2-Methylcitrate Dehydratase, a New Enzyme Functioning at the Methylcitric Acid Cycle of Propionate Metabolism

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-D_s-2-methylisocitrate, citrate, cis- or trans-aconitate, threo-D_s-, threo-D_L- or erythro-L_s-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-D_s-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-D_s-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.