Influential factor contributing to the isoform‐specific inhibition by ATP of human mitochondrial NAD(P)⁺‐dependent malic enzyme

Abstract

Malic enzymes are a family of oxidative decarboxylases that catalyze the oxidation of L-malate to pyruvate with accompanying reduction of NAD(P) + to NAD(P)H and release of CO 2 [1][2][3][4][5][6][7]. The enzyme requires a divalent metal ion (Mn 2+ or Mg 2+ ) for enzyme catalysis [8,9]. In addition, the metal ion is 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 k cat,NADP and decreasing K m,NADP . Furthermore, the cooperative binding of malate becomes less significant in human m-NAD-ME after mutation of Lys346. The h value for the wildtype 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.Abbreviations c-NADP-ME, cytosolic NADP + -dependent malic enzyme; k cat,NAD, catalytic constant using NAD + as the cofactor; k cat,NADP, catalytic constant using NADP + as the cofactor; K i,ATP(NAD), inhibition constant of ATP for NAD + ; K i,ATP(NADP), inhibition constant of ATP for NADP + ; ME, malic enzyme; m-NAD-ME, mitochondrial NAD(P) + -dependent malic enzyme; m-NADP-ME, mitochondrial NADP + -dependent malic enzyme.

Engineering of the Cofactor Specificities and Isoform-specific Inhibition of Malic Enzyme

“…However, the double mutant K346S/Q362K shows slight improvement in decreasing K m,NADP and increasing k cat,NADP , suggesting that Ser-346 plays a supplementary role in cofactor preference of the human m-NAD(P)-ME isoform. We have also suggested that the role of Lys-346 in human NAD(P)-ME is associated with the isoform-specific ATP inhibition (35). In this paper, we further demonstrate that human enzyme with the K346S mutation (K346S, K346S/Y347K, K346S/Q362K, and K346S/Y347K/Q362K) is much less inhibited by ATP and has a larger K i,ATP value than those enzymes without K346S (Fig.…”

“…Recent works on the Ascaris enzyme indicate that changing this and other residues in the cofactor-binding site have only detrimental effects on cofactor binding (33,34). Lys-346 in human m-NAD(P)-ME has minor effects on determining the cofactor preference, but the positive charge of this residue has been identified as having a significant impact on the isoform-specific ATP inhibition (35). Residue 347 also displays an isoform-specific distribution as a Lys in c-NADP-ME but as Tyr or Phe in m-NAD(P)-ME (Fig.…”

“…Our previous studies have suggested that Lys-346 is important for ATP inhibition of human m-NAD(P)-ME (35). With NAD ϩ as the cofactor, the K346S enzyme became less inhibited by ATP (Fig.…”

Functional Roles of the Tetramer Organization of Malic Enzyme

“…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 abolished by fumarate (9 -12). Mutagenesis and kinetic studies demonstrated that ATP is an active-site inhibitor, although it also binds to the exo sites in the tetramer interface (13)(14)(15). Structural studies also revealed an allosteric binding site for fumarate residing at the dimer interface.…”

“…1A). In the ME family, Ascaris suum and human m-NAD(P)-ME were found to be activated by fumarate (11,(15)(16)(17)31). However, the relationship between enzyme regulation and subunit-subunit interaction is still unclear.…”