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

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Abstract
The sequence acetyl‐CoA → acetyl‐O‐enzyme → 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.