Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme)

Abstract

Human mitochondrial NAD(P)+-dependent malate dehydrogenase (decarboxylating) (malic enzyme) can be specifically and allosterically activated by fumarate. X-ray crystal structures have revealed conformational changes in the enzyme in the absence and in the presence of fumarate. Previous studies have indicated that fumarate is bound to the allosteric pocket via Arg67 and Arg91. Mutation of these residues almost abolishes the activating effect of fumarate. However, these amino acid residues are conserved in some enzymes that are not activated by fumarate, suggesting that there may be additional factors controlling the activation mechanism. In the present study, we tried to delineate the detailed molecular mechanism of activation of the enzyme by fumarate. Site-directed mutagenesis was used to replace Asp102, which is one of the charged amino acids in the fumarate binding pocket and is not conserved in other decarboxylating malate dehydrogenases. In order to explore the charge effect of this residue, Asp102 was replaced by alanine, glutamate or lysine. Our experimental data clearly indicate the importance of Asp102 for activation by fumarate. Mutation of Asp102 to Ala or Lys significantly attenuated the activating effect of fumarate on the enzyme. Kinetic parameters indicate that the effect of fumarate was mainly to decrease the K(m) values for malate, Mg2+ and NAD+, but it did not notably elevate kcat. The apparent substrate K(m) values were reduced by increasing concentrations of fumarate. Furthermore, the greatest effect of fumarate activation was apparent at low malate, Mg2+ or NAD+ concentrations. The K(act) values were reduced with increasing concentrations of malate, Mg2+ and NAD+. The Asp102 mutants, however, are much less sensitive to regulation by fumarate. Mutation of Asp102 leads to the desensitization of the co-operative effect between fumarate and substrates of the enzyme.

Figure 1. Crystal structure and fumarate binding pocket of human m-NAD-MDH

(A) Crystal structure of the enzyme in complex with NAD⁺, pyruvate, Mn²⁺ and fumarate (PDB code 1pj3). The active- and fumarate-site regions are highlighted with white boxes. Blue denotes NAD⁺, green denotes pyruvate, and red denotes Mn²⁺ in the active site; yellow denotes fumarate in the dimer interface. This figure was generated with PyMOL (DeLano Scientific LLC, San Carlos, CA, U.S.A.). (B) Fumarate binding ligands of human m-NAD-MDH are shown as a LIGPLOT diagram [29]. The bold bonds belong to the fumarate, the thin bonds belong to the hydrogen-bonded residues from the enzyme, and the dashed lines represent the hydrogen bonds. Spoked arcs represent hydrophobic contacts.

Figure 2. Activation of human m-NAD-MDH by fumarate

The assay mixture contained 15 mM malate, 10 mM MgCl₂ and 1 mM NAD⁺. ●, WT enzyme; ▼, D102E; ○, D102A; ▽, D102K.

Figure 3. Substrate and cofactor concentration-dependence of the activation of human m-NAD-MDH by fumarate

The fumarate concentration was fixed at 3 mM to achieve maximal activation of the enzyme under the indicated conditions. (A) Initial rate of the WT enzyme with (●; v_t) or without (○; v₀) 3 mM fumarate at various malate concentrations. The concentrations of MgCl₂ and NAD⁺ were fixed at 10 mM and 1.0 mM respectively. In (B)–(D), each point was obtained from v_t divided by v₀ at various concentrations of malate, MgCl₂ and NAD⁺ respectively. The non-varied substrate concentrations in (B)–(D) were 15, 10 and 1.0 mM for L-malate, MgCl₂ and NAD⁺ respectively. ●, WT enzyme; ▼, D102E; ○, D102A; ▽, D102K.

Figure 4. Activation by fumarate of human m-NAD-MDH at various malate concentrations

The initial velocities were measured at 50 mM Tris/HCl (pH 7.4), 10 mM MgCl₂ and 1.0 mM NAD⁺. The malate concentrations used were, from top to bottom, 0.25, 0.5, 1, 2, 3, 5, 10 and 15 mM. (A) WT; (B) D102E; (C) D102A; (D) D102K.

Figure 5. Fumarate binding region of human m-NAD-MDH

(A) Sequence alignments around the fumarate binding site of the 35 decarboxylating MDHs with amino acid sequences available. Amino acid sequences of the enzymes were searched by Blast [30] and alignments were generated by Clustal W [31]. The results are expressed by sequence logos with error bars [32]. The amino acid residues highlighted are charged residues in the fumarate binding region; blue for positively and red for negatively charged amino acids. (B) Local structural representation of the charged amino acid residues at the fumarate (Fum) binding region. Blue indicates basic residues (Lys and Arg) and red indicates acidic residues (Asp and Glu). This figure was generated with PyMOL (DeLano Scientific LLC). (C) Superimposition of the closed form I (PDB code 1do8; in pink) and closed form II (PDB code 1pj4) structures, indicating the location and conformation of Asp¹⁰² in the fumarate binding pocket. The green dashed lines representing hydrogen bonds between amino acid residues and fumarate (in yellow) were generated by Swiss-Pdb Viewer [33].