Structure of binary and ternary complexes of zinc and cobalt carboxypeptidase A as determined by X-ray absorption fine structure

Carboxypeptidase A, ZnCPD, is typical of a wide range of exo- and endo-metalloproteases that have three protein ligands and a water molecule bound to a catalytic zinc atom and a glutamate residue in the active site that likely acts in conjunction with the Zn-bound water to bring about catalysis. Such enzymes generally have bell-shaped pH-activity profiles (EH(2) reversible arrow EH reversible arrow E) where the concentration of the catalytic species EH is regulated by PKEH2 and PKEH. The present X-ray absorption fine structure, XAFS, study has determined the structure and pH behavior of the binary and ternary product complexes in order to examine the role of the Zn-bound water in catalysis. Increasing the pH from 7 to 10 of the ZnCPD . L-Phe complex results in the same type of progressive spectral changes in the near-edge XAFS spectrum as is seen for ZnCPD, but the changes are complete by more than 1 pH value below that observed for ZnCPD. The results are in agreement with kinetic studies that show E binds the protonated form of L-Phe more tightly than EH, thus in effect decreasing the value of pK(EH). The XAFS results show the: average interatomic distance, R, for the zinc ligands of the EH . L-Phe complex decreases by 0.02 Angstrom upon formation of the E . L-Phe complex, essentially identical to that obtained for the EH and E forms of the native enzyme. Addition of azide to ZnCPD . L-Phe at pH 7 markedly changes the zinc coordination sphere from 4 N/O atoms at 2.021 +/- 0.06 Angstrom and 1.4 +/- 0.5 N/O atoms at 2.54 +/- 0.5 Angstrom to 3.9 N/O atoms at 1.995 +/- 0.006 Angstrom. The decrease in R of about 0.03 Angstrom in both the Zn- and CoCPD . L-Phe . N-3(-) complexes is likely due to the ligand exchange from a neutral water to an anion. The XAFS spectra of the ternary complex is pH independent from 7 to 9, in agreement with the ionization of the water being the source of the spectral changes in the free enzyme and its binary L-Phe complex. The enzyme . azide L-Phe complex is likely bound in a manner analogous to that expected for a post-transition state in a biproduct complex for peptide hydrolysis-that is, the carboxylate anion of the peptide bound to the Zn and the protonated form of L-Phe H-bonded to the catalytic Glu-270 carboxylate. The XAFS results on the ''spectroscopically silent'' ZnCPD are compared to nuclear magnetic resonance and electron absorption studies on CoCPD.