Properties of the thioesterase component obtained by limited trypsinization of the fatty acid synthetase multienzyme complex.
The fatty acid synthetase from lactating rat mammary gland is shown to consist of two polyfunctional polypeptides of similar molecular weight (about 220,000); a 4'-phosphopantetheine residue is covalently bound to one, or both subunits. Limited trypsinization of the fatty acid synthetase releases on enzymatically active thioesterase component which has been purified and its properties studied. The thioesterase sediments in the ultracentrifuge as a single component of molecular weight 32,000; its sedimentation coefficient is 2.9 x 10-(13) s its diffusion coefficient 5.0 x 10-(7) cm2 s-(1). The thioesterase also elutes from a column of Sephadex G-75 as a single, symmetrical peak of constant specific activity. However, electrophoresis of the denatured thioesterase in the presence of sodium dodecyl sulfate reveals that the enzyme has been partially nicked during isolation. The kinetic data of the enzyme reaction were studied using palmityl-CoA as a model substrate. Solvent pH was found to affect both Vmax and Km (Km = 0.5 micron at pH 6.6, 2.5 micron at pH 8.0) wereas solvent ionic strength affected Vmax but no Km. The thioesterases from the fatty acid synthetases of rat liver and lactating mammary gland have identical physical properties, identical amino acid compositions, and are immunologically indistinguishable. Both thioesterases hydrolyze long chain, in preference to short chain, thioesters of CoA, an observation consistent with their role in regulation of the chain-terminating step in fatty acid synthesis by the parent multienzyme complexes.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D064429:Fatty Acid Synthases / Q000378:metabolism; D005260:Female; D005779:Immunodiffusion; D007700:Kinetics; D008321:Mammary Glands, Animal / Q000201:enzymology; D008970:Molecular Weight; D011247:Pregnancy; D011487:Protein Conformation; D051381:Rats; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D014357:Trypsin
D000596:Amino Acids; D064429:Fatty Acid Synthases; D013869:Thiolester Hydrolases; D014357:Trypsin