Evidence that the acyl‐O‐esters are intermediates in the catalysis

The sequence acetyl-CoA leads to acetyl-O-enzyme leads to acetyl-S-acyl carrier protein has for the first time been demonstrated directly with a multifunctional (mammalian) fatty acid synthase. This was achieved by blocking of the active-site thiols of rabbit mammary fatty acid synthase with iodoacetamide. The modified enzyme was incubated with [14C]acetyl-CoA to form acetyl-O-enzyme, and acetyl-CoA was removed rapidly by centrifuge desalting. We were then able to demonstrate transfer of the acetyl group from [14C]acetyl-O-enzyme to the pantetheine thiol in a fragment of rabbit mammary fatty acid synthase containing the phosphopantetheine group, and to E. coli acyl carrier protein.