Analysis of a new crystal form of procarboxypeptidase B: further insights into the catalytic mechanism.
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.
Departament de Bioquímica i Biologia Molecular, Facultat de Biociències, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain.
Daniel
D
Fernández
Ester
E
Boix
Irantzu
I
Pallarès
Francesc X
FX
Avilés
Josep
J
Vendrell
D000595:Amino Acid Sequence; D000818:Animals; D043424:Carboxypeptidase B / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013552:Swine; D015032:Zinc / Q000737:chemistry
D043424:Carboxypeptidase B; D015032:Zinc