, activated TAFIa rapidly degrades in
solution and cannot be used for crystallographic studies. In contrast, porcine pancreatic CpB is stable at room temperature and is available from commercial sources. Both pancreatic CpB and TAFIa are
zinc-based
exopeptidases, and the
proteins share a 47% sequence identity. The homology improves considerably in the active site where nearly all of the residues are conserved. The inhibitors used in this study were designed to mimic a C-terminal
arginine residue, one of the natural substrates of TAFIa. The X-ray structures show that the
thiol group chelates the active site
zinc, the
carboxylic acid forms a
salt bridge to Arg145, and the
guanidine group forms two hydrogen bonds to Asp255. A meta-substituted phenyl was introduced into our inhibitors to reduce conformational freedom. This modification vastly improved the selectivity of compounds against other
exopeptidases that cleave basic residues. Comparisons between structures indicate that selectivity derives from the interaction between the
guanidine group in the inhibitors and an acidic active site residue. The location of this acidic residue is not conserved in the various
carboxypeptidases.