pH dependence of the kinetic parameters for oxalacetate decarboxylation and pyruvate reduction reactions catalyzed by malic enzyme

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Abstract
Both chicken liver NADP-malic enzyme and Ascaris suum NAD-malic enzyme catalyze the metal-dependent decarboxylation of oxalacetate. Both enzymes catalyze the reaction either in the presence or in the absence of dinucleotide. The presence of dinucleotide increases the affinity of oxalacetate for the chicken liver NADP-malic enzyme, but this information could not be obtained in the case of A. suum NAD-malic enzyme because of the low affinity of free enzyme for NAD. The kinetic mechanism for oxalacetate decarboxylation by the chicken liver NADP-malic enzyme is equilibrium ordered at pH values below 5.0 with NADP adding to enzyme first. The Ki for NADP increases by a factor of 10 per pH unit below pH 5.0. An enzyme residue is required protonated for oxalacetate decarboxylation (by both enzymes) and pyruvate reduction (by the NAD-malic enzyme), but the .beta.-carboxyl of oxalacetate must be unprotonated for reaction (by both enzymes). The pK of the enzyme residue of the chicken liver NADP-malic enzyme decreases from a value of 6.4 in the absence of NADP to about 5.5 with Mg2+ and 4.8 with Mn2+ in the presence of NADP. The pK value of the enzyme residue required protonated for either oxalacetate decarboxylation or pyruvate reduction for the A. suum NAD-malic enzyme is about 5.5-6.0. Although oxalacetate binds equally well to protonated and unprotonated forms of the NADP-enzyme, the NAD-enzyme requires that oxalacetate or pyruvate selectively bind to the protonated form of the enzyme. Both enzymes prefer Mn2+ over Mg2+ for oxalacetate decarboxylation. The NAD-malic enzyme also prefers Mn2+ for pyruvate reduction, and the rate of this reaction is 1/600th the rate of the oxidative decarboxylation of malate (using Mg2+ as the divalent metal ion). The kinetic mechanism for pyruvate reduction is rapid equilibrium ordered with NADH adding first. A comparison of the absolute values of Vmax for the oxidative decarboxylation of L-malate and the decarboxylation of oxalacetate suggests that decarboxylation of the oxalacetate intermediate is the main rate-determining step in the malic enzyme reaction. Since the NAD-malic enzyme from A. suum catalyzes the metal-dependent decarboxylation of oxalacetate and reduction of pyruvate, the present classification for this enzyme (EC 1.1.1.39) should be changed to EC 1.1.1.38.