Studies on the Mechanism of Fatty Acid Synthesis: XXIII. THE ACYL BINDING SITES OF THE PIGEON LIVER FATTY ACID SYNTHETASE

Abstract

Abstract The covalent binding of acetyl, malonyl, and hexanoyl groups to the pigeon liver fatty acid synthetase has been investigated. Performic acid oxidation of the acyl synthetases has revealed that the synthetase possesses two types of acyl binding sites, these being thiol and nonthiol in nature. From studies on radioactive peptic peptides obtained from 14C-acetyl synthetase and 14C-malonyl synthetase, it has been established that there are two thiol binding sites. One of these thiol sites is the sulfhydryl group of the 4'-phosphopantetheine moiety of the synthetase. Both acetyl and malonyl groups bind to this sulfhydryl group. The second thiol binding site is the sulfhydryl group of a cysteine residue. This sulfhydryl binds acetyl groups but not malonyl groups. The nonthiol binding site or sites has been tentatively identified as being the hydroxyl group of a serine residue. Prior treatment of the synthetase with high concentrations of N-ethylmaleimide inhibits all binding of acetyl and malonyl groups, while prior treatment with low concentrations of N-ethylmaleimide results in inhibition of thiol binding but has little effect on the nonthiol binding of these acyl groups. Prior treatment of the synthetase with iodoacetamide has no effect on the binding of acetyl and malonyl groups to the 4'-phosphopantetheine sulfhydryl but inhibits acetyl binding to the sulfhydryl of the cysteine residue. From these studies the order of acetyl binding to the synthetase appears to be binding first at the nonthiol site with successive transfers to the 4'-phosphopantetheine sulfhydryl and the cysteine sulfhydryl. The binding of malonyl groups follows the same sequence, except that malonyl groups are not transferred from the 4'-phosphopantetheine sulfhydryl to the cysteine sulfhydryl.