Conformational Changes, Inactivation, and Dissociation of Pigeon Liver Fatty Acid Synthetase Complex

Abstract

Abstract The pigeon liver fatty acid synthetase complex (s020,w = 14.0 S) is inactivated and dissociated into half-molecular weight subunits (s020,w = 9.0 S) in the presence of low ionic strength buffers. The rates of inactivation and dissociation of the complex are dependent upon the ionic strength, pH, and temperature of the medium. At low ionic strengths (0.01 or less) and mildly alkaline pH (8.35), the rates of inactivation and dissociation of the complex into subunits are coincident and the rate of inactivation is independent of protein concentration over a 62-fold range. At higher ionic strengths (above 0.02) and in the presence of 2-mercaptoethanol, complex, inactive enzyme complex, and inactive subunits are found. Increasing the pH above neutrality at constant ionic strength increases the rate of inactivation as well as dissociation of the complex. The inactivation rate is also increased as the pH is lowered below 7.0, but the rate of dissociation of the complex is quite slow; the enzyme thus exists mainly as inactive complex. Temperature also has a marked effect on the rate of inactivation and dissociation of the complex. The rate of inactivation and dissociation in Tris-glycine buffer, pH 8.35 (µ = 0.008) is nearly 10-fold greater at 0° than at room temperature. The lowest rate of inactivation is found at 18–25°. Above 25° the rate of inactivation again increases as the temperature rises. The fatty acid synthetase complex (s020,w = 14.0 S) appears to undergo a transition to an enzymatically at low ionic strength. The active intermediate is then further converted to either an inactive enzyme complex or to inactive subunits (s020,w = 9.0 S). There is no requirement for the oxidation of —SH groups of the enzyme prior to its dissociation to subunits. However, the subunits obtained in the presence of dithiothreitol (conditions under which there is no loss of —SH groups of the enzyme) do not transfer the acetyl group from the 4'-phosphopantetheine to the cysteine site. Hence, these subunits have no activity for fatty acid synthesis. The results presented in this paper suggest that hydrophobic forces and a diminution of electrostatic repulsions are factors which contribute to the stability of the complex in high ionic strength buffers at neutral pH. A lowering of the temperature weakens the hydrophobic interactions and a decrease in the ionic strength increases the electrostatic repulsion between the subunits. These cumulative effects lead ultimately to dissociation of the complex to two subunits.