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

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Abstract
A rapid assay was described for homocitrate synthase (EC 4.1.3.21) of the lysine biosynthetic pathway of S. cerevisiae. The .alpha.-ketoglutarate-dependent cleavage of acetyl-coenzyme A 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 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.degree. C, respectively, and was inhibited by metal-chelating and sulfhydryl-binding agents.