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Proc Natl Acad Sci U S A. Aug 1980; 77(8): 4544–4548.
PMCID: PMC349880

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

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.

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Selected References

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