Yeast fatty acid synthetase: structure-function relationship and nature of the beta-ketoacyl synthetase site.

Yeast fatty acid synthetase consists of two multifunctional proteins, alpha and beta, which are arranged in a complex of alpha(6)beta(6). 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 (t((1/2)) approximately 7 sec) with two juxtapositioned active sulfhydryl groups. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of the dibromopropanone-inhibited synthetase shows that the beta subunit is intact and the alpha subunit nearly absent with a concomitant appearance of oligomers with an estimated molecular weight of 0.4-1.2 x 10(6). These results indicate that the alpha subunits are crosslinked by this bifunctional reagent. Because the active centers of dibromopropanone are 5 A apart, it is concluded that the alpha subunits are closely packed so that the reacting thiols of the adjacent alpha subunits are within 5 A 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 alpha subunits are the "plates" and the beta subunits therefore are the "arches." Assay of the partial reactions shows that dibromopropanone inhibits the beta-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 beta-ketoacyl synthetase of one alpha subunit and the pantetheine-SH of the acyl carrier protein moiety of an adjacent alpha subunit. Thus, the enzymically active center of the beta-ketoacyl synthetase consists of an acyl group attached to the cysteine-SH of one alpha subunit (plate) and a malonyl group attached to the pantetheine-SH of an adjacent alpha subunit. This arrangement appears to be necessary for the coupling of the acyl and beta-carbon of the malonyl group to occur to yield CO(2) and the beta-ketoacyl product.