The architecture of the animal fatty acid synthetase. II. Separation of the core and thioesterase functions and determination of the N-C orientation of the subunit.

Chicken fatty acid synthetase is cleaved by alpha-chymotrypsin into two fragments of molecular weight 230,000 and 33,000. These fragments may be easily separated by ammonium sulfate fractionation and gel filtration to yield pure preparations. The large 230,000-Da fragment contains all of the core activities of the fatty acid synthetic sequence i.e. acetyl and malonyl transacylases, condensing enzyme, beta-ketoacyl and enoyl reductases, the dehydratase, and the acyl carrier protein. The smaller 33,000-Da fragment retains the thioesterase activity which catalyzes the release of the completed acyl chains from the complex. Antibodies against the purified thioesterase fragment cross-react with analogous (Mr 33,000) peptides released from the complex by other proteases, as well as with all proteolytic intermediates that were predicted by peptide mapping to contain the thioesterase segment (Mattick, J. S., Tsukamoto, Y., Nickless, J., and Wakil, S. J. (1983) J. Biol. Chem. 258, 15291-15299). Amino acid sequence analyses demonstrate that the thioesterase domain is located at the carboxyl terminus of the synthetase monomer, thereby orienting the proteolytic (and functional) sites within the complex with respect to the direction of transcription and translation.