Fatty Acid Synthetase of Chicken Liver: REVERSIBLE DISSOCIATION INTO TWO NONIDENTICAL SUBCOMPLEXES OF SIMILAR SIZE

Abstract

Abstract The synthetic reaction catalyzed by the chicken liver fatty acid synthetase has a bell-shaped pH rate profile with a maximum at pH 6.7 and gives a discontinuous biphasic Arrhenius plot. The activation energy at low temperature is 28,900 cal per mole, which changes to 11,300 cal per mole at temperatures above 15°. The amino acid composition of the purified enzyme was found to be very similar to other animal synthetases. Rabbit antibody to chicken liver fatty acid synthetase partially cross-reacts with the pigeon liver enzyme, but does not precipitate the rat and hamster liver enzymes. The chicken liver enzyme which has an s20, w of 12 S dissociates at low ionic strength in Tris-glycine buffer, pH 8.3, into an essentially homogeneous 8 S species, which has a molecular weight of 253,000 by equilibrium sedimentation. The dissociated enzyme contains a mixture of equivalent amounts of two subcomplexes of similar size, but differing in net charge and probably in the composition of enzyme components. The molecular weight (Msd), diffusion coefficient, frictional ratio (f/f0), and Stokes' radius (α) of the native and dissociated enzymes were found to be 500,800, 240,000; 2.44, 3.24; 1.63, 1.59; and 8.61, 6.70, respectively. Dissociation of this enzyme by the above method is a reversible process, accompanied by extensive oxidation of thiol groups. Reactivation of the resulting subcomplexes has been shown to follow a two-step reaction sequence: (a) reassociation of these half-molecules into an inactive enzyme in 0.2 m potassium phosphate buffer at neutral pH; (b) reduction of this inactive enzyme by dithiothreitol to yield the active enzyme. Dissociation, however, is not dependent on the oxidation of thiol groups, occurring just as readily when these groups are protected by dithiothreitol. The subcomplexes obtained in this manner are fully reduced and reconstitute in neutral phosphate buffer directly into the active enzyme in the absence of dithiothreitol. Synthetase activity, however, requires both fully reduced protein thiols and intact quaternary structure.