Comparative Studies of the Pigeon Liver Fatty Acid Synthetase Complex and Its Subunits

Abstract

Abstract The fatty acid synthetase complex of pigeon liver has been dissociated (greater than 95%) into half-molecular weight subunits by dialyzing against Tris-glycine buffer, pH 8.3, containing 1 mm 2-mercaptoethanol or 1 mm dithiothreitol. The dissociated enzyme has very little activity for the synthesis of palmitic acid. However, it can be reassociated by dialysis against 0.2 m potassium phosphate, pH 7.0, containing 10 mm dithiothreitol. The reassociated enzyme has the same sedimentation coefficient and the same specific activity for fatty acid synthesis as the untreated complex. Comparisons have been made between the complex and the mixture of subunits for each of the partial reactions of fatty acid synthesis. Acetyl- and malonyl-CoA-pantetheine transacylase activities are, respectively, 30 and 50% lower in the dissociated enzyme than in the complex. The Km(app) values for the complex and the subunits are quite similar for the reduction of S-acetoacetyl-N-acetyl cysteamine, dehydration of β-hydroxybutyryl-N-acetyl cysteamine, and the deacylation of palmityl-CoA. The Vmax values for these three reactions are also either identical for both enzyme species or approximately 30% lower for the dissociated units. The Vmax/Km(app) value for the reduction of crotonyl-N-acetyl cysteamine by the subunits is at least 50% of the value obtained with undissociated enzyme. The most significant difference between the two enzyme species is the absence of the condensation-CO2 exchange reaction (between malonyl-CoA, caproyl-CoA (hexanoyl-CoA), coenzyme-A, and NaH14CO3) in the dissociated enzyme. The transfer of acetyl and malonyl groups (from their CoA esters) to the different sites on the complex and dissociated enzyme has also been compared. The covalent binding of acetyl groups to the cysteine-SH site, which is essential for the condensation reaction, is eliminated in the dissociated enzyme, whereas the extent of transfer to the hydroxyl and the 4'-phosphopantetheine-SH sites is either unchanged or is enhanced. The covalent binding of malonyl groups to the hydroxyl of the dissociated enzyme is unaffected, whereas transfer to the 4'-phosphopantetheine is appreciably reduced. The dissociation process does not appear to have drastically altered the spatial arrangement around the active site of all of the component enzymes except those associated with the transfer of the acetyl group to the cysteine-SH site, and possibly those involved in the condensation-CO2 exchange reaction.