title: Refined structure of bovine carboxypeptidase A at 1.25 A resolution.
abstract: The crystal structure of the bovine zinc metalloproteinase carboxypeptidase A (CPA) has been refined to 1.25 A resolution based on room-temperature X-ray synchrotron data. The significantly improved structure of CPA at this resolution (anisotropic temperature factors, R factor = 10.4%, R(free) = 14.5%) allowed the modelling of conformational disorders of side chains, improved the description of the protein solvent network (375 water molecules) and provided a more accurate picture of the interactions between the active-site zinc and its ligands. The calculation of standard uncertainties in individual atom positions of the refined model of CPA allowed the deduction of the protonation state of some key residues in the active site and confirmed that Glu72 and Glu270 are negatively charged in the resting state of the enzyme at pH 7.5. These results were further validated by theoretical calculations that showed significant reduction of the pK(a) of these side chains relative to solution values. The distance between the zinc-bound solvent molecule and the metal ion is strongly suggestive of a neutral water molecule and not a hydroxide ion in the resting state of the enzyme. These findings could support both the general acid/general base mechanism, as well as the anhydride mechanism suggested for CPA.
authors.affiliation: Department of Inorganic Chemistry and the Laboratory for Structural Chemistry and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Kilshtain-Vardi
authors.forename: Meir
authors.initials: M
authors.lastname: Glick
authors.forename: Harry M
authors.initials: HM
authors.lastname: Greenblatt
authors.forename: Amiram
authors.initials: A
authors.lastname: Goldblum
authors.forename: Gil
authors.initials: G
authors.lastname: Shoham
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002268:Carboxypeptidases / Q000737:chemistry*; D043422:Carboxypeptidases A; D002417:Cattle; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011487:Protein Conformation; D011522:Protons; D055672:Static Electricity; D017356:Synchrotrons; D014965:X-Rays; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D011522:Protons; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D015032:Zinc