Cyclic AMP-dependent and -independent protein kinases in HeLa cells

Malate saturation isotherms for the NAD⁺ malic enzyme determined at widely differing, but saturating, concentrations (8, 80, 160 mm) of magnesium show the same response to malate concentration only when velocity is plotted against the concentration of free malate^2?. This identification of the ionized malic acid as the true substrate for this enzyme, together with the observation that the complex of Mg with malate has no influence on the reaction rate even at very high concentrations, indicates that the metal ion activator of the enzyme must also bind in the ionized form. A kinetic analysis shows that, with respect to magnesium and malate, the malic enzyme catalyzes a rapid equilibrium reaction of the intersecting type. Either Mg²⁺ or malate^2? may bind first but the fact that the K_m's for both Mg²⁺ and malate^2? are smaller than the respective K_i's suggests that, when either metal ion or malate is present on the enzyme, the other is bound more tightly than when it binds to the free enzyme. This demonstration of the nature of the true substrates for this enzyme has implications for studies of the NAD⁺ malic enzyme in which conditions influencing the amount of free magnesium and malate, e.g., changes in pH, addition of weak acid effectors etc., are involved.