The coenzyme A requirement of mammalian fatty acid synthetase: Evidence for involvement in the elongation of acyl-enzyme thioesters

Abstract

We have confirmed that coenzyme A is required for rat fatty acid synthetase activity (T. C. Linn, M. J. Stark, and P. A. Srere, 1980, J. Biol. Chem. 255, 1388–1392) . When rat liver or mammary gland fatty acid synthetase was assayed in the presence of a CoA-scavenging system such as ATP citrate lyase, almost complete inhibition of fatty acid synthesis was observed. The inhibition was reversed by addition of CoA or pantetheine, but not by addition of N-acetylcysteamine or other thiols. In the absence of CoA, the rate of elongation of acyl moieties on both native fatty acid synthetase and fatty acid synthetase lacking the chain-terminating thioesterase I component (trypsinized fatty acid synthetase) was reduced 100-fold. All of the palmitate synthesized slowly by the CoA-depleted native multienzyme was released, by the thioesterase I component, as the free fatty acid; only shorter-chainlength acyl moieties remained bound to the enzyme. The acyl- S-multienzyme thioesters formed by the trypsinized fatty acid synthetase in the absence of CoA contained saturated moieties of chain length C 6-C 16; addition of CoA promoted elongation of the acyl- S-multienzyme thioesters without release from the enzyme. The transfer of acetyl and malonyl moieties from CoA to the multienzyme, the reduction of S-acetoacetyl- N-acetylcysteamine and S-crotonyl- N-acetylcysteamine, and the dehydration of S-β-hydroxybutyryl- N-acetylcysteamine, reactions catalyzed by the fatty acid synthetase, were not dependent on the presence of CoA. The hydrolysis of acyl- S-multienzyme catalyzed by thioesterase I, the resident chain-terminating component of the fatty acid synthetase, and thioesterase II, a monofunctional mammary gland chain-terminating enzyme, was also independent of CoA availability as was hydrolysis of an acyl- S-pantetheine pentapeptide isolated from the multienzyme. On the basis of these observations we conclude that CoA is required for the elongation of acyl moieties on the fatty acid synthetase but not for their release from the multienzyme.