The Cu²⁺‐Alkaline Phosphatase of <i>Escherichia coli</i>

Abstract

The Cu 2+ ‐alkaline phosphatase is considerably less active than the Zn 2+ ‐ and Co 2+ ‐enzymes. It also differs from the native Zn 2+ ‐alkaline phosphatase by its sensitivity to nucleophiles and ionic strength. However, in spite of these differences, there remains an important common property, the considerable analogy between the kinetics parameters ( K m and V max ) for substrates with very different chemical structures. The Cu 2+ ‐enzyme, like the Zn 2+ ‐enzyme, is phosphorylated on both sites at acidic pH with the substrate AMP or the product inorganic phosphate. No phosphorylation occurs at alkaline pH where phosphorylation of the active centers is the rate‐limiting step. Only non‐covalent complexes containing one mole of inorganic phosphate per mole of enzyme could be isolated. The binding of another phosphate on the second site appears to be very difficult and to occur only at high concentrations of inorganic phosphate. Anticooperativity, an important property of the Zn 2+ ‐phosphatase is preserved in the Cu 2+ ‐phosphatase. Electron paramagnetic resonance indicates that the 4 coppers of the Cu 2+ ‐alkaline phosphatase belong to 2 different classes. There are 2 tight metal sites of type I and 2 loose metal sites of type II. Orthophosphate binding to the Cu 2+ ‐phosphatase alters both the visible and the electron paramagnetic resonance spectra. Electron paramagnetic resonance provides direct evidence for the participation of the coppers of type I in substrate binding. This is further evidence that each active site in the metallo‐phosphatases contains 2 metal atoms, one of type I and one of type II.