Release of two thioesterase domains from fatty acid synthetase by limited digestion with trypsin

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Abstract
Limited digestion, with trypsin, of the fatty acid synthetase from rat mammary gland releases an enzymically active thioesterase component that, under denaturing conditions, consists of 2 major spp. of MW 35,000 and 17,500 and a minor species, MW 15,000. The 17,500 and 15,000 MW species originate from the 35,000 MW speces as a result of nicking by trypsin. The nicked polypeptides are enzymically active. The fatty acid synthetase is inhibited by [1,3-14C]DFP, which binds to, and inactivate, 2 thioesterase active sites. When the [1,3-14C]DFP-labeled fatty acid synthetase is subjected to limited digestion with trypsin, all of the radioactivity is recovered in the isolated thioesterase component, i.e., in the 35,000 MW polypeptide and its nicked products. Since the isolated thioesterase binds only 1 DFP residue per 35,000 MW polypeptide, it is concluded that the fatty acid synthetase has 2 thioesterase domains, both of which are removed by limited trypsin treatment.