Abstract

α-Acetolactate synthase (α-ALS) of Enterobacter cloacae ATCC 27613 was purified to homogeneity by ammonium sulphate precipitation, Sephadex G-200 gel filtration and hydroxyapatite affinity chromatography. The molecular weight of the enzyme was found to be 60 kDa by SDS–polyacrylamide gel electrophoresis and ∼200 kDa by gel filtration through Sephadex G-200, showing that the enzyme is a homotrimer. The Km and Vmax of the enzyme were 20 mM and 200 μmol min−1 mg (protein)−1 respectively. The enzyme was optimally active at pH 6.0–8.0, 37 °C and showed concentration-dependent sensitivity to cofactors viz. FAD, NADP and NADPH and branched chain amino acids: leucine, isoleucine and valine. Substances like sodium formate, sodium acetate and sodium propionate, sugars and the selected intermediates of glycolytic pathway inhibited the enzyme. Glycerol, BSA and pyruvate-TPP stabilized the α-ALS. The enzyme showed the properties of both a catabolic as well as an anabolic α-ALS.

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