Interactions of Zn 2+ and Mg 2+ with rabbit liver fructose 1,6-bisphosphatase

Abstract

Purified rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) binds 12 mol of Zn 2+/mol, presumably 3 per subunit. Zn 2+ binds to the first set of 4 sites with high affinity and shows positive cooperativity; half-saturation of these sites was at a concentration of 0.03 μ m. The dissociation constant for Zn 2+ binding to the second set of sites is 0.35 μ m. Binding to the third set of sites requires the presence of the substrate, fructose 1,6-bisphosphate, and shows low affinity, with K d ≅ 2.5 μM. Mg 2+, the activating cation, alters the binding of Zn 2+ to all three sets of sites and together both cations appear to modulate the catalytic activity. When the enzyme is activated with 0.2 m m Mg 2+, filling of the first set of high-affinity sites by Zn 2+ results in inhibition of catalytic activity; both Zn 2+ binding and inhibition by Zn 2+ are prevented when the concentration of Mg 2+ is raised to 0.1 m, or when EDTA is added. The inhibitory effect of Zn 2+ is also partially reversed by raising the concentration of Zn 2+ and at high concentrations (10 μ m) Zn 2+ can substitute for Mg 2+ as the activating cation. A tentative model is presented in which the first set of sites is considered to be involved in inhibition by Zn 2+ and the third set in activation by Mg 2+ or Zn 2+; the second set of sites of intermediate affinity appears to modulate the catalytic activity between the high and low range, depending on whether these sites are filled by Zn 2+ or by Mg 2+.