2-Methylcitrate Dehydratase,a New Enzyme Functioning at the Methylcitric Acid Cycle of Propionate Metabolism |
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Authors: | Takeshi Tabuchi Hiroyuki Aoki Hiroo Uchiyama Tadaatsu Nakahara |
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Institution: | Institute of Applied Biochemistry, The University of Tsukuba, Sakura-mura, Ibaraki 305, Japan |
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Abstract: | 2-Methylcitrate dehydratase (2-methylcitrate hydro-lyase), a new enzyme functioning at the methylcitric acid cycle of propionyl-CoA oxidation, was present in the cell-free extract of Yarrowia (Saccharomycopsis) lipolytica. The enzyme was separated from the usual aconitate hydratase (EC 4.2.1.3) of the yeast with DEAE-Sephadex A-50 column chromatography. The enzyme was able to catalyze a reversible reaction between 2-methylcitrate and 2-methyl-cis-aconitate, but showed no activity on threo-ds-2-methylisocitrate, citrate, cis- or trans-aconitate, threo-ds-, threo-DL- or erythro-ls-isocitrate, DL-homocitrate or other hydroxy-acids tested.In contrast, the other enzyme fraction separated as aconitate hydratase by chromatography showed no activity on synthetic 2-methylcitrate, but was able to catalyze strongly a reversible reaction between 2-methyl-cis-aconitate and threo-ds-2-methylisocitrate.From these findings, the previously proposed cycle sequence was revised at the following broken arrows: propionyl-CoA+oxaloacetate → (CoASH+) 2-methylcitrate ? 2-methyl-cis-aconitate ? threo-ds-2-methylisocitrate → pyruvate+succinate (→→oxaloacetate).2-Methylcitrate dehydratase showed maximum activity at pH 6.5 to 7.0 and at 25 to 40°C. The enzyme was stable at temperatures up to 40°C and at pH 6.5 to 7.5, but labile in Tris-HCl buffer. The synthesis of this enzyme was constitutive in this yeast, although it was slightly repressed by glucose. |
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