Identification of the sites of binding of acetyl and malonyl groups to the pigeon liver fatty acid synthetase complex

Abstract

Peptic digestion of 14C-acetyl-labeled pigeon liver fatty acid synthetase complex liberates three groups of radioactive peptides (designated A 2, B 1, and B 2). A similar treatment of 14C-malonyl-labeled enzyme yields only two groups of radioactive peptides (A 2 and B 1). The present paper reports the results of experiments designed to provide information on the structures of the acetyl- and malonyl-binding sites on the fatty acid synthetase complex. Each of the peptides, labeled with radioactive acetyl or malonyl groups, was purified and then subjected to amino acid analysis. In addition, experiments were performed on the identity of the sites binding acetyl and malonyl groups to each of the peptides. Iodoacetamide selectively inhibits the binding of acetyl groups to the sites of binding on the A 2 and B 2 peptides and the binding of malonyl groups to the sites on the A 2 peptides; but it enhances the binding of acetyl and malonyl groups to the sites on the B 1 peptides. Hydroxylamine liberates these compounds from each peptide. However, the concentration of hydroxylamine required to release radioactive hydroxamates differs for the A 2, B 1 and B 2 peptides. The acyl groups of acetyl- and malonyl-A 2 and acetyl-B 2 peptides were quantitatively cleaved from the peptides at lower concentrations (3 mg/ml) than were required to split acetyl and malonyl groups from B 1 peptides (30–100 mg/ml). Performic acid treatment liberated acetate and malonate from A 2 and B 2 peptides but it did not release these compounds when they were bound to B 1 peptides. These data suggest that sites on the A 2 and B 2 peptides bind acety] and malonyl groups through thioester linkage and that the site on the B 1 peptide binds these groups as more stable oxygen esters. 4′-Phosphopantetheine was identified as the site of binding of acetyl and malonyl groups in the A 2 peptides by formation of the S-carboxymethyl cysteamine derivative from acetyl- and malonyl-labeled A 2 peptides. Amino acid analyses showed the presence of cysteine in the B 2 peptide; threonine and serine were found in both acetyl- and malonyl-B 1 peptides. It is suggested that the B 2 site may be cysteine and that the B 1 site may be an aliphatic hydroxyamino acid.