65Zn(II), 115mCd(II), 60Co(II), and mg(II) binding to alkaline phosphatase of Escherichia coli. Structural and functional effects.

Zn(II), Cd(II), Co(II) and Mg(II) binding to apoalkaline phosphatase of Escherichia coli and the relative stabilities of the resulting metalloenzyme complexes have been measured by equilibrium dialysis and metal exchange reactions using gamma-emitting isotopes of these metals. At millimolar concentrations of these metal ions the alkaline phosphatase dimer binds three pairs of metal ions (A, B, and C sites). One of these pairs dialyzes readily without detectable change in the structure or function of the enzyme (C site). Of the remaining two pairs, the binding affinity of both for Zn(II) and Cd(II) is increased by formation of the phosphoenzyme intermediates. Cd(II) is bound less tightly to both A and B sites than Zn(II), and at pH 6.5 Cd(II) is induced to bind to the B sites by formation of the phosphate complexes. Mg(II), 5-10 mM, competes successfully with the IIB metal ions for the second or lower affinity pair of binding sites (B sites), although Mg(II) is a relatively poor competitor on an equimolar basis, especially for Cd(II). Binding of metal ions to the apoenzyme appears to be a cooperative process involving conformational changes in the protein which are not readily reversible. The initial binding of a pair of Zn(II) or Cd(II) ions to the apoenzyme is characterized by equilibrium constants of 10(-5) to 10(-7) M for Zn(II) and 10(-4) to 10(-5) M for Cd(II). Following the cooperative binding of all three pairs of metal ions, however, re-establishment of equilibrium by dialysis indicates binding constants of less than 10(-8) M for Zn(II) and less than 10(-6) M for Cd(II) at the sites of greatest affinity (A sites). Binding of Mg(II) or Cd(II) to the B site, once the A site is occupied, increases the phosphorylation rate of the Cd(II) enzyme by 20-fold. In the presence of saturating concentrations of Mg(II) complete activity is restored to the apoenzyme by 2 Zn(II) ions. In the absence of Mg(II) as many as 6 Zn(II) ions may be required before complete restoration is achieved. Roles for the A and B site metal ions in the catalytic mechanism are discussed.