Biosynthesis of branched-chain amino acids in Schizosaccharomyces pombe: properties of acetohydroxy acid synthetase.
The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (K(i) = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system.
D000085:Acetates / Q000378:metabolism; D000596:Amino Acids / Q000096:biosynthesis*; D000645:Ammonium Sulfate; D001203:Ascomycota / Q000201:enzymology* / Q000378:metabolism; D000440:Butanols / Q000378:metabolism; D002074:Butanones / Q000378:metabolism; D002262:Carboxy-Lyases / Q000378:metabolism; D002850:Chromatography, Gel; D005246:Feedback; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine / Q000096:biosynthesis; D007700:Kinetics; D007773:Lactates / Q000378:metabolism; D008628:Mercury / Q000494:pharmacology; D007652:Oxo-Acid-Lyases / Q000302:isolation & purification / Q000378:metabolism*; D011479:Protamines; D011773:Pyruvates; D013835:Thiamine Pyrophosphate / Q000378:metabolism; D014633:Valine / Q000096:biosynthesis / Q000494:pharmacology
D000085:Acetates; D000596:Amino Acids; D000440:Butanols; D002074:Butanones; D007773:Lactates; D011479:Protamines; D011773:Pyruvates; D007532:Isoleucine; D005182:Flavin-Adenine Dinucleotide; D002262:Carboxy-Lyases; D007652:Oxo-Acid-Lyases; D008628:Mercury; D014633:Valine; D005947:Glucose; D005990:Glycerol; D013835:Thiamine Pyrophosphate; D000645:Ammonium Sulfate