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

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 k_cat. 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.