EXAFS STUDY OF THE ACTIVE-SITE OF ALKALINE-PHOSPHATASE FROM ESCHERICHIA-COLI

The structure of the active site of the enzyme alkaline phosphatase (AP) from E. coli has been investigated by EXAFS spectroscopy. The study used two derivatives of the enzyme, Zn2AE2BE2CAP and Zn2ACO2BE2CAP, the symbols referring to the fact that each active center of the enzyme dimer, consisting of two identical subunits, contains three metal binding sites (E refers to empty). Thus EXAFS has examined the environment of the Zn(II) ion in the A site of the enzyme in the absence and presence of a metal ion in the B site, 3.9 angstrom away. The analysis of the data has been performed by comparison with model compounds among which Zn(imidazole)2(acetate)2 shows a very close approximation to the structure of the zinc environment in the enzyme. From these findings the coordination of the metal at site A in the active site of AP appears to be composed of two histidine nitrogens plus four oxygen donors at an average distance of 2.04(2) angstrom from the Zn(II) ion. The oxygen donors appear to be made up of a carboxylate group acting as a bidentate ligand and the oxygens of two coordinated water molecules. Neither the ligands nor the coordination geometry around the Zn(II) ion at the A site change upon binding of a metal ion to the B site.