The Functional Properties of the Zn2+-and Co2+-Alkaline Phosphatases of Escherichia coli. Labelling of the Active Site with Pyrophosphate, Complex Formation with Arsenate, and Reinvestigation of the Role of the Zinc Atoms

Abstract

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.