Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1989 Feb;55(2):323–329. doi: 10.1128/aem.55.2.323-329.1989

Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids.

D P Wiesenborn 1, F B Rudolph 1, E T Papoutsakis 1
PMCID: PMC184109  PMID: 2719476

Abstract

Coenzyme A (CoA) transferase from Clostridium acetobutylicum ATCC 824 was purified 81-fold to homogeneity. This enzyme was stable in the presence of 0.5 M ammonium sulfate and 20% (vol/vol) glycerol, whereas activity was rapidly lost in the absence of these stabilizers. The kinetic binding mechanism was Ping Pong Bi Bi, and the Km values at pH 7.5 and 30 degrees C for acetate, propionate, and butyrate were, respectively, 1,200, 1,000, and 660 mM, while the Km value for acetoacetyl-CoA ranged from about 7 to 56 microM, depending on the acid substrate. The Km values for butyrate and acetate were high relative to the intracellular concentrations of these species; consequently, in vivo enzyme activity is expected to be sensitive to changes in those concentrations. In addition to the carboxylic acids listed above, this CoA transferase was able to convert valerate, isobutyrate, and crotonate; however, the conversion of formate, n-caproate, and isovalerate was not detected. The acetate and butyrate conversion reactions in vitro were inhibited by physiological levels of acetone and butanol, and this may be another factor in the in vivo regulation of enzyme activity. The optimum pH of acetate conversion was broad, with at least 80% of maximal activity from pH 5.9 to greater than 7.8. The purified enzyme was a heterotetramer with subunit molecular weights of about 23,000 and 25,000.

Full text

PDF
323

Images in this article

326Image
on p.326

Selected References

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

  1. Andrews P. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem J. 1964 May;91(2):222–233. doi: 10.1042/bj0910222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barker H. A., Jeng I. M., Neff N., Robertson J. M., Tam F. K., Hosaka S. Butyryl-CoA:acetoacetate CoA-transferase from a lysine-fermenting Clostridium. J Biol Chem. 1978 Feb 25;253(4):1219–1225. [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Gilbert H. F., Lennox B. J., Mossman C. D., Carle W. C. The relation of acyl transfer to the overall reaction of thiolase I from porcine heart. J Biol Chem. 1981 Jul 25;256(14):7371–7377. [PubMed] [Google Scholar]
  5. Hartmanis M. G. Butyrate kinase from Clostridium acetobutylicum. J Biol Chem. 1987 Jan 15;262(2):617–621. [PubMed] [Google Scholar]
  6. Huang L., Forsberg C. W., Gibbins L. N. Influence of External pH and Fermentation Products on Clostridium acetobutylicum Intracellular pH and Cellular Distribution of Fermentation Products. Appl Environ Microbiol. 1986 Jun;51(6):1230–1234. doi: 10.1128/aem.51.6.1230-1234.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Huang L., Gibbins L. N., Forsberg C. W. Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions. Appl Environ Microbiol. 1985 Oct;50(4):1043–1047. doi: 10.1128/aem.50.4.1043-1047.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jones D. T., Woods D. R. Acetone-butanol fermentation revisited. Microbiol Rev. 1986 Dec;50(4):484–524. doi: 10.1128/mr.50.4.484-524.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Rudolph F. B., Fromm H. J. Use of isotope competition and alternative substrates for studying the kinetic mechanism of enzyme action. I. Experiments with hexokinase and alcohol dehydroeenase. Biochemistry. 1970 Nov 24;9(24):4660–4665. doi: 10.1021/bi00826a007. [DOI] [PubMed] [Google Scholar]
  11. STADTMAN E. R. The coenzyme A transphorase system in Clostridium kluyveri. J Biol Chem. 1953 Jul;203(1):501–512. [PubMed] [Google Scholar]
  12. STERN J. R. Optical properties of aceto-acetyl-S-coenzyme A and its metal chelates. J Biol Chem. 1956 Jul;221(1):33–44. [PubMed] [Google Scholar]
  13. Sramek S. J., Frerman F. E. Escherichia coli coenzyme A-transferase: kinetics, catalytic pathway and structure. Arch Biochem Biophys. 1975 Nov;171(1):27–35. doi: 10.1016/0003-9861(75)90003-x. [DOI] [PubMed] [Google Scholar]
  14. Sramek S. J., Frerman F. E. Purification and properties of Escherichia coli coenzyme A-transferase. Arch Biochem Biophys. 1975 Nov;171(1):14–26. doi: 10.1016/0003-9861(75)90002-8. [DOI] [PubMed] [Google Scholar]
  15. Tung K. K., Wood W. A. Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii. J Bacteriol. 1975 Dec;124(3):1462–1474. doi: 10.1128/jb.124.3.1462-1474.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. VALENTINE R. C., WOLFE R. S. Purification and role of phosphotransbutyrylase. J Biol Chem. 1960 Jul;235:1948–1952. [PubMed] [Google Scholar]
  17. Wiesenborn D. P., Rudolph F. B., Papoutsakis E. T. Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis. Appl Environ Microbiol. 1989 Feb;55(2):317–322. doi: 10.1128/aem.55.2.317-322.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Yan Run-Tao, Zhu Chang-Xi, Golemboski Christine, Chen Jiann-Shin. Expression of Solvent-Forming Enzymes and Onset of Solvent Production in Batch Cultures of Clostridium beijerinckii ("Clostridium butylicum"). Appl Environ Microbiol. 1988 Mar;54(3):642–648. doi: 10.1128/aem.54.3.642-648.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES