Elementary steps in the reaction mechanism of chicken liver fatty acid synthase. Acylation of specific binding sites.

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The mechanism of acyl enzyme formation from acyl-CoA derivatives was studied for chicken liver fatty acid synthase in 0.1 M potassium phosphate (pH 7.0) and 1 mM EDTA at 23 degrees C. Three mechanistically important acyl-binding sites exist: a cysteine, 4'-phosphopantetheine, and a hydroxyl (serine). The cysteine was specifically labeled with iodoacetamide, and chemical modification of this labeled enzyme with chloroacetyl-CoA resulted in additional covalent labeling of 4'-phosphopantetheine. Reaction of the enzyme with acetyl-CoA results in 47% oxyester formation, whereas with malonyl-CoA and butyryl-CoA, 57 and 80% are oxyesters, respectively, as judged by treatment of the denatured enzyme with hydroxylamine. Limited proteolysis with trypsin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the reactive hydroxyl and cysteine are on the same peptide. Butyryl-CoA is a relatively poor primer for steady state fatty acid synthesis, probably because transfer from the hydroxyl-binding site to 4'-phosphopantetheine is inefficient. Quenched flow studies indicate that the rate constants for transfer of acetyl from enzyme-bound acetyl-CoA to native, iodoacetamide-labeled, and iodoacetamide-chloroacetyl-labeled enzyme are 43, 110, and 150 s-1. These results can be interpreted in terms of a random acylation of the hydroxyl, 4'-phosphopantetheine, and cysteine by enzyme-bound acetyl-CoA with rate constants of 150 s-1, less than 110 s-1, and less than 43 s-1, respectively. Alternatively the latter two rate constants could be characteristic of intramolecular transfer between enzyme acylation sites. Structural constraints apparently prevent all three acylation sites from being occupied simultaneously. The rate of deacetylation of the acetylated enzyme by enzyme-bound CoA also is most rapid for the iodoacetamide-chloroacetyl-labeled enzyme.