XAFS STUDIES OF CARBOXYPEPTIDASE-A - DETECTION OF A STRUCTURAL ALTERATION IN THE ZINC COORDINATION SPHERE COUPLED TO THE CATALYTICALLY IMPORTANT ALKALINE PK(A)

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 0) at an average distance of 2.024 +/- 0.006 angstrom, and a smaller distribution of 1.3 atoms (N or 0) at 2.54 +/- 0.05 angstrom from the zinc ion (from the least square fitting analysis). At pH 9.9, the larger distribution contains four atoms at a 0.022 angstrom shorter distance (2.002 angstrom) from the zinc, while the smaller distribution contains 0.7 atom at 2.52 +/- 0.06 angstrom. The smaller distribution can be attributed mainly to the contribution of the epsilon2-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. The higher shell comparison shows that the two histidine ligands His 69 and His 196 remain unchanged at the pHs examined. The plot of the normalized spectral changes at the absorption edge as a function of pH conforms to a theoretical pH-titration curve with a pK(a) 9.49 at -4-degrees-C. This value corresponds to that obtained by extrapolation of values of pK(EH) by kinetic analysis of the carboxypeptidase-A catalyzed hydrolysis of tripeptides (Auld & Vallee (1971) Biochemistry 10, 2892). The pH-dependent shorter average metal-ligand distance and the increased first shell structural disorder is most easily explained by one of the other two metal ligands (the water molecule or the epsilon1-oxygen of Glu 72) to the metal moving 0.09 +/- 0.03 angstrom closer to the zinc ion at alkaline pH values. Such a situation could be due to either direct ionization of the metal bound water or an ionization-linked alteration in the coordination of Glu 72. As discussed herein, we favor assigning the alkaline pK(a) in the kinetic profiles, pK(EH), to the ionization of the metal bound water molecule.