Mammalian fatty acid synthetase is a structurally and functionally symmetrical dimer

• Published in: European journal of biochemistry Vol. 152; no. 3; pp. 547 - 555
• Main Authors: SMITH, Stuart, STERN, Alan, RANDHAWA, Zafar I., KNUDSEN, Jens
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-11-1985; Blackwell
• Subjects: Acetone - analogs & derivatives; Alkylation; Analytical, structural and metabolic biochemistry; Animals; Binding Sites - drug effects; Biological and medical sciences; Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fatty Acid Synthases - isolation & purification; Female; Fundamental and applied biological sciences. Psychology; Hydrolysis; Lactation; Ligases; Liver - enzymology; Mammary Glands, Animal - enzymology; Molecular Weight; Pantetheine - analogs & derivatives; Pantetheine - analysis; Pregnancy; Rats; Trypsin; Ligase; Stoichiometry; Vertebrata; Mammalia; Molecular structure; Enzyme; Active site; Reaction mechanism; Dimer; Fatty acid synthetase; Catalyst activity; Symmetry
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1985.tb09230.x

We have explored a comprehensive experimental approach to determine whether the two condensing‐enzyme active centers of the mammalian fatty acid synthetase are simultaneously functional. Our strategy involved utilization of trypsinized fatty acid synthetase, which is a nicked homodimer composed of two pairs of 125 + 95‐kDa polypeptides. These core polypeptides lack the chain‐terminating thioesterase domains but retain all other functional domains of the native enzyme and can assemble long‐chain acyl moieties at a rate equal to that of the native enzyme. The 4′‐phosphopantetheine content of these enzyme preparations, estimated from the amount of β‐alanine present, from the amount of taurine formed by performic acid oxidation and from the amount of carboxymethylcysteamine formed by alkylation with iodo[2‐14C]acetate, was typically 0.86 mol/mol 95‐kDa polypeptide. The stoichiometry of long‐chain acyl‐enzyme synthesis, measured with radiolabeled precursors, indicated that 0.84 mol acyl‐chains were assembled/mol 95‐kDa polypeptide. When the small amount of apoenzyme present is taken into account, this stoichiometry translates to 1.94 acyl chains per holoenzyme dimer. The 125‐kDa polypeptide of one subunit could be cross‐linked to the 95‐kDa polypeptide of the other subunit by 1,3‐dibromo‐2‐propanone yielding a single molecular species of 220 kDa. Cross‐linking was accompanied by a loss of condensing‐enzyme activity. This result is consistent with a structurally symmetrical model for the animal fatty acid synthetase [J. K. Stoops and S. J. Wakil (1981) J. Biol. Chem. 256, 5128–5133] in which the juxtaposed 4′‐phosphopantetheine and cysteine thiols of opposing subunits that form the two potential catalytic centers for condensing activity are readily susceptible to cross‐linking. Both half‐maximal cross‐linking and 50% inhibition of activity were observed with 1 mol 1,3‐dibromo‐2‐propanone bound/mol enzyme. After assembly of long‐chain acyl moieties on the 4′‐phosphopantetheine residues, no vacant condensing‐enzyme active sites were demonstrable either by cross‐linking with 1,3‐dibromo‐2‐propanone or by formation of carboxymethylcysteamine on treatment with iodoacetate.
These results are consistent with a structurally and functionally symmetrical model for the mammalian fatty acid synthetase in which the two condensation sites are simultaneously active.