Isolation of a functional transferase component from the rat fatty acid synthase by limited trypsinization of the subunit monomer. Formation of a stable functional complex between transferase and acyl carrier protein domains.
Limited trypsinization of rat fatty acid synthase monomers results in cleavage at sites protected in the native dimer. A 47,000-Da polypeptide containing the transferase component was isolated from the digest and its location in the multifunctional polypeptide established. Both acetyl and malonyl moieties are transferred stoichiometrically from CoA ester to this polypeptide and each can replace the other, confirming that a single common site is utilized in the loading of these substrates onto the fatty acid synthase. Transferase activity of the 47,000-Da polypeptide decreases with increasing acyl donor chain length (malonyl = acetyl greater than butyryl greater than hexanoyl greater than octanoyl). Activity is inhibited by certain thiol-directed reagents, and protection is afforded by substrate suggesting the presence of a sensitive cysteine residue near the substrate binding site. The transferase was also able to utilize as acyl acceptor the Escherichia coli acyl carrier protein and the acyl carrier protein domain of the multifunctional fatty acid synthase. When the fatty acid synthase monomer was trypsinized under milder conditions, the 47,000-Da transferase domain could be isolated in association with the 8,000-Da acyl carrier protein domain. The transferase was capable of translocating substrate moieties from CoA ester donors to the associated acyl carrier protein. The results provide the first direct evidence that, in the head-to-tail oriented fatty acid synthase homodimer, functional communication between the transferase domain located near the end of one polypeptide and the acyl carrier protein domain located at the opposite end of the other polypeptide is facilitated by a stable physical interaction between these domains.
Children's Hospital Oakland Research Institute, California 94609.
V S
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Rangan
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A
Witkowski
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Smith
D000213:Acyl Carrier Protein / Q000378:metabolism*; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D064429:Fatty Acid Synthases / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051381:Rats; D013379:Substrate Specificity; D014166:Transferases / Q000302:isolation & purification*; D014357:Trypsin / Q000378:metabolism*
D000213:Acyl Carrier Protein; D014166:Transferases; D064429:Fatty Acid Synthases; D014357:Trypsin