The cytosolic malic enzyme from pigeon liver is very sensitive to the metal-catalysed oxidation systems. Our previous studies using the Cu2+-ascorbate as the oxidation system showed that the enzyme was oxidized and cleaved at several positions, including Asp141. The recently resolved crystal structure of pigeon liver malic enzyme revealed that Asp141 was near to the metal-binding site, but was not a direct metal ligand. However, Asp141 is located next to Phe236, which directly follows the metal ligands Glu234 and Asp235. Mutation at Asp141 caused a drastic effect on the metal-binding affinity of the enzyme. Since Asp141 and Phe236 are highly conserved in most species of malic enzyme, we used a double-mutant cycle to study the possible interactions between these two residues. Four single mutants [D141A (Asp141-->Ala), D141N, F236A and F236L] and four double mutants (D141A/F236A, D141N/F236A, D141A/F236L and D141N/F236L), plus the wild-type enzyme were successfully cloned, expressed and purified to homogeneity. The secondary, tertiary and quaternary structures of these mutants, as assessed by CD, fluorescence and analytical ultracentrifuge techniques, were similar to that of the wild-type enzyme. Initial velocity experiments were performed to derive the various kinetic parameters, which were used to analyse further the free energy change and the coupling energy (DeltaDeltaG(int)) between any two residues. The dissociation constants for Mn2+ ( K (d,Mn)) of the D141A and F236A mutants were increased by approx. 6- and 65-fold respectively, compared with that of the wild-type enzyme. However, the K (d,Mn) for the double mutant D141A/F236A was only increased by 150-fold. A coupling energy of -2.12 kcal/mol was obtained for Asp141 and Phe236. We suggest that Asp141 is involved in the second sphere of the metal-binding network of the enzyme.