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

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 (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.

Figure 1

Structure and sequence alignment of human m-NADP-ME. (a) The crystal structure of human m-NADP-ME shows the dimer interface between the AB or CD dimers, the tetramer interface between the AD or BC dimers and the four active sites and the exosite with ATP in each monomer (PDB code: 1PJ4). (b) The overall binding resides of the exosite, with an NADH-binding mode (PDB code: 1PJ2) and an ATP-binding mode (PDB code: 1GZ4). These figures were generated by using PyMOL.

Figure 2

Continuous sedimentation coefficient distribution of human m-NAD(P)-ME with non-nucleotide ligands. The enzyme concentrations were 1.6, 4.7, and 14.2 μM in the phosphate buffer. (a) Apoenzyme; (b) L-malate (20 mM); (c) pyruvate (8 mM); (d) MgCl₂ (2 mM); and (e) fumarate (0.2 mM).

Figure 3

Continuous sedimentation coefficient distribution of m-NAD(P)-ME with different nucleotide ligands. The enzyme concentrations were 1.6, 4.7, and 14.2 μM in the phosphate buffer with L-malate (20 mM), pyruvate (8 mM), MgCl₂ (2 mM) or fumarate (0.2 mM). (a–h) with NAD⁺ (0.4 mM); (i–p) with ATP (0.5 mM); and (q–x) ADP (with 1 mM).

Figure 4

Continuous sedimentation coefficient distribution of human m-NAD(P)-ME WT and the exosite mutants. The enzyme concentrations were fixed at 1.6, 4.7, and 14.2 μM in PBS. The size distribution plots of apoenzyme (ligand-free), enzyme with NAD⁺ (0.4 mM), and enzyme with ATP (0.5 mM) were shown on the upper, middle, and lower panels, respectively. (a) ME2_WT; (b) ME2_H154V; (c) ME2_K156A; (d) ME2_G192A; (e) ME2_R194N; (f) ME2_R197E; (g) ME2_R197D.

Figure 5

Continuous sedimentation coefficient distribution of human m-NAD(P)-ME WT and the exosite mutants. The enzyme concentrations were fixed at 1.6, 4.7, and 14.2 μM in PBS. The size distribution plots of apoenzyme (ligand-free), enzyme with NAD⁺ (0.4 mM), and enzyme with ATP (0.5 mM) were shown on the upper, middle, and lower panels, respectively. (a) ME2_N482G; (b) ME2_N482A; (c) ME2_R542V; (d) ME2_Y552F; (e) ME2_R556Q; (f) ME2_R556V.