Phosphate Binding to Alkaline Phosphatase

The metal ion dependence of a2P binding to Escherichia coli alkaline phosphatase has been studied by means of equilibrium dialysis. Whereas the apoenzyme does not bind phosphate (no sites with K < 5 X 10-5 M), the addition of 2 Zn(I1) cations per molecule induces the tight binding of 1 phosphate dianion (K = 6 X 1OF M). The magnitude of this binding constant is affected by ionic strength, pH, and protein concentration. Of the first transition and IIB metal ions, Mn(II), Co(II), Zn(II), and Cd(H) all induce the tight binding of one phosphate per dimer, but only Zn(I1) and Co(I1) induce enzymatic activity. Cu(I1) is less effective, while Ni(I1) and Hg(I1) are ineffective in inducing phosphate binding. The formation of the phosphoryl enzyme, as measured by treatment of the 32P-protein mixture with perchloric acid, is also metal ion-dependent. The Zn(I1) enzyme forms the phosphoryl enzyme only at low pH, a maximum of 0.6 mole per mole of dimer being observed at pH 5. The apoenzyme forms no phosphoryl enzyme at any pH. In marked contrast, Cd(H) induces the formation of significant equilibrium concentrations of phosphoryl enzyme in the alkaline pH range, reaching a maximum stoichiometry of 1 mole per mole of dimer at pH 6.5. Isolation of a phosphorylated peptide from the Cd(I1) enzyme shows that the same seryl residue is phosphorylated in the Cd(I1) protein as in the native Zn(I1) enzyme. Mn(I1) and Co(H) also induce the formation of significant amounts of phosphoryl enzyme in the alkaline pH region. Phosphorylation of the Cd(H) enzyme is relatively slow, k = -3 x lo-” set-I, but once formed the Cd(I1) phosphoryl enzyme does not break down as shown by its failure to catalyze the exchange of I*0 from H2180 into inorganic phosphate. The data suggest that the metal ion plays an important role in formation and breakdown of the phosphoryl enzyme as well as in the binding of phosphate.