Characterization of NADP+-Isocitrate Dehydrogenase from Immature Pod

NADP'-isocitrate dehydrogenase (threo-DS-isocitrate: NADP' oxidoreductase [decarboxylating]; EC 1.1.1.42) (IDH) from pod walls of chickpea (Cicer arietinum L.) was purified 192-fold using ammonium sulfate fractionation, ion exchange chromatography on DEAE-Sephadex A-50, and gel filtration through Sephadex G-200. The purified enzyme, having a molecular weight of about 126,000, exhibited a broad pH optima from 8.0 to 8.6. It was quite stable at 4°C and had an absolute requirement for a divalent cation, either Mg2" or Mn2, for its activity. Typical hyperbolic kinetics was obtained with increasing concentrations of NADP', DL-isocitrate, Mn2+, and Mg2+. Their K,. values were 15, 110, 15, and 192 micromolar, respectively. The enzyme activity was inhibited by sulfhydryl reagents. Various amino acids, amides, organic acids, nucleotides, each at a concentration of 5 millimolar, had no effect on the activity of the enzyme. The activity was not influenced by adenylate energy charge but decreased linearly with increasing ratio of NADPH to NADP+. Initial velocity studies indicated kinetic mechanism to be sequential. NADPH inhibited the forward reaction competitively with respect to NADP+ at fixed saturating concentration of isocitrate, whereas 2-oxoglutarate inhibited the enzyme noncompetitively at saturating concentrations of both NADP+ and isocitrate, indicating the reaction mechanism to be random sequential. Results suggest that the activity of NADP+-IDH in situ is likely to be controlled by intracellular NADPH to NADP+ ratio as well as by the concentration of various substrates and