Activation of rabbit muscle fructose diphosphatase by EDTA and the effect of divalent cations

Abstract Rabbit muscle fructose diphosphatase, purified to homogeneity, was found to require EDTA for activity at neutral, but not alkaline, pH. In the presence of EDTA the enzyme shows a single optimum at pH 7.2 with MgCl 2 , and two optima at pH 7.5 and 9.5 with MnCl 2 . The absence of the alkaline pH optimum with Mg 2+ may be attributed to inhibition by this cation at alkaline pH, which is observed when the enzyme is tested in the presence of both Mn 2+ and Mg 2+ . In addition, inhibition by excess substrate and KF is observed only in the presence of Mg 2+ . The concentration of EDTA required for half-maximum activity at pH 7.2 varies with the nature and concentration of the divalent cation employed. With MgCl 2 (10 m m ) half-maximum activity was observed with 1.5 μ m EDTA; with MnCl 2 (0.1 m m ) the concentration of EDTA required was 0.02 m m . When the concentration of EDTA exceeded that of the divalent cation, the reaction was inhibited. Therefore, it is concluded that in addition to the absolute requirement for free Mn 2+ or Mg 2+ , the EDTA-divalent cation complex is required for optimal activity at neutral pH. The affinity of the enzyme for EDTA-divalent cation complex is much higher with Mg 2+ , as compared to Mn 2+ . Zn 2+ and Pb 2+ , and to a lesser extent Ca 2+ , were found to be strongly inhibitory; inhibition by Ca 2+ was competitive with Mg 2+ and Mn 2+ . The sensitivity to inhibition by AMP is much greater in the presence of Mg 2+ , as compared with Mn 2+ . The results suggest that the conformations induced by Mg 2+ and Mn 2+ are different, with the former being more sensitive to effectors than the conformation induced by Mn 2+ .