Purification and characterization of the NADP-linked malate dehydrogenase (decarboxylating) from Mangifera indica

The NADP-linked malate dehydrogenase (decarboxylating) (l-malate: NADP⁺ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40), known as ‘malic’ enzyme, has been isolated and purified to apparent homogeneity from the mango fruit, Mangifera indica, by means of extraction, gel chromatography with Sephadex G-200 and anion-exchange chromatography with DEAE-Sephacel. A 16-fold purification with a 49% yield was obtained. The enzyme was physically characterized and its homogeneity determined by polyacrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and analytical chromatography. An average molecular weight of 258 000 was obtained for the enzyme as well as a Stokes radius of 54.0 · 10⁻⁸ cm and a diffusion coefficient of 3.96 · 10⁻⁷ cm² · s⁻¹. A frictional ratio, $\text{f/f}{}_0$ of 1.28 indicated the globular character of the enzyme. The native enzyme consists of four subunits with an average molecular weight of 64 900. The enzyme thus appears to be an oligomeric protein with an apparently homogeneous quarternary structure. The pH optimum for the decarboxylation reaction varied between 6.8 and 7.5, depending on the type of buffer and increased with increasing malate concentration. No cooperativity could be detected between the malate binding sites in the presence of Mn²⁺ as cofactor. It would seen as if Mn²⁺ elicits a positive allosteric effect on the enzyme. Increasing Mn²⁺ concentrations lead to an increase in the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for l(-)malate from 666 μM at 1.0 mM Mn²⁺ to 1.08 mM at 5.0 mM Mn²⁺. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value determined at pH 7.1 for Mn²⁺ was 14.3 μM. An approximate $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of 16 μM was found for NADP⁺ with some indication of cooperativity between the nucleotide binding sites. The enzyme activity was much more sensitive to regulation when Mg²⁺ served as cation. The allosteric activator, succinate, removed the sigmoidicity observed in the velocity-malate saturation curve and lowered to Hill coefficient from 1.5 to 1.0 Differences were found in the response of the enzyme to certain metabolites depending on whether Mg²⁺ or Mn²⁺ served as cofactor. It appears as if Mg²⁺ and Mn²⁺ stabilize two structurally distinct forms of the enzyme which vary in catalytic and regulatory properties.