Analysis of a new crystal form of procarboxypeptidase B: Further insights into the catalytic mechanism

Abstract

A new triclinic crystal structure form of porcine pancreatic procarboxypeptidase B (PCPB) was obtained at higher resolution than the previously known tetragonal crystal structure. This new crystal polymorph has allowed for a corrected, accurate assignment of residues along the polypeptide chain based on the currently available gene sequence information and crystallographic data. The present structure shows unbound PCPB in a distinct molecular packing as compared to the previous benzamidine complexed form. Its catalytically important Tyr248 residue is oriented and hydrogen-bonded to solvent water molecules, and locates the furthest away from the catalytic zinc ion as compared to previous structures. A relatively long stretch of residues flanking Tyr248 and guarding the access to the catalytic zinc ion was found to be sequentially unique to the M14 family of peptidases. Predictions from a normal mode analysis indicated that this stretch of residues belongs to a rigid subdomain in the protein structure. The specific presence of a tyrosyl residue at the most exposed position in this region would allow for a delicate balance between extreme hydrophobicity and hydrophilicity, and affect substrate binding and the kinetic efficiency of the enzyme.

The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids

“…In metallocarboxypeptidases, the residue at position 255 directly interacts with the C-terminal side chain of the substrates (reviewed in Arolas et al. , 2007 ; Fernández et al. , 2010 ).…”

Structural and Functional Analysis of the Complex between Citrate and the Zinc Peptidase Carboxypeptidase A

“…The latter residue has been identified as a key player in the CPA catalyzed mechanism by site-specific mutagenesis studies [24]. The conformational change suffered by Tyr248 upon ligand binding is well documented: the aromatic ring switches by 120° its location between the solvent exposed “open” to the “closed” conformation with the phenolic hydroxyl pointing to the catalytic zinc [41, 42]. In this paper, Tyr248 is in the closed conformation expected to occur when the active site of the enzyme is occupied.…”

Normal Mode Analysis as a Routine Part of a Structural Investigation

“…The greatest number of studies used NMA to characterize the general flexibility and domain movements of the protein under study [132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148]. The use of NMA in these studies runs from the simple to the sophisticated.…”