Degradative acetolactate synthase of Bacillus subtilis: purification and properties

J Bacteriol. 1975 Mar;121(3):917-22. doi: 10.1128/JB.121.3.917-922.1975.

Abstract

A degradative acetolactate synthase (acetolactate pyruvate-lyase [carboxylating], EC 4.1.3.18) from Bacillus subtilis has been partially purified and characterized. The synthesis of the enzyme was induced by growth of cells in minimal medium plus isobutyrate or acetate. The enzyme was partially purified by ammonium sulfate fractionation, gel filtration, and hydroxyapatite chromatography. The pH optimum of the purified enzyme was 7.0 in phosphate buffer. When assayed in phosphate buffer (pH 7.0), activity was stimulated by acetate and inhibited by sulfate. When assayed in acetate buffer (pH 5.8), activity was inhibited both by sulfate and phosphate. Michaelis-Menten kinetics was observed when the enzyme was assayed in phosphate buffer (pH 6.0 or 7.0), and inhibition by sulfate was competitive and activation by acetate was noncompetitive. When assayed in acetate buffer (pH 5.8), nonlinear Lineweaver-Burk plots were obtained; inhibition by phosphate appeared to be competitive and that by sulfate was of the mixed type. The approximate molecular weight of the purified enzyme was 250,000 as determined by gel filtration.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetates / pharmacology
  • Ammonium Sulfate
  • Bacillus subtilis / enzymology*
  • Butanones
  • Butyrates / pharmacology
  • Cell-Free System
  • Chemical Precipitation
  • Chromatography
  • Chromatography, Gel
  • Enzyme Induction
  • Fatty Acids / pharmacology
  • Hydrogen-Ion Concentration
  • Hydroxyapatites
  • Kinetics
  • Molecular Weight
  • Oxo-Acid-Lyases* / isolation & purification
  • Oxo-Acid-Lyases* / metabolism
  • Phosphates / pharmacology
  • Pyruvates / pharmacology
  • Sulfates / pharmacology

Substances

  • Acetates
  • Butanones
  • Butyrates
  • Fatty Acids
  • Hydroxyapatites
  • Phosphates
  • Pyruvates
  • Sulfates
  • Oxo-Acid-Lyases
  • Ammonium Sulfate