Open AccessThe Functional Properties of the Zn 2+ ‐and Co 2+ ‐Alkaline Phosphatases of Escherichia coli
Author(s) -
Petitclerc C.,
Lazdunski C.,
Chappelet D.,
Moulin A.,
Lazdunski M.
Publication year - 1970
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1970.tb00290.x
Subject(s) - arsenate , pyrophosphate , chemistry , zinc , enzyme , alkaline phosphatase , phosphatase , arsenite , inorganic chemistry , stereochemistry , biochemistry , arsenic , organic chemistry
The labelling of the Zn 2+ and Co 2+ ‐phosphatase with [ 32 P]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 [ 32 P]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 [ 32 P]pyrophosphate in the Zn 2+ as in the Co 2+ ‐phosphatase. Both sites are reactive at acidic pH. (b) The binding of two arsenate molecules to the Co 2+ ‐phosphatase at pH 8.0 has an anticooperative character which we found previously in 1969 for the binding of orthophosphate to the Zn 2+ phosphatase. (c) The incubation of the enzyme at pH 8.5 in high concentrations of [ 74 As]arsenate gives rise to a complex containing 2 moles of arsenate per mole of dimer. One [ 74 As]arsenate is loosely bound and easily exchangeable for unlabelled arsenate; the other [ 74 As]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 [ 32 P]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 Zn 2+ ‐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.