The Effects of Reduced Nicotinamide Adenine Dinucleotide Phosphate, Its Structural Analogues, and Coenzyme A and Its Derivatives on the Rate of Dissociation, Conformation, and Enzyme Activity of the Pigeon Liver Fatty Acid Synthetase Complex

Abstract

Abstract The fatty acid synthetase complex of pigeon liver is completely dissociated to half-molecular weight subunits of either negligible or no enzyme activity in the presence of 35 mm glycine, 5 mm Tris, 1 mm EDTA, and 1.0 mm 2-mercaptoethanol at pH 8.3 (µ = 0.008). This dissociation and loss of enzyme activity can be prevented by the addition of 0.2 m KCl or 20 µm NADPH to the above incubation medium. The addition of NADH at either low (20 µm) or high (1.0 mm) concentrations has no effect on the rate of dissociation of the complex, whereas adenosine 2'-monophosphoric acid at 1.0 mm protects the enzyme slightly against inactivation. Adenosine 3'-monophosphoric acid has no protective effect. Reduced nicotinamide mononucleotide (1.0 mm), either alone or in association with adenosine 2'-monophosphoric acid (1.0 mm), also has no effect. Fructose 1,6-diphosphate (0.5 mm and 2.0 mm) considerably enhances the stability of the fatty acid synthetase complex at a low ionic strength. This effect is not due to an increase in ionic strength contributed by fructose 1,6-diphosphate. The latter compound does not compete with NADPH for binding sites on the enzyme. In contrast to NADPH, coenzyme A and dephospho-CoA increase the rate of inactivation of the complex possibly as a result of the formation of mixed disulfides with sulfhydryl groups of the enzyme. The sedimentation coefficient of the fatty acid synthetase complex in low ionic strength buffer is increased by NADPH (from 11.8 S in the absence to 12.9 S in the presence of 0.3 mm NADPH). This effect appears to result from a partial alteration in the structure of the complex. The structure of the enzyme also appears to be modified by increasing the pH, even at high ionic strengths. The slope of plots of the reciprocal of the sedimentation coefficient against protein concentration is increased 4-fold in going from pH 7.0 to pH 8.3 in 0.2 m potassium phosphate. The results reported in this paper are discussed in relation to the stability and integrity of the enzyme complex and the type of forces which may hold the subunits of the complex together. The possibility of NADPH and CoA acting as metabolic regulators of fatty acid synthesis through their effect on the stability of the complex has been considered.