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

Publisher Website
Google Scholar
Abstract
Yeast fatty acid synthetase consists of 2 multifunctional proteins, .alpha. and .beta., which are arranged in a complex of .alpha.6.beta.6. EM studies of this complex led to a model for the synthetase as an ovate structure consisting of an equatorial plate-like structure to which 6 arches are equally distributed on either side. The bifunctional reagent 1,3-dibromo-2-propanone inhibits the synthetase by reacting rapidly (half life .apprxeq. 7 s) with 2 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 is nearly absent with a concomitant appearance of oligomers with an estimated MW of 0.4-1.2 .times. 106. The .alpha. subunits may be crosslinked by this bifunctional reagent. Because the active centers of dibromopropanone are 5 .ANG. apart, it is concluded that the .alpha. subunits are closely packed so that the reacting thiols of the adjacent .alpha. subunits are within 5 .ANG. of each other. Because the plate-like structures in the model are the only components that are arranged closely enough to satisy this requirement, it is proposed that the .alpha. subunits are the plates and the .beta. subunits are therefore the arches. Assay of the partial reactions shows that dibromopropanone inhibits the .beta.-ketoacyl synthetase reaction but none of the 6 other partial reactions, indicating that the site of action of the bifunctional reagent is the condensing reaction. Pretreatment of the synthetase with acetyl-CoA or iodoacetamide prevented dibromopropanone from interacting at this site and obviated the formation of the crosslinked oligomer. The 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 CO2 and the .beta.-ketoacyl product.