Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1989 Oct;171(10):5430–5435. doi: 10.1128/jb.171.10.5430-5435.1989

Isolation and characterization of acetyl-coenzyme A synthetase from Methanothrix soehngenii.

M S Jetten 1, A J Stams 1, A J Zehnder 1
PMCID: PMC210380  PMID: 2571608

Abstract

In Methanothrix soehngenii, acetate is activated to acetyl-coenzyme A (acetyl-CoA) by an acetyl-CoA synthetase. Cell extracts contained high activities of adenylate kinase and pyrophosphatase, but no activities of a pyrophosphate:AMP and pyrophosphate:ADP phosphotransferase, indicating that the activation of 1 acetate in Methanothrix requires 2 ATP. Acetyl-CoA synthetase was purified 22-fold in four steps to apparent homogeneity. The native molecular mass of the enzyme from M. soehngenii estimated by gel filtration was 148 kilodaltons (kDa). The enzyme was composed of two subunits with a molecular mass of 73 kDa in an alpha 2 oligomeric structure. The acetyl-CoA synthetase constituted up to 4% of the soluble cell protein. At the optimum pH of 8.5, the Vmax was 55 mumol of acetyl-CoA formed per min per mg of protein. Analysis of enzyme kinetic properties revealed a Km of 0.86 mM for acetate and 48 microM for coenzyme A. With varying amounts of ATP, weak sigmoidal kinetic was observed. The Hill plot gave a slope of 1.58 +/- 0.12, suggesting two interacting substrate sites for the ATP. The kinetic properties of the acetyl-CoA synthetase can explain the high affinity for acetate of Methanothrix soehngenii.

Full text

PDF
5435

Images in this article

5433Image
on p.5433

Selected References

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

  1. Aceti D. J., Ferry J. G. Purification and characterization of acetate kinase from acetate-grown Methanosarcina thermophila. Evidence for regulation of synthesis. J Biol Chem. 1988 Oct 25;263(30):15444–15448. [PubMed] [Google Scholar]
  2. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Brown T. D., Jones-Mortimer M. C., Kornberg H. L. The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J Gen Microbiol. 1977 Oct;102(2):327–336. doi: 10.1099/00221287-102-2-327. [DOI] [PubMed] [Google Scholar]
  4. Grahame D. A., Stadtman T. C. Carbon monoxide dehydrogenase from Methanosarcina barkeri. Disaggregation, purification, and physicochemical properties of the enzyme. J Biol Chem. 1987 Mar 15;262(8):3706–3712. [PubMed] [Google Scholar]
  5. Grahame D. A., Stadtman T. C. In vitro methane and methyl coenzyme M formation from acetate: evidence that acetyl-CoA is the required intermediate activated form of acetate. Biochem Biophys Res Commun. 1987 Aug 31;147(1):254–258. doi: 10.1016/s0006-291x(87)80114-6. [DOI] [PubMed] [Google Scholar]
  6. Hutten T. J., Bongaerts H. C., van der Drift C., Vogels G. D. Acetate, methanol and carbon dioxide as substrates for growth of Methanosarcina barkeri. Antonie Van Leeuwenhoek. 1980;46(6):601–610. doi: 10.1007/BF00394016. [DOI] [PubMed] [Google Scholar]
  7. Jetten M. S., Stams A. J., Zehnder A. J. Purification and characterization of an oxygen-stable carbon monoxide dehydrogenase of Methanothrix soehngenii. Eur J Biochem. 1989 May 1;181(2):437–441. doi: 10.1111/j.1432-1033.1989.tb14744.x. [DOI] [PubMed] [Google Scholar]
  8. Josse J. Constitutive inorganic pyrophosphatase of Escherichia coli. 1. Purification and catalytic properties. J Biol Chem. 1966 May 10;241(9):1938–1947. [PubMed] [Google Scholar]
  9. Keltjens J. T., van Erp R., Mooijaart R. J., van der Drift C., Vogels G. D. Inorganic pyrophosphate synthesis during methanogenesis from methylcoenzyme M by cell-free extracts of Methanobacterium thermoautotrophicum (strain delta H). Eur J Biochem. 1988 Mar 1;172(2):471–476. doi: 10.1111/j.1432-1033.1988.tb13912.x. [DOI] [PubMed] [Google Scholar]
  10. Kenealy W. R., Zeikus J. G. One-carbon metabolism in methanogens: evidence for synthesis of a two-carbon cellular intermediate and unification of catabolism and anabolism in Methanosarcina barkeri. J Bacteriol. 1982 Aug;151(2):932–941. doi: 10.1128/jb.151.2.932-941.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Krzycki J. A., Lehman L. J., Zeikus J. G. Acetate catabolism by Methanosarcina barkeri: evidence for involvement of carbon monoxide dehydrogenase, methyl coenzyme M, and methylreductase. J Bacteriol. 1985 Sep;163(3):1000–1006. doi: 10.1128/jb.163.3.1000-1006.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Krzycki J. A., Wolkin R. H., Zeikus J. G. Comparison of unitrophic and mixotrophic substrate metabolism by acetate-adapted strain of Methanosarcina barkeri. J Bacteriol. 1982 Jan;149(1):247–254. doi: 10.1128/jb.149.1.247-254.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Krzycki J. A., Zeikus J. G. Characterization and purification of carbon monoxide dehydrogenase from Methanosarcina barkeri. J Bacteriol. 1984 Apr;158(1):231–237. doi: 10.1128/jb.158.1.231-237.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Lovley D. R., White R. H., Ferry J. G. Identification of methyl coenzyme M as an intermediate in methanogenesis from acetate in Methanosarcina spp. J Bacteriol. 1984 Nov;160(2):521–525. doi: 10.1128/jb.160.2.521-525.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Matin A., Veldkamp H. Physiological basis of the selective advantage of a Spirillum sp. in a carbon-limited environment. J Gen Microbiol. 1978 Apr;105(2):187–197. doi: 10.1099/00221287-105-2-187. [DOI] [PubMed] [Google Scholar]
  17. Nelson M. J., Ferry J. G. Carbon monoxide-dependent methyl coenzyme M methylreductase in acetotrophic Methosarcina spp. J Bacteriol. 1984 Nov;160(2):526–532. doi: 10.1128/jb.160.2.526-532.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Oberlies G., Fuchs G., Thauer R. K. Acetate thiokinase and the assimilation of acetate in methanobacterium thermoautotrophicum. Arch Microbiol. 1980 Dec;128(2):248–252. doi: 10.1007/BF00406167. [DOI] [PubMed] [Google Scholar]
  19. ROSE I. A., GRUNBERG-MANAGO M., KOREY S. R., OCHOA S. Enzymatic phosphorylation of acetate. J Biol Chem. 1954 Dec;211(2):737–756. [PubMed] [Google Scholar]
  20. Schaupp A., Ljungdahl L. G. Purification and properties of acetate kinase from Clostridium thermoaceticum. Arch Microbiol. 1974;100(2):121–129. doi: 10.1007/BF00446312. [DOI] [PubMed] [Google Scholar]
  21. Skrabanja A. T., van der Hijden H. T., De Pont J. J. Transport ratios of reconstituted (H+ + K+)-ATPase. Biochim Biophys Acta. 1987 Oct 16;903(3):434–440. doi: 10.1016/0005-2736(87)90050-2. [DOI] [PubMed] [Google Scholar]
  22. Smith M. R., Mah R. A. Acetate as sole carbon and energy source for growth of methanosarcina strain 227. Appl Environ Microbiol. 1980 May;39(5):993–999. doi: 10.1128/aem.39.5.993-999.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Smith P. H., Mah R. A. Kinetics of acetate metabolism during sludge digestion. Appl Microbiol. 1966 May;14(3):368–371. doi: 10.1128/am.14.3.368-371.1966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Terlesky K. C., Barber M. J., Aceti D. J., Ferry J. G. EPR properties of the Ni-Fe-C center in an enzyme complex with carbon monoxide dehydrogenase activity from acetate-grown Methanosarcina thermophila. Evidence that acetyl-CoA is a physiological substrate. J Biol Chem. 1987 Nov 15;262(32):15392–15395. [PubMed] [Google Scholar]
  25. Terlesky K. C., Nelson M. J., Ferry J. G. Isolation of an enzyme complex with carbon monoxide dehydrogenase activity containing corrinoid and nickel from acetate-grown Methanosarcina thermophila. J Bacteriol. 1986 Dec;168(3):1053–1058. doi: 10.1128/jb.168.3.1053-1058.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Thauer R. K. Citric-acid cycle, 50 years on. Modifications and an alternative pathway in anaerobic bacteria. Eur J Biochem. 1988 Oct 1;176(3):497–508. doi: 10.1111/j.1432-1033.1988.tb14307.x. [DOI] [PubMed] [Google Scholar]
  27. Zehnder A. J., Huser B. A., Brock T. D., Wuhrmann K. Characterization of an acetate-decarboxylating, non-hydrogen-oxidizing methane bacterium. Arch Microbiol. 1980 Jan;124(1):1–11. doi: 10.1007/BF00407022. [DOI] [PubMed] [Google Scholar]

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

RESOURCES