Fatty acid biosynthesis: IX. Fatty acid synthesis from enzymically and chemically acetylated rabbit mammary gland fatty acid synthetase

Abstract

1. I. [I- 14C]Acetate was covalently bound to rabbit mammary gland fatty acid synthetase by enzymic transacylation from [I- 14C]acetyl-CoA. Per mole of enzyme 2 moles of acetate were bound to thiol groups and up to I mole of acetate was bound to non-thiol groups. 2. 2. The acetyl-fatty acid synthetase complex was isolated free from acetyl-CoA. It was rapidly hydrolysed at 30°C, but hydrolysis was greatly diminished at o°C and triacetic lactone synthesis occurred. In the presence of malonyl-CoA and NADPH, all the acetate bound to fatty acid synthetase was incorporated into long-chain fatty acids. Hydrolysis of bound acetate and incorporation of bound acetate into fatty acids were inhibited to the same extent by guanidine hydrochloride. 3. 3. Acetate was also covalently bound to fatty acid synthetase by chemical acetylation with [I- 14C]acetic anhydride in the absence of CoASH. A total of 60 moles of acetate were bound per mole of fatty acid synthetase, with 20–25 moles being bound to thiol groups per mole of enzyme. Acylation did not inhibit enzyme activity. The majority of the bound acetate was stable to hydrolysis at o °C. Out of the 60 moles of acetate bound per mole of acetylated enzyme, up to 20 moles were incorporated into fatty acids in the presence of malonyl-CoA and NADPH. 4. 4. Due to the rapid hydrolysis of acetyl-fatty acid synthetase prepared from both acetyl-CoA and acetic anhydride, direct carboxylation of acetyl-fatty acid synthetase to form malonyl-fatty acid synthetase could not be demonstrated by CO 2 fixation under the experimental conditions used. With acetic anhydride as acetyl donor, there was a low rate of incorporation of acetate into fatty acids in the presence of NADPH but absence of added malonyl-CoA. This could be due to direct carboxylation of the acetate bound to fatty acid synthetase or, if any CoA were associated with the fatty acid synthetase, to carboxylation of acetyl-CoA formed by chemical acetylation of this CoA.