Biosynthesis of lysine in Saccharomyces cervisiae: properties and spectrophotometric determination of homocitrate synthase activity.
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.
D015086:2,6-Dichloroindophenol / Q000378:metabolism; D000105:Acetyl Coenzyme A / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002950:Citrate (si)-Synthase / Q000378:metabolism; D002951:Citrates / Q000096:biosynthesis; D005971:Glutamates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids / Q000378:metabolism; D008239:Lysine / Q000096:biosynthesis*; D009154:Mutation; D010071:Oxaloacetates / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013696:Temperature
D002614:Chelating Agents; D002951:Citrates; D005971:Glutamates; D007656:Ketoglutaric Acids; D010071:Oxaloacetates; D013439:Sulfhydryl Reagents; D000105:Acetyl Coenzyme A; D015086:2,6-Dichloroindophenol; D002950:Citrate (si)-Synthase; D007652:Oxo-Acid-Lyases; D008239:Lysine