Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1988 Mar 15;250(3):789–796. doi: 10.1042/bj2500789

Purification and characterization of [acyl-carrier-protein] acetyltransferase from Escherichia coli.

P N Lowe 1, S Rhodes 1
PMCID: PMC1148925  PMID: 3291856

Abstract

A multi-step procedure has been developed for the purification of [acyl-carrier-protein] acetyltransferase from Escherichia coli, which allows the production of small amounts of homogeneous enzyme. The subunit Mr was estimated to be 29,000 and the native Mr was estimated to be 61,000, suggesting a homodimeric structure. The catalytic properties of the enzyme are consistent with a Bi Bi Ping Pong mechanism and the existence of an acetyl-enzyme intermediate in the catalytic cycle. The enzyme was inhibited by N-ethylmaleimide and more slowly by iodoacetamide in reactions protected by the substrate, acetyl-CoA. However, the enzyme was apparently only weakly inhibited by the thiol-specific reagent methyl methanethiosulphonate. The nature of the acetyl-enzyme intermediate is discussed in relationship to that found in other similar enzymes from E. coli, yeast and vertebrates.

Full text

PDF
789

Images in this article

Selected References

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

  1. ALBERTS A. W., MAJERUS P. W., TALAMO B., VAGELOS P. R. ACYL-CARRIER PROTEIN. II. INTERMEDIARY REACTIONS OF FATTY ACID SYNTHESIS. Biochemistry. 1964 Oct;3:1563–1571. doi: 10.1021/bi00898a030. [DOI] [PubMed] [Google Scholar]
  2. Anderson C. W., Baum P. R., Gesteland R. F. Processing of adenovirus 2-induced proteins. J Virol. 1973 Aug;12(2):241–252. doi: 10.1128/jvi.12.2.241-252.1973. [DOI] [PMC free article] [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. Caughey I., Kekwick R. G. The characteristics of some components of the fatty acid synthetase system in the plastids from the mesocarp of avocado (Persea americana) fruit. Eur J Biochem. 1982 Apr;123(3):553–561. doi: 10.1111/j.1432-1033.1982.tb06568.x. [DOI] [PubMed] [Google Scholar]
  5. Fulco A. J. Fatty acid metabolism in bacteria. Prog Lipid Res. 1983;22(2):133–160. doi: 10.1016/0163-7827(83)90005-x. [DOI] [PubMed] [Google Scholar]
  6. Hayashi T., Yamamoto O., Sasaki H., Okazaki H., Kawaguchi A. Inhibition of fatty acid synthesis by the antibiotic thiolactomycin. J Antibiot (Tokyo) 1984 Nov;37(11):1456–1461. doi: 10.7164/antibiotics.37.1456. [DOI] [PubMed] [Google Scholar]
  7. Joshi V. C., Wakil S. J. Studies on the mechanism of fatty acid synthesis. XXVI. Purification and properties of malonyl-coenzyme A--acyl carrier protein transacylase of Escherichia coli. Arch Biochem Biophys. 1971 Apr;143(2):493–505. doi: 10.1016/0003-9861(71)90234-7. [DOI] [PubMed] [Google Scholar]
  8. Lowe P. N., Rowe A. F. Probing the active sites of aspartate: 2-oxoglutarate aminotransferases from Trichomonas vaginalis and pig heart cytoplasm using substrate analogues. Comp Biochem Physiol B. 1987;88(1):223–227. doi: 10.1016/0305-0491(87)90104-0. [DOI] [PubMed] [Google Scholar]
  9. Lynen F. The role of biotin-dependent carboxylations in biosynthetic reactions. Biochem J. 1967 Feb;102(2):381–400. doi: 10.1042/bj1020381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Morino Y., Tanase S., Watanabe T., Kagamiyama H., Wada H. Large-scale preparation of cytosolic and mitochondrial asparatate aminotransferases from pig heart. J Biochem. 1977 Sep;82(3):847–852. doi: 10.1093/oxfordjournals.jbchem.a131760. [DOI] [PubMed] [Google Scholar]
  11. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  12. Nishida I., Kawaguchi A., Yamada M. Effect of thiolactomycin on the individual enzymes of the fatty acid synthase system in Escherichia coli. J Biochem. 1986 May;99(5):1447–1454. doi: 10.1093/oxfordjournals.jbchem.a135614. [DOI] [PubMed] [Google Scholar]
  13. Oishi H., Noto T., Sasaki H., Suzuki K., Hayashi T., Okazaki H., Ando K., Sawada M. Thiolactomycin, a new antibiotic. I. Taxonomy of the producing organism, fermentation and biological properties. J Antibiot (Tokyo) 1982 Apr;35(4):391–395. doi: 10.7164/antibiotics.35.391. [DOI] [PubMed] [Google Scholar]
  14. Penefsky H. S. Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase. J Biol Chem. 1977 May 10;252(9):2891–2899. [PubMed] [Google Scholar]
  15. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  16. Roberts D. D., Lewis S. D., Ballou D. P., Olson S. T., Shafer J. A. Reactivity of small thiolate anions and cysteine-25 in papain toward methyl methanethiosulfonate. Biochemistry. 1986 Sep 23;25(19):5595–5601. doi: 10.1021/bi00367a038. [DOI] [PubMed] [Google Scholar]
  17. Rock C. O., Cronan J. E., Jr Acyl carrier protein from Escherichia coli. Methods Enzymol. 1981;71(Pt 100):341–351. doi: 10.1016/0076-6879(81)71043-7. [DOI] [PubMed] [Google Scholar]
  18. Shimakata T., Stumpf P. K. Purification of plant acetyl-CoA:acyl carrier protein transacylase. Methods Enzymol. 1986;122:53–59. doi: 10.1016/0076-6879(86)22148-5. [DOI] [PubMed] [Google Scholar]
  19. Shimakata T., Stumpf P. K. The purification and function of acetyl coenzyme A:acyl carrier protein transacylase. J Biol Chem. 1983 Mar 25;258(6):3592–3598. [PubMed] [Google Scholar]
  20. Volpe J. J., Vagelos P. R. Mechanisms and regulation of biosynthesis of saturated fatty acids. Physiol Rev. 1976 Apr;56(2):339–417. doi: 10.1152/physrev.1976.56.2.339. [DOI] [PubMed] [Google Scholar]
  21. Wakil S. J., Stoops J. K., Joshi V. C. Fatty acid synthesis and its regulation. Annu Rev Biochem. 1983;52:537–579. doi: 10.1146/annurev.bi.52.070183.002541. [DOI] [PubMed] [Google Scholar]
  22. Williamson I. P., Wakil S. J. Studies on the mechanism of fatty acid synthesis. XVII. Preparation and general properties of acetyl coenzyme A and malonyl coenzyme A-acyl carrier protein transacylases. J Biol Chem. 1966 May 25;241(10):2326–2332. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES