Crystal Structures of Potent Thiol-Based Inhibitors Bound to Carboxypeptidase B^,

Abstract

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.

Molecular dynamics and free energy studies on the carboxypeptidases complexed with peptide/small molecular inhibitor: Mechanism for drug resistance

“…It is known that zinc ion directly involved in catalysis plays an important role in maintaining the physiological function of the MCPs, which may affect the protein-inhibitor interactions (Adler et al, 2005;Bayés et al, 2005;Estébanez-Perpiñá et al, 2001;Rees and Lipscomb, 1982). Up to now, three methods have been developed for tacking "missing parameter" problem regarding zinc ion of metalloprotein in MD simulations researches, namely nonbonded, semibonded and bonded models (Lin and Wang, 2010).…”

Substrate Specificity of Human Carboxypeptidase A6

“…In recent years more has been learned about the enzymatic specificity, gene expression, and physiological roles of other members of this subfamily. X-ray crystal structures have been solved for five of the nine members of this subfamily (3,(12)(13)(14)(15)(16)(17). CPA2 and CPB1 are pancreatic metallocarboxypeptidases that function in the digestion of food (18).…”

Structural insights into the broad substrate specificity of carboxypeptidase T from Thermoactinomyces vulgaris

“…Two contacts mediated by single water molecules were found between the guanidine group of GEMSA and CpT (water molecules 597 and 788, Fig. ), compared to four between the guanidine group of the ligand and CpB (PDB ID http://www.rcsb.org/pdb/search/structidSearch.do?structureId=1ZG8) . These water molecules are engaged in formation of the hydrogen bond network between protein and the guanidine residue and thereby facilitate binding of the ligand.…”