Abstract
A specific isocitrate lyase (ICL) activity of 0.17 U (mg protein)-1 was detected in cultures of the riboflavin-producing fungus Ashbya gossypii during growth on soybean oil. Enzyme activity was not detectable during growth on glucose [<0.005 U (mg protein)-1], indicating a regulation. The enzyme was purified 108-fold by means of ammonium sulphate fractionation, gel filtration and cation-exchange chromatography. SDS-PAGE of the purified protein showed a homogeneous band with an M r of 66000. The M r of 254000 determined by gel-filtration chromatography indicated a tetrameric structure of the native protein. The enzyme was found to have a pH optimum for the isocitrate cleavage of 7.0, and the K m for threo-DL-isocitrate was determined as 550 μ. Enzyme activity was Mg2+- dependent. In regulation studies ICL was weakly inhibited by central metabolites. A concentration of 10 mM phosphoenolpyruvate or 6-phosphogluconate revealed a residual activity of more than 40%. On the other hand, oxalate (K i: 4 μM) and itaconate (K i: 170 μM) showed a strong inhibition and may therefore be interesting as antimetabolites.
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