Abstract
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 12μm and 4.4μm, 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 1 mm. 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.
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