Purification and Characterization of NADP^+-Linked Isocitrate Dehydrogenase from Paecilomyces varioti(Biological Chemistry)

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NADP^+-linked isocitrate dehydrogenase (EC 1.1.1.42) was purified to homogeneity from a crude extract of acetate-grown Paecilomyces varioti AHU 9417 with high malic acid productivity, by ammonium sulfate fractionation and column chromatography on DEAE-Sepharose CL-6B, Matrex Blue-A and Agarose-hexane-NADP^+. The molecular weight of the enzyme was 99,000 and the subunit molecular weight was 51,000. The optimum pH for the reaction was 8.4. The Km values for threo-Ds-isocitrate and NADP^+ were calculated to be 12 μM and 4.4 μM, respectively. Adenine nucleotides had little effect on enzyme activity. Of metabolic intermediates related to the TCA and glyoxylate cycles, α-ketoglutarate inhibited the activity for isocitrate competitively and oxalacetate, non-competitively. Addition of other organic acids such as succinate, fumarate, and pyruvate brought no or slight inhibition at the concentration of 1mM. However, when oxalacetate and glyoxylate were added simultaneously, the enzyme activity was strongly inhibited at the level of a few μM of each compound. From these observations and previous knowledge of isocitrate lyase, the regulation of the TCA cycle and the glyoxylate cycle is discussed.
社団法人日本農芸化学会の論文
1986-06-23