P-31 NMR OF ALKALINE-PHOSPHATASE - DEPENDENCE OF PHOSPHATE BINDING STOICHIOMETRY ON METAL-ION CONTENT

Alkaline phosphatase of Escherichia coli (a dimeric Zn metalloenzyme of identical subunits) containing either 2 catalytic Zn(II) ions or 2 catalytic Cd(II) ions shows absolute negative cooperativity of phosphate binding as determined by 31P NMR of enzyme-bound phosphate species. With excess phosphate at millimolar concentrations, only 1 mol of noncovalent phosphate complex, E.cntdot.P (31P resonance at 5 ppm) is formed by the 2 Zn(II) enzyme at pH 8; 1 mol of the covalent intermediate, E-P (31P resonance at 8 ppm) is formed by the 2 Cd(II) enzyme at pH 6.5. Enzymes containing an additional pair of structural Zn(II) or Cd(II) ions and 2 Mg(II) ions form 2 mol of E.cntdot.P or E-P, respectively, as detected by the characteristic 31P resonances for these 2 intermediates. The change in phosphate binding stoichiometry induced by the pair of structural metal ions was confirmed by [32P]-phosphate labeling studies of the 2 Cd(II) and 4 Cd(II) enzymes carried out at enzyme concentrations identical with those of the NMR samples. Addition of 2 Cd(II) ions to the apodiphosphoryl enzyme, prepared by treatment of the 4 Cd(II) enzyme with chelating agents, results in dephosphorylation of one of the active sites, confirming that negative cooperativity of phosphate binding is an equilibrium property of the 2 metal enzyme. The varying content of structural metal ions in the enzyme as a function of changing pH, dilution and preparative technique appears to account for the variation in phosphate binding stoichiometry reported for this enzyme. Metal ions in the environment may act as allosteric activators of alkaline phosphatase in addition to the essential role of the 2 catalytic Zn(II) ions in the mechanisms of action.