Biosynthesis of lysine in Saccharomyces cerevisiae: properties and spectrophotometric determination of homocitrate synthase activity

Abstract

A rapid assay is described for homocitrate synthase (EC 4.1.3.21) of the lysine biosynthetic pathway of Saccharomyces cerevisiae. The α-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 a citrate synthaseless glutamate auxotroph glu3, and the activity was inhibited by 5 mML-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 °C, respectively, and was inhibited by metal-chelating and sulfhydryl-binding agents.