Inactivation of 3-oxoacyl synthetase activity of pigeon liver fatty acid synthetase by S-(4-bromo-2,3-dioxobutyl)-coenzyme A.

Abstract

Pigeon liver fatty acid synthetase was inactivated by treatment with S-(4-bromo-2,3-dioxobutyl)-CoA. This inactivation is fast, irreversible and dependent on both time and inhibitor concentration. It is also specific and selective as only the 3-oxoacyl synthetase and acetyl-CoA transacylase component activities, out of the seven component activities possessed by the fatty acid synthetase complex, were inhibited. The binding of bromo-dioxobutyl-CoA to the enzyme is covalent as evidenced by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The modified enzyme showed characteristic absorption peaks at 266 nm and 315 nm that are identical to the absorption maxima of pure inhibitor. All the available evidence suggests that the mechanism of inhibition of fatty acid synthetase by S-(4-bromo-2,3-dioxobutyl)-CoA involved its binding to critical thiols, namely the cysteine — SH of the condensing enzyme and cysteamine — SH of the 4′-phosphopantetheine prosthetic group. Both subsrates, acetyl-CoA and malonyl-CoA, also protected the enzyme against inhibition by S-(4-bromo-2,3-dioxobutyl)-CoA. Comparison of high-voltage electrophoretic patterns of peptic peptides obtained from [1-14C]acetyl-labeled native and S-(4-bromo-2,3-dioxobutyl)-CoA-inactivated fatty acid synthetase confirmed the involvement of both cysteine and 4′-phosphopantetheine — SH groups in the binding with S-(4-bromo-2,3-dioxobutyl)-CoA. Scatchard analysis of the results of binding studies has established that 4 mol of the inhibitor are bound/mol of the enzyme complex, which indicates the presence of two moles condensing activities and two moles 4′-phosphopantetheine prosthetic groups/mole of enzyme. Our data do not support a half-site reactivity for the fatty acid synthetase as proposed earlier [Clements et al. (1979) Biochem. Biophys. Res. Commun. 86, 278–284].