Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1972 Jun;110(3):1118–1126. doi: 10.1128/jb.110.3.1118-1126.1972

α-Isopropylmalate Synthase from Yeast: Purification, Kinetic Studies, and Effect of Ligands on Stability1

E H Ulm a, R Böhme a, G Kohlhaw a
PMCID: PMC247535  PMID: 5079061

Abstract

α-Isopropylmalate synthase, the first specific enzyme in leucine biosynthesis, was purified approximately 100-fold from extracts of Saccharomyces sp. (strain 60615), the most effective step being specific elution with the feedback inhibitor leucine from a hydroxyapatite column. In the early steps of purification, special care was taken to protect the synthase against proteolytic activities. The apparent molecular weight of the enzyme as determined from gel filtration on a calibrated column was 137,000 in the absence and 121,000 in the presence of leucine. Inhibition by leucine was specific and strongly pH-dependent, with the leucine concentration necessary for half-maximal inhibition increasing about 10-fold as the pH increased from 7.5 to 8.5. Within this pH range, catalytic activity remained almost unchanged. The apparent Km values for the two substrates were found to be 16 μm for α-ketoisovalerate and 9 μm for acetyl-coenzyme A. K+ was required for full activity, the apparent Ka value being 2 mm. Leucine inhibition was of the mixed type, resulting in decreased Vmax and increased apparent Km values forboth substrates. Whereas no cooperative effects were observed with either substrate, positive cooperativity was seen with leucine in the presence of saturating substrate concentrations. Leucine and, to a lesser extent, α-ketoisovalerate stabilized the purified enzyme against heat-inactivation. The presence of acetyl-coenzyme A, on the other hand, accelerated the inactivation. In subsequent experiments, coenzyme A was recognized as the actual inactivating ligand, being effective even at lower temperatures and in concentrations which were estimated to be in the range of the enzyme concentration.

Full text

PDF
1118

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BURNS R. O., UMBARGER H. E., GROSS S. R. THE BIOSYNTHESIS OF LEUCINE. III. THE CONVERSION OF ALPHA-HYDROXY-BETA-CARBOXYISOCAPROATE TO ALPHA-KETOISOCAPROATE. Biochemistry. 1963 Sep-Oct;2:1053–1058. doi: 10.1021/bi00905a024. [DOI] [PubMed] [Google Scholar]
  2. Bollon A. P., Magee P. T. Involvement of threonine deaminase in multivalent repression of the isoleucine-valine pathway in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1971 Sep;68(9):2169–2172. doi: 10.1073/pnas.68.9.2169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bussey H., Umbarger H. E. Biosynthesis of branched-chain amino acids in yeast: regulation of synthesis of the enzymes of isoleucine and valine biosynthesis. J Bacteriol. 1969 May;98(2):623–628. doi: 10.1128/jb.98.2.623-628.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clark J. F., Jakoby W. B. Yeast aldehyde dehydrogenase. 3. Preparation of three homogeneous species. J Biol Chem. 1970 Nov 25;245(22):6065–6071. [PubMed] [Google Scholar]
  5. HALVORSON H. Studies on protein and nucleic acid turnover in growing cultures of yeast. Biochim Biophys Acta. 1958 Feb;27(2):267–276. doi: 10.1016/0006-3002(58)90333-0. [DOI] [PubMed] [Google Scholar]
  6. Kohlhaw G., Boatman G. Cross-linking of Salmonella isopropylmalate synthesis with dimethyl suberimidate: evidence for antagonistic effects of leucine and acetyl-CoA on the guaternary structure. Biochem Biophys Res Commun. 1971 May 21;43(4):741–746. doi: 10.1016/0006-291x(71)90678-4. [DOI] [PubMed] [Google Scholar]
  7. Kohlhaw G., Leary T. R., Umbarger H. E. Alpha-isopropylmalate synthase from Salmonella typhimurium. Purification and properties. J Biol Chem. 1969 Apr 25;244(8):2218–2225. [PubMed] [Google Scholar]
  8. Kohlhaw G. Number and reactivity of the sulfhydryl groups of alpha-isopropylmalate synthase of Salmonella typhimurium. Biochim Biophys Acta. 1970 Jul 15;212(1):58–64. doi: 10.1016/0005-2744(70)90178-6. [DOI] [PubMed] [Google Scholar]
  9. Leary T. R., Kohlhaw G. Dissociation of alpha-isopropylmalate synthase from Salmonella typhimurium by its feedback inhibitor leucine. Biochem Biophys Res Commun. 1970 May 11;39(3):494–501. doi: 10.1016/0006-291x(70)90605-4. [DOI] [PubMed] [Google Scholar]
  10. Ramel A. H., Rustum Y. M., Jones J. G., Barnard E. A. Yeast Hexokinase. IV. Multiple forms of hexokinase in the yeast cell. Biochemistry. 1971 Sep 14;10(19):3499–3508. doi: 10.1021/bi00795a003. [DOI] [PubMed] [Google Scholar]
  11. Sasaki R., Sugimoto R., Chiba H. Yeast phosphoglyceric acid mutase-modifying enzyme. Arch Biochem Biophys. 1966 Jul;115(1):53–61. doi: 10.1016/s0003-9861(66)81037-8. [DOI] [PubMed] [Google Scholar]
  12. Satyanarayana T., Umbarger H. E., Lindegren G. Biosynthesis of branched-chain amino acids in yeast: correlation of biochemical blocks and genetic lesions in leucine auxotrophs. J Bacteriol. 1968 Dec;96(6):2012–2017. doi: 10.1128/jb.96.6.2012-2017.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Satyanarayana T., Umbarger H. E., Lindegren G. Biosynthesis of branched-chain amino acids in yeast: regulation of leucine biosynthesis in prototrophic and leucine auxotrophic strains. J Bacteriol. 1968 Dec;96(6):2018–2024. doi: 10.1128/jb.96.6.2018-2024.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Schulze I. T., Colowick S. P. The modification of yeast hexokinases by proteases and its relationship to the dissociation of hexokinase into subunits. J Biol Chem. 1969 May 10;244(9):2306–2316. [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES