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 homodim er has been addressed by using immobilized subunits which cannot reass ociate under the conditions of assay. Ketoreductase activity, assessed with the model substrate S-acetoacetyl-N-acetylcysteamine, was identi cal in immobilized monomers and dimers, exhibiting normal Michaelis-Me nten kinetics with K(m) values in the millimolar range. When acetoacet yl-CoA was used as a substrate, however, biphasic kinetics were observ ed in the case of the dimer, with estimated K(m) values in the micro- and milli-molar ranges, but only the high-K(m) reaction was observed w ith the monomer. Thus when the ketoreductase activities of the monomer and dimer are assessed with acetoacetyl-CoA at concentrations suffici ent to saturate only the low-K(m) reaction, it appears that the ketore ductase activity towards acetoacetyl-CoA is lost upon dissociation. Re duction of acetoacetyl-CoA via the low-K(m) pathway is CoA-dependent, indicating that acetoacetyl-CoA can react with the dimer by two mechan isms: a high-K(m) pathway analogous to that utilized by model substrat es and a low-K(m) pathway in which substrate and product are transferr ed between acyl-CoA and acyl-enzyme forms. The results indicate that t he ketoreductase activity per se is unaffected by subunit dissociation and are consistent with a model in which the transfer of substrate fr om CoA ester to the acyl-carrier-protein domain necessitates juxtaposi tion of the transferase active-site serine residue of one subunit and the phosphopantetheine moiety of the adjacent subunit.