Characterization of fatty acid synthase monomers restrained from reassociating by immobilization to a solid support.
The controversial question as to whether the ketoreductase activity of the animal fatty acid synthase is lost on dissociation of the homodimer has been addressed by using immobilized subunits which cannot reassociate under the conditions of assay. Ketoreductase activity, assessed with the model substrate S-acetoacetyl-N-acetylcysteamine, was identical in immobilized monomers and dimers, exhibiting normal Michaelis-Menten kinetics with Km values in the millimolar range. When acetoacetyl-CoA was used as a substrate, however, biphasic kinetics were observed in the case of the dimer, with estimated Km values in the micro- and milli-molar ranges, but only the high-Km reaction was observed with the monomer. Thus when the ketoreductase activities of the monomer and dimer are assessed with acetoacetyl-CoA at concentrations sufficient to saturate only the low-Km reaction, it appears that the ketoreductase activity towards acetoacetyl-CoA is lost upon dissociation. Reduction of acetoacetyl-CoA via the low-Km pathway is CoA-dependent, indicating that acetoacetyl-CoA can react with the dimer by two mechanisms: a high-Km pathway analogous to that utilized by model substrates and a low-Km pathway in which substrate and product are transferred between acyl-CoA and acyl-enzyme forms. The results indicate that the ketoreductase activity per se is unaffected by subunit dissociation and are consistent with a model in which the transfer of substrate from CoA ester to the acyl-carrier-protein domain necessitates juxtaposition of the transferase active-site serine residue of one subunit and the phosphopantetheine moiety of the adjacent subunit.
Petithory
J R
JR
Children's Hospital Oakland Research Institute, CA 94609.
Smith
S
S
D002384:Catalysis; D003065:Coenzyme A / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000378:metabolism*; D064429:Fatty Acid Synthases / Q000378:metabolism*; D007700:Kinetics; D010170:Palmitoyl-CoA Hydrolase / Q000037:antagonists & inhibitors; D013379:Substrate Specificity
D004800:Enzymes, Immobilized; D064429:Fatty Acid Synthases; D010170:Palmitoyl-CoA Hydrolase; D003065:Coenzyme A