Inhibition of the functional interaction between fatty acid synthetase and thioesterase II by modification of a single cysteine thiol on the thioesterase.
Medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II), a discrete, monomeric, serine active-site enzyme, modifies the product specificity of the de novo lipogenic pathway by hydrolyzing the thioester bond linking the growing acyl chain to the 4'-phosphopantetheine of the fatty acid synthetase. The mechanism of interaction of thioesterase II and fatty acid synthetase has been studied by probing the thioesterase with sulfhydryl-modifying reagents. Modification of a single cysteine thiol with 5,5'-dithiobisnitrobenzoate destroyed the ability of thioesterase II to catalyze hydrolysis of S-acyl fatty acid synthetase thioesters but had no effect on the ability of the enzyme to hydrolyze the model substrate, decanoyl-S-pantetheine. The inhibition was readily reversed on removal of the thionitrobenzoate moiety from the thioesterase with dithiothreitol. The results of kinetic experiments indicated that loss of the capacity of the thioesterase to hydrolyze the natural substrate could be attributed to an inability of the 5,5'-dithiobisnitrobenzoate-modified enzyme to bind to the fatty acid synthetase. Modification of the same cysteine thiol with methyl methanethiolsulfonate did not affect the ability of thioesterase II to hydrolyze either the natural or model substrates. The results are interpreted to indicate that cysteine thiol, remote from the catalytic active-site serine residue, is present on a binding domain of the thioesterase which interfaces with the fatty acid synthetase. Modification of this thiol with the large thionitrobenzoate moiety, but not with the small CH3S- moiety, inhibits the functional interaction either by steric hindrance or by perturbation of the polypeptide configuration at the binding domain.
D000818:Animals; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D003545:Cysteine / Q000032:analysis; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D064429:Fatty Acid Synthases / Q000378:metabolism*; D005260:Female; D006868:Hydrolysis; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D013438:Sulfhydryl Compounds / Q000032:analysis; D013869:Thiolester Hydrolases / Q000378:metabolism*
D013438:Sulfhydryl Compounds; C014674:methyl methanethiosulfonate; D004228:Dithionitrobenzoic Acid; D008741:Methyl Methanesulfonate; D064429:Fatty Acid Synthases; D013869:Thiolester Hydrolases; C022738:thioesterase II; D003545:Cysteine