Distribution of reaction intermediates on chicken liver fatty acid synthase

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Abstract
The distributions of covalent intermediates in the reaction cycle catalyzed by chicken liver fatty acid synthase were studied. In isotope-trapping experiments, 30% of [1-14C]acetyl-enzyme and 6.7% of [2-14C]malonyl-enzyme are converted to long-chain fatty acids, indicating the initiation reaction is partially random. The 3-hydroxybutyryl intermediate is located on the tryptic peptide which contains the 4''-phosphopantetheine (> 90%), while the C4-C18 saturated intermediates are distributed both on this peptide and on the peptide that contains the active cysteine. The ratio of intermediates on the 2 peptides is about unity for chain lengths < C14, but the amount on the active cysteine progressively decreases for chain lengths of C14, C16 and C18 with trypsinized enzyme. The distributions of carbon chain lengths for the saturated or 3-keto intermediates when acetoacetyl-labeled trypsinized enzyme is incubated with limiting malonyl coenzyme A or NADPH, respectively, show large fractions both of unreacted enzyme and of C16 or longer intermediates. The initial condensation and reduction steps apparently are slower than the analogous reactions with longer chain length intermediates. The 3-keto intermediate comprises over 70% of each chain length intermediate detected when NAPDH is the limiting substrate, indicating the reduction of the 3-keto intermediates is at least 2 times slower than the reduction of the unsaturated intermediates.

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