The Functional Properties of the Zn2+‐and Co2+‐Alkaline Phosphatases of Escherichia coli

The labelling of the Zn2+ and Co2+-phosphatase with [32P]pyrophosphate was studied at different pH. The formation of complexes between arsenate and the enzyme has been investigated using a variety of techniques: kinetics, equilibrium dialysis, isolation of the complex, determination of its activity for [32P]pyrophosphate. The mechanism of the alkaline phosphatase is obviously very peculiar. Its main features are: (a) Only one of the two sites can be phosphorylated at alkaline pH from [32P]pyrophosphate in the Zn2+ as in the Co2+-phosphatase. Both sites are reactive at acidic pH. (b) The binding of two arsenate molecules to the Co2+-phosphatase at pH 8.0 has an anticooperative character which we found previously in 1969 for the binding of orthophosphate to the Zn2+ phosphatase. (c) The incubation of the enzyme at pH 8.5 in high concentrations of [74As]arsenate gives rise to a complex containing 2 moles of arsenate per mole of dimer. One [74As]arsenate is loosely bound and easily exchangeable for unlabelled arsenate; the other [74As]arsenate is very firmly bound and not exchangeable. A detailed study of this complex suggests that it is an hybrid complex containing one covalently bound and one noncovalently bound arsenate. One never observes the formation of a diarsenylated enzyme. The labelling of the phosphatase molecule with [32P]pyrophosphate provides an easy all-or-none assay which was used for a revaluation of the number and of the role of the zinc atoms in the enzyme. The 4 g atoms of zinc per mole of protein belong to 2 different families. The 2 sites of type I bind zinc tightly, while the 2 sites of type II bind it more loosely. Only the Zn2+-phosphatase with 4 g atoms of zinc per mole of protein is an active enzyme. It is suggested that each active site contains 2 zinc atoms, one of type I and one of type II.