Dual roles of Lys(57) at the dimer interface of human mitochondrial NAD(P)+-dependent malic enzyme.

Human m-NAD(P)-ME [mitochondrial NAD(P)+-dependent ME (malic enzyme)] is a homotetramer, which is allosterically activated by the binding of fumarate. The fumarate-binding site is located at the dimer interface of the NAD(P)-ME. In the present study, we decipher the functional role of the residue Lys57, which resides at the fumarate-binding site and dimer interface, and thus may be involved in the allosteric regulation and subunit-subunit interaction of the enzyme. In the present study, Lys57 is replaced with alanine, cysteine, serine and arginine residues. Site-directed mutagenesis and kinetic analysis strongly suggest that Lys57 is important for the fumarate-induced activation and quaternary structural organization of the enzyme. Lys57 mutant enzymes demonstrate a reduction of Km and an elevation of kcat following induction by fumarate binding, and also display a much higher maximal activation threshold than WT (wild-type), indicating that these Lys57 mutant enzymes have lower affinity for the effector fumarate. Furthermore, mutation of Lys57 in m-NAD(P)-ME causes the enzyme to become less active and lose co-operativity. It also increased K0.5,malate and decreased kcat values, indicating that the catalytic power of these mutant enzymes was significantly impaired following mutation of Lys57. Analytical ultracentrifugation analysis demonstrates that the K57A, K57S and K57C mutant enzymes dissociate predominantly into dimers, with some monomers present, whereas the K57R mutant forms a mixture of dimers and tetramers, with a small amount of the enzyme in monomeric form. The dimeric form of these Lys57 mutants, however, cannot be reconstituted into tetramers with the addition of fumarate. Modelling structures of the Lys57 mutant enzymes show that the hydrogen bond network in the dimer interface where Lys57 resides may be reduced compared with WT. Although the fumarate-induced activation effects are partially maintained in these Lys57 mutant enzymes, the mutant enzymes cannot be reconstituted into tetramers through fumarate binding and cannot recover their full enzymatic activity. In the present study, we demonstrate that the Lys57 residue plays dual functional roles in the structural integrity of the allosteric site and in the subunit-subunit interaction at the dimer interface of human m-NAD(P)-ME.