Previous work from this laboratory (L. J. Libertini and S. Smith, 1979, Arch. Biochem. Biophys. 192, 47-60) has shown that thioesterase II, a mammary gland-specific enzyme, modifies the product specificity of the fatty acid synthetase multienzyme by removing acyl chains attached to the multienzyme through thioester linkage. In this study, two experimental approaches were used to identify the site of action of thioesterase II. (i) The two thioesterase I domains were removed from the fatty acid synthetase with trypsin. The modified multienzyme was incubated with [2-14C]malonyl-CoA, acetyl-CoA, and NADPH to form [14C]acyl-multienzyme thioesters, which were shown to be susceptible to hydrolysis by thioesterase II. Following peptic digestion and [14C]acyl-peptide purification, the site of attachment of the acyl moiety to the modified multienzyme was identified as a pantetheine thiol. (ii) The ability of thioesterase II to hydrolyze a number of cysteine- and cysteamine-containing thioesters was compared. Cysteamine thioesters were good substrates for thioesterase II, which showed the following preference: S-decanoyl-pantetheine > S-decanoyl-CoA > S-decanoyl N-acetylcysteamine. None of the cysteine thioesters were effective substrates for thioesterase II. These two lines of evidence clearly indicate that the site of action of thioesterase II is the thioester bond linking the acyl moiety to the 4′-phosphopantetheine component of the fatty acid synthetase. © 1979.
