Abstract
A rapid assay is described for homocitrate synthase (EC 4.1.3.21) of the lysine biosynthetic pathway of Saccharomyces cerevisiae. The alpha-ketoglutarate-dependent cleavage of acetyl-coA was measured spectrophotometrically as decrease in absorbance at 600 nm in the presence of 2,6-dichlorophenol-indophenol and enzyme from the wild type strain X2180. This activity was also present in citrate synthaseless glutamate auxotroph glu3, and the activity was inhibited by 5 mM L-lysine. Radioactive homocitric acid was obtained from a reaction mixture containing [1-14C]acetyl-coA. Homocitrate synthase activity was dependent upon time, both substrates, and enzyme. The activity exhibited a pH and temperature optimum of 7.5-8.0 and 32 degrees C, respectively, and was inhibited by metal-chelating and sulfhydryl-binding agents.
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