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Human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity, ATP inhibition and substrate cooperativity. The determinant of ATP inhibition in malic enzyme isoforms has not yet been identified. Sequence alignment of nucleotide-binding sites of ME isoforms revealed that Lys346 is conserved uniquely in m-NAD-ME. In other ME isoforms, this residue is serine. As the inhibitory effect of ATP is more pronounced on m-NAD-ME than on other ME isoforms, we have examined the possible role of Lys346 by replacing it to alanine, serine or arginine. Our kinetic data indicate that the K346S mutant enzyme displays a shift in its cofactor preference from NAD+ to NADP+ upon increasing kcat,NADP and decreasing Km, NADP. Furthermore, the cooperative binding of malate becomes less significant in human m-NAD-ME after mutation of Lys346. The h value for the wild-type is close to 2, but those of the K346 mutants are approximately 1.5. The K346 mutants can also be activated by fumarate and the cooperative effect can be abolished by fumarate, suggesting that the allosteric property is retained in these mutants. Our data strongly suggest that Lys346 in human m-NAD-ME is required for ATP inhibition. Mutation of Lys346 to Ser or Ala causes the enzyme to be much less sensitive to ATP, similar to cytosolic NADP-dependent malic enzyme. Substitution of Lys to Arg did not change the isoform-specific inhibition of the enzyme by ATP. The inhibition constants of ATP are increased for K346S and K346A, but are similar to those of the wild-type for K346R, suggesting that the positive charge rather than group specificity is required for binding affinity of ATP. Thus, ATP inhibition is proposed to be determined by the electrostatic potential involving the positive charge on the side chain of Lys346.