Role of the divalent metal ion in the NAD:malic enzyme reaction: An ESEEM determination of the ground state conformation of malate in the E:Mn: Malate complex

Tipton, Peter A.; Quinn, Thomas P.; Peisach, Jack; Cook, Paul F.

doi:10.1002/pro.5560050818

Cited by 7 publications

(6 citation statements)

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“…In the functionally related enzyme ICDH, which also has a two step catalytic mechanism, one of the aspartate ligands of the cation was proposed as the general base and a tyrosine or lysine residue was proposed as the general acid [56]. The situation with ME is more complicated, as a water-mediated interaction between the cation and the substrate is expected [4,58]. This might increase the distance between the aspartate/glutamate ligands of the cation and malate, making them less likely to function as the general base.…”

Section: Discussionmentioning

confidence: 99%

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Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases

Bhargava

et al. 1999

Structure

144

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Background: Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate and CO(2) with the concomitant reduction of NAD(P)(+) to NAD(P)H. They are widely distributed in nature and have important biological functions. Human mitochondrial NAD(P)(+)-dependent malic enzyme (mNAD-ME) may have a crucial role in the metabolism of glutamine for energy production in rapidly dividing cells and tumors. Moreover, this isoform is unique among malic enzymes in that it is a cooperative enzyme, and its activity is controlled allosterically. Results: The crystal structure of human mNAD-ME has been determined at 2.5 Å resolution by the selenomethionyl multiwavelength anomalous diffraction method and refined to 2.1 Å resolution. The structure of the monomer can be divided into four domains; the active site of the enzyme is located in a deep cleft at the interface between three of the domains. Three acidic residues (Glu255, Asp256 and Asp279) were identified as ligands for the divalent cation that is required for catalysis by malic enzymes. Conclusions: The structure reveals that malic enzymes belong to a new class of oxidative decarboxylases. The tetramer of the enzyme appears to be a dimer of dimers. The active site of each monomer is located far from the tetramer interface. The structure also shows the binding of a second NAD(+) molecule in a pocket 35 Å away from the active site. The natural ligand for this second binding site may be ATP, an allosteric inhibitor of the enzyme.

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Section: Discussionmentioning

confidence: 99%

“…Malic enzyme (ME; EC 1.1.1.40) catalyzes the oxidative decarboxylation of L-malate to pyruvate with the concomitant reduction of the cofactor NAD + or NADP + [1][2][3][4][5]. In addition to NAD(P) + , the enzyme also requires divalent cations (Mg 2+ or Mn 2+ ) as cofactors.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases

Bhargava

et al. 1999

Structure

144

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“…Malic enzyme (ME) is a homotetramer with four independent catalytic sites. This enzyme catalyzes an oxidative decarboxylation of L-malate (MAL) to pyruvate (PYR) concomitant with the reduction of NAD(P) + to NAD(P)H 1,2 . The enzyme family displays conserved sequences in nature, from bacteria to mammals, indicating their importance for biological function 3 .…”

Section: Introductionmentioning

confidence: 99%

Functional Roles of Metabolic Intermediates in Regulating the Human Mitochondrial NAD(P)+-Dependent Malic Enzyme

Hsieh

Shih

Kuo

et al. 2019

Sci Rep

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Human mitochondrial NAD(P) + -dependent malic enzyme (m-NAD(P)-ME) has a dimer of dimers quaternary structure with two independent allosteric sites in each monomer. Here, we reveal the different effects of nucleotide ligands on the quaternary structure regulation and functional role of the human m-NAD(P)-ME exosite. In this study, size distribution analysis was utilized to investigate the monomer-dimer-tetramer equilibrium of m-NAD(P)-ME in the presence of different ligands, and the monomer-dimer ( K d,12 ) and dimer-tetramer ( K d,24 ) dissociation constants were determined with these ligands. With NAD + , the enzyme formed more tetramers, and its K d,24 (0.06 µM) was 6-fold lower than the apoenzyme K d,24 (0.34 µM). When ATP was present, the enzyme displayed more dimers, and its K d,24 (2.74 µM) was 8-fold higher than the apoenzyme. Similar to the apoenzyme, the ADP-bound enzyme was present as a tetramer with a small amount of dimers and monomers. These results indicate that NAD + promotes association of the dimeric enzyme into tetramers, whereas ATP stimulates dissociation of the tetrameric enzyme into dimers, and ADP has little effect on the tetrameric stability of the enzyme. A series of exosite mutants were created using site-directed mutagenesis. Size distribution analysis and kinetic studies of these mutants with NAD + or ATP indicated that Arg197, Asn482 and Arg556 are essential for the ATP binding and ATP-induced dissociation of human m-NAD(P)-ME. In summary, the present results demonstrate that nucleotides perform discrete functions regulating the quaternary structure and catalysis of m-NAD(P)-ME. Such regulation by the binding of different nucleotides may be critically associated with the physiological concentrations of these ligands.

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

2008

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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.

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Role of the divalent metal ion in the NAD:malic enzyme reaction: An ESEEM determination of the ground state conformation of malate in the E:Mn: Malate complex

Cited by 7 publications

References 44 publications

Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases

Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases

Functional Roles of Metabolic Intermediates in Regulating the Human Mitochondrial NAD(P)+-Dependent Malic Enzyme

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

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Role of the divalent metal ion in the NAD:malic enzyme reaction: An ESEEM determination of the ground state conformation of malate in the E:Mn: Malate complex

Cited by 7 publications

References 44 publications

Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases

Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases

Functional Roles of Metabolic Intermediates in Regulating the Human Mitochondrial NAD(P)+-Dependent Malic Enzyme

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

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