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[摘要]：Human m-NAD(P)-ME [mitochondrial NAD(P)(+)-dependent ME (malic enzyme)] is a homotetramer, which is allosterically activated by the binding of fumarate. Ther 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 Lys(57), 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, Lys(57) is replaced with alanine, cysteine, serine and arginine residues. Site-directed mutagenesis and kinetic analysis strongly suggest that Lys(57) is important for the fumarate-induced activation and quaternary structural organization of the enzyme. Lys(57) mutant enzymes demonstrate a reduction of K-m and an elevation of k(eat) following induction by fumarate binding, and also display a much higher maximal activation threshold than WT (wild-type), indicating that these Lys(57) mutant enzymes have lower affinity for the effector fumarate. Furthermore, mutation of Lys(57) in m-NAD(P)-ME causes the enzyme to become less active and lose co-operativity. It also increased K-0.5,K-malate and decreased k(cat) values, indicating that the catalytic power of these mutant enzymes was significatly impaired following mutatin of Lys(57). Analytical ultracentrifugation analysis demonstrates that the K57A, K57S and K57C mutant enzymes dissociate predominatly 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 Lys(57) mutants, however, cannot be reconstituted into tetramers with the addition of fumarate. Modelling structures of the Lys(57) mutant enzymes show that the hydrogen bond network in the dimer interface where Lys(57) resides may be reduced compared with WT. Although the fumarate-induced activation effects are partially maintained in these Lys(57) 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 Lys(57) 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. |
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