Abstract
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 (Kd,12) and dimer-tetramer (Kd,24) dissociation constants were determined with these ligands. With NAD+, the enzyme formed more tetramers, and its Kd,24 (0.06 µM) was 6-fold lower than the apoenzyme Kd,24 (0.34 µM). When ATP was present, the enzyme displayed more dimers, and its Kd,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.
Highlights
Malic enzyme (ME) is a homotetramer with four independent catalytic sites
The enzyme m-NAD(P)-ME has a dimer of dimers quaternary structure with four identical subunits, and catalysis of the enzyme is regulated by the substrate L-malate, cofactor NAD(P)+, divalent cation Mg2+, activator fumarate, and inhibitor ATP when these ligands are bound to their respective sites at the dimer or tetramer interfaces (Fig. 1a)
The results of the present study demonstrate that different nucleotides have disparate effects on regulating the dynamics of the quaternary structure of m-NAD(P)-ME (Figs 2 and 3)
Summary
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) H1,2. The m-NADP-ME isoform distributes mainly in brain, muscle, and heart tissues This enzyme isoform participates in fatty acid biosynthesis and is important for insulin secretion in pancreatic β-cells[1,6]. Tumor cells can use glutamine and glutamate as an alternative energy source[10,11,12], and m-NAD(P)-ME may play an important role in glutamine metabolism[7,10,11,13,14,15] For this reason, m-NAD(P)-ME is considered a cancer drug target. The m-NAD(P)-ME and c-NADP-ME enzymes are found to associate with p53-dependent senescence, and MEs and p53 play reciprocal roles in regulating the fate of the cells[19] Both m-NAD(P)-ME knockdown and treatment with dimethyl-malate (DMM) mimicking the m-NAD(P)-ME knockdown phenotype suppress lung tumor growth in vivo[20]. Human m-NAD(P)-ME has a complex kinetic regulatory mechanism and is bound to its substrate L-malate cooperatively, activated by fumarate allosterically and inhibited by ATP23,25,26
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