Effect of pH on the role of Mg2+ and Mn2+ on Phycomyces isocitrate lyase kinetics.

Mg2+ and Mn2+ function with the same partial mixed-type activation/inhibition mechanism, in which the metal isocitrate complex is the true substrate of Phycomyces isocitrate lyase. Binding of Mg2+ or Mn2+ to the activation site normally contributes significantly to the mechanism of catalysis. Whereas both ions activate catalysis at pH 7.3, at pH 8.5, Mg2+ ions behaved as inhibitors (beta < 1) and Mn2+ ions continued to function as activators. The binding of Mg2+ or Mn2+ to the activator site is virtually independent of the pH value. The affinity of the non-activated form of the enzyme for the Mg(2+)-isocitrate complex decreased (Ksa increased 20-fold) as pH was raised, but for Mn2+ ions the affinity of the activated enzyme for the Mn(2+)-isocitrate complex decreased 86-fold. The ion moiety of the metal-ion-isocitrate complex appears to be involved in the formation of the active enzyme-substrate complex from the non-activated enzyme.