Dibromopropanone cross-linking of the phosphopantetheine and active-site cysteine thiols of the animal fatty acid synthase can occur both inter- and intrasubunit - Reevaluation of the side-by-side, antiparallel subunit model

The objective of this study was to test a new model for the homodimeric ani mal FAS which implies that the condensation reaction can be catalyzed by th e aminoterminal beta-ketoacyl synthase domain in cooperation with the penul timate carboxyl-terminal acyl carrier protein domain of either subunit, Tre atment of animal fatty acid synthase dimers with dibromopropanone generates three new molecular species with decreased electrophoretic mobilities; non e of these species are formed by fatty acid synthase mutant dimers lacking either the active-site cysteine of the beta-ketoacyl synthase domain (C161A ) or the phosphopantetheine thiol of the acyl carrier protein domain (S2151 A), A double affinity-labeling strategy was used to isolate dimers that car ried one or both mutations on one or both subunits; the heterodimers were t reated with dibromopropanone and analyzed by a combination of sodium dodecy l sulfate/polyacrylamide gel electrophoresis, Western blotting, gel filtrat ion, and matrix-assisted laser desorption mass spectrometry, Thus the two s lowest moving of these species, which accounted for 45 and 15% of the total , were identified as doubly and singly cross-linked dimers, respectively, w hereas the fastest moving species, which accounted for 35% of the total, wa s identified as originating from internally cross-linked subunits, These re sults show that the two polypeptides of the fatty acid synthase are oriente d such that head-to-tail contacts are formed both between and within subuni ts, and provide the first structural evidence in support of the new model.