Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1980 Aug;77(8):4544–4548. doi: 10.1073/pnas.77.8.4544

Yeast fatty acid synthetase: Structure—function relationship and nature of the β-ketoacyl synthetase site

James K Stoops 1, Salih J Wakil 1
PMCID: PMC349880  PMID: 7001460

Abstract

Yeast fatty acid synthetase consists of two multifunctional proteins, α and β, which are arranged in a complex of α6β6. Electron microscopic studies of this complex led to a model for the synthetase as an ovate structure consisting of an equatorial plate-like structure to which six arches are equally distributed on either side. The bifunctional reagent 1,3-dibromo-2-propanone inhibits the synthetase by reacting rapidly (t½ ≈7 sec) with two juxtapositioned active sulfhydryl groups. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the dibromopropanone-inhibited synthetase shows that the β subunit is intact and the α subunit nearly absent with a concomitant appearance of oligomers with an estimated molecular weight of 0.4-1.2 × 106. These results indicate that the α subunits are crosslinked by this bifunctional reagent. Because the active centers of dibromopropanone are 5 Å apart, it is concluded that the α subunits are closely packed so that the reacting thiols of the adjacent α subunits are within 5 Å of each other. Furthermore, because the plate-like structures in our model are the only components that are arranged closely enough to satisfy this requirement, it is proposed that the α subunits are the “plates” and the β subunits therefore are the “arches.” Assay of the partial reactions shows that dibromopropanone inhibits the β-ketoacyl synthetase reaction but none of the six other partial reactions, indicating that the site of action of the bifunctional reagent is the condensing reaction. This conclusion was supported by the finding that pretreatment of the synthetase with acetyl-CoA or iodoacetamide prevented dibromopropanone from interacting at this site and obviated the formation of the crosslinked oligomer. These observations and other lead us to propose that a site of action of the dibromopropanone is the active cysteine-SH of the β-ketoacyl synthetase of one α subunit and the pantetheine-SH of the acyl carrier protein moiety of an adjacent α subunit. Thus, the enzymically active center of the β-ketoacyl synthetase consists of an acyl group attached to the cysteine-SH of one α subunit (plate) and a malonyl group attached to the pantetheine-SH of an adjacent α subunit. This arrangement appears to be necessary for the coupling of the acyl and β-carbon of the malonyl group to occur to yield CO2 and the β-ketoacyl product.

Keywords: arches (β subunits), plates (α subunits), bifunctional reagent, cysteine-SH, pantetheine-SH

Full text

PDF
4544

Images in this article

4546Image
on p.4546
4546Image
on p.4546
4546Image
on p.4546
4546Image
on p.4546
4547Image
on p.4547

Selected References

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

  1. Bryce C. F., Crichton R. R. Gel filtration of proteins and peptides in the presence of 6M guanidine hydrochloride. J Chromatogr. 1971 Dec 23;63(2):267–280. doi: 10.1016/s0021-9673(01)85639-9. [DOI] [PubMed] [Google Scholar]
  2. Fish W. W., Mann K. G., Tanford C. The estimation of polypeptide chain molecular weights by gel filtration in 6 M guanidine hydrochloride. J Biol Chem. 1969 Sep 25;244(18):4989–4994. [PubMed] [Google Scholar]
  3. Oesterhelt D., Bauer H., Kresze G. B., Steber L., Lynen F. Reaction of yeast fatty acid synthetase with iodoacetamide. 1. Kinetics of inactivation and extent of carboxamidomethylation. Eur J Biochem. 1977 Sep 15;79(1):173–180. doi: 10.1111/j.1432-1033.1977.tb11795.x. [DOI] [PubMed] [Google Scholar]
  4. Shapiro A. L., Viñuela E., Maizel J. V., Jr Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels. Biochem Biophys Res Commun. 1967 Sep 7;28(5):815–820. doi: 10.1016/0006-291x(67)90391-9. [DOI] [PubMed] [Google Scholar]
  5. Stoops J. K., Arslanian M. J., Aune K. C., Wakil S. J. Further evidence for the multifunctional enzyme characteristic of the fatty acid synthetases of animal tissues. Arch Biochem Biophys. 1978 Jun;188(2):348–359. doi: 10.1016/s0003-9861(78)80019-8. [DOI] [PubMed] [Google Scholar]
  6. Stoops J. K., Arslanian M. J., Oh Y. H., Aune K. C., Vanaman T. C., Wakil S. J. Presence of two polypeptide chains comprising fatty acid synthetase. Proc Natl Acad Sci U S A. 1975 May;72(5):1940–1944. doi: 10.1073/pnas.72.5.1940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Stoops J. K., Awad E. S., Arslanian M. J., Gunsberg S., Wakil S. J., Oliver R. M. Studies on the yeast fatty acid synthetase. Subunit composition and structural organization of a large multifunctional enzyme complex. J Biol Chem. 1978 Jun 25;253(12):4464–4475. [PubMed] [Google Scholar]
  8. Stoops J. K., Ross P., Arslanian M. J., Aune K. C., Wakil S. J., Oliver R. M. Physicochemical studies of the rat liver and adipose fatty acid synthetases. J Biol Chem. 1979 Aug 10;254(15):7418–7426. [PubMed] [Google Scholar]
  9. Stoops J. K., Wakil S. J. The isolation of the two subunits of yeast fatty acid synthetase. Biochem Biophys Res Commun. 1978 Sep 14;84(1):225–231. doi: 10.1016/0006-291x(78)90286-3. [DOI] [PubMed] [Google Scholar]
  10. Wieland F., Siess E. A., Renner L., Verfürth C., Lynen F. Distribution of yeast fatty acid synthetase subunits: three-dimensional model of the enzyme. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5792–5796. doi: 10.1073/pnas.75.12.5792. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES