Functional role of fumarate site Glu59 involved in allosteric regulation and subunit-subunit interaction of human mitochondrial NAD(P)⁺-dependent malic enzyme

• Published in: The FEBS journal 2009-02, Vol.276 (4), p.983-994
• Main Authors: Hsieh, Ju-Yi, Chiang, Yu-Hsiu, Chang, Kuan-Yu, Hung, Hui-Chih
• Format: Article
• Language: English
• Subjects: Allosteric Regulation; Amino Acid Sequence; Amino Acid Substitution; analytical ultracentrifugation; Binding sites; Biochemistry; electrostatic interaction; Enzyme Activation; Enzymes; Fumarates - metabolism; Humans; Kinetics; Malate Dehydrogenase - chemistry; Malate Dehydrogenase - genetics; Malate Dehydrogenase - metabolism; Malates - metabolism; malic enzyme; Mitochondrial Proteins - chemistry; Mitochondrial Proteins - metabolism; Models, Molecular; Molecular Sequence Data; Molecular structure; mutagenesis; Mutation; Protein Multimerization; Protein Subunits - chemistry; Protein Subunits - metabolism; Recombinant Proteins - chemistry
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2008.06834.x

Here we report on the role of Glu59 in the fumarate-mediated allosteric regulation of the human mitochondrial NAD(P)⁺-dependent malic enzyme (m-NAD-ME). In the present study, Glu59 was substituted by Asp, Gln or Leu. Our kinetic data strongly indicated that the charge properties of this residue significantly affect the allosteric activation of the enzyme. The E59L enzyme shows nonallosteric kinetics and the E59Q enzyme displays a much higher threshold in enzyme activation with elevated activation constants, KA,Fum and αKA,Fum. The E59D enzyme, although retaining the allosteric property, is quite different from the wild-type in enzyme activation. The KA,Fum and αKA,Fum of E59D are also much greater than those of the wild-type, indicating that not only the negative charge of this residue but also the group specificity and side chain interactions are important for fumarate binding. Analytical ultracentrifugation analysis shows that both the wild-type and E59Q enzymes exist as a dimer-tetramer equilibrium. In contrast to the E59Q mutant, the E59D mutant displays predominantly a dimer form, indicating that the quaternary stability in the dimer interface is changed by shortening one carbon side chain of Glu59 to Asp59. The E59L enzyme also shows a dimer-tetramer model similar to that of the wild-type, but it displays more dimers as well as monomers and polymers. Malate cooperativity is not significantly notable in the E59 mutant enzymes, suggesting that the cooperativity might be related to the molecular geometry of the fumarate-binding site. Glu59 can precisely maintain the geometric specificity for the substrate cooperativity. According to the sequence alignment analysis and our experimental data, we suggest that charge effect and geometric specificity are both critical factors in enzyme regulation. Glu59 discriminates human m-NAD-ME from mitochondrial NADP⁺-dependent malic enzyme and cytosolic NADP⁺-dependent malic enzyme in fumarate activation and malate cooperativity.