title: XAFS studies of carboxypeptidase A: detection of a structural alteration in the zinc coordination sphere coupled to the catalytically important alkaline pKa.
abstract: X-ray absorption fine structure (XAFS) spectra of carboxypeptidase A (ZnCPD) show progressive spectral changes particularly in the near edge region when the pH is changed from neutral to alkaline values. Both least square fitting and radial distribution function (RDF) data analysis yield two distributions of atoms in the first coordination shell of ZnCPD at all pH values and at both 150 and 297 K. Direct comparison of the first and higher coordination shells of ZnCPD reveals structural differences between pH 7.0 and pH 9.9. At pH 7.0, the zinc ion has four ligand atoms (N or O) at an average distance of 2.024 +/- 0.006 A, and a smaller distribution of 1.3 atoms (N or O) at 2.54 +/- 0.05 A from the zinc ion (from the least square fitting analysis). At pH 9.9, the larger distribution contains four atoms at a 0.022 A shorter distance (2.002 A) from the zinc, while the smaller distribution contains 0.7 atoms at 2.52 +/- 0.06 A. The smaller distribution can be attributed mainly to the contribution of the epsilon 2-oxygen of Glu 72 and to the atoms farther away for which the contribution cannot be fully separated from the first shell peak. The structural changes of ZnCPD at intermediate pHs are consistent with the changes observed at pH 7.0 and pH 9.9. The XAFS Debye-Waller factor shows an increased structural disorder for the four atom distribution at the alkaline pH.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts 02115.
authors.forename: K
authors.initials: K
authors.lastname: Zhang
authors.forename: D S
authors.initials: DS
authors.lastname: Auld
mesh_terms: D000818:Animals; D002268:Carboxypeptidases / Q000737:chemistry*; D043422:Carboxypeptidases A; D002384:Catalysis; D002417:Cattle; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D015032:Zinc / Q000737:chemistry*
chemical_list: D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D015032:Zinc