Malic enzyme has a dimer of dimers quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In addition, the enzyme has distinct active sites within each subunit. The mitochondrial NAD(P) ؉ -dependent malic enzyme (m-NAD(P)-ME) isoform behaves cooperatively and allosterically and exhibits a quaternary structure in dimertetramer equilibrium. The cytosolic NADP ؉ -dependent malic enzyme (c-NADP-ME) isoform is noncooperative and nonallosteric and exists as a stable tetramer.In this study, we analyze the essential factors governing the quaternary structure stability for human c-NADP-ME and m-NAD(P)-ME. Site-directed mutagenesis at the dimer and tetramer interfaces was employed to generate a series of dimers of c-NADP-ME and m-NAD(P)-ME. Size distribution analysis demonstrated that human c-NADP-ME exists mainly as a tetramer, whereas human m-NAD(P)-ME exists as a mixture of dimers and tetramers. Kinetic data indicated that the enzyme activity of c-NADP-ME is not affected by disruption of the interface. There are no significant differences in the kinetic properties between AB and AD dimers, and the dimeric form of c-NADP-ME is as active as tetramers. In contrast, disrupting the interface of m-NAD(P)-ME causes the enzyme to be less active than wild type and to become less cooperative for malate binding; the k cat values of mutants decreased with increasing K d,24 values, indicating that the dissociation of subunits at the dimer or tetramer interfaces significantly affects the enzyme activity. The above results suggest that the tetramer is required for a fully functional m-NAD(P)-ME. Taken together, the analytical ultracentrifugation data and the kinetic analysis of these interface mutants demonstrate the differential role of tetramer organization for the c-NADP-ME and m-NAD(P)-ME isoforms. The regulatory mechanism of m-NAD(P)-ME is closely related to the tetramer formation of this isoform.Malic enzymes catalyze a reversible oxidative decarboxylation of L-malate to yield pyruvate and CO 2 with reduction of NAD(P) ϩ to NAD(P)H. This reaction requires a divalent metal ion (Mg 2ϩ or Mn 2ϩ ) for catalysis (1-3). Malic enzymes are found in a broad spectrum of living organisms that share conserved amino acid sequences and structural topology; such shared characteristics reveal a crucial role for the biological functions of these enzymes (4, 5). In mammals, malic enzymes have been divided into three isoforms according to their cofactor specificity and subcellular localization as follows: cytosolic NADP ϩ -dependent (c-NADP-ME), 2 mitochondrial NADP ϩ -dependent (m-NADP-ME), and mitochondrial NAD(P) ϩ -dependent (m-NAD(P)-ME). The m-NAD(P)-ME isoform displays dual cofactor specificity; it can use both NAD ϩ and NADP ϩ as the coenzyme, but NAD ϩ is more favored in a physiological environment (6 -8). Dissimilar to the other two isoforms, m-NAD(P)-ME binds malate cooperatively, and it can be allosterically activated by fumarate; the sigmoidal kinetics observed with cooperativity is abo...