DIFFERENTIAL AFFINITY LABELING OF THE 2 SUBUNITS OF THE HOMODIMERIC ANIMAL FATTY-ACID SYNTHASE ALLOWS ISOLATION OF HETERODIMERS CONSISTING OF SUBUNITS THAT HAVE BEEN INDEPENDENTLY MODIFIED

To explore the domain interactions that are required for catalytic act ivity of the multifunctional, homodimeric fatty acid synthase (FAS), w e have formulated a strategy that allows isolation of modified dimers containing independently mutated subunits, Either a hexahistidine or a FLAG octapeptide tag was incorporated into the FAS at either the amin o terminus, within an internal noncatalytic domain, or at the carboxyl terminus, The presence of the tags had no effect on the activity of t he wild-type FAS. His-tagged dimers were mixed with FLAG-tagged dimers , and the subunits were randomized to produce a mixture of His-tagged homodimers, FLAG-tagged homodimers, and doubly tagged heterodimers. Th e doubly tagged heterodimers could be purified to homogeneity by chrom atography on an anti-FLAG immunoaffinity column followed by a metal io n chelating column, This procedure for isolation of FAS heterodimers w as utilized to determine whether the two centers for fatty acid synthe sis in the FAS dimer can function independently of each other, Doubly tagged heterodimers, consisting of one wild-type subunit and one subun it in which the thioesterase activity had been eliminated, either by m utation or by treatment with phenylmethanesulfonyl fluoride, have 50% of the wild-type thioesterase activity and, in the presence of substra tes, accumulate a long chain fatty acyl moiety on the modified subunit , thus blocking further substrate turnover at this center, Nevertheles s, the ability of the heterodimer to synthesize fatty acids is also 50 % of the wild-type FAS, demonstrating that an individual center for fa tty acid synthesis has the same activity when paired with either a fun ctional or nonfunctional catalytic center.