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.