1. By dissociation and subsequent reassociation of appropriate pairs of mutant fatty acid synthetases, hybrid multienzyme complexes were obtained whose overall fatty acid synthetase activities were restored to a considerable extent. The complementation thus achieved in vitro could be both intragenic and intergenic and was, in all cases studied, in agreement with the known complementation characteristics of fatty acid synthetase (fas) mutants in vivo. 2. Similarly, the method of reversible dissociation could be used to reactivate a wild-type fatty acid synthetase which had undergone considerable loss of activity due to prolonged storage. It is believed that only those subunits which have retained their native conformation are used in the reassociation of the complex. 3. Specific component enzyme activities were determined with a variety of different mutant fatty acid synthetases and with several hybrid enzymes obtained from them by complementation in vivo or in vitro. The results indicate that the activities of most fatty acid synthetase functional domains are influenced by homologous and/or heterologous subunit interactions within the alpha(6)beta(6) oligomeric complex. Thus. mutational inactivation of any one of the active sites in subunit beta simultaneously also alters the other four catalytic sites of this subunit. The lack of complementation occasionally observed between certain mutants of two different fas 1 complementation groups may be explained by this effect. Furthermore, alpha/beta interactions are indicated by the fact that enoylreductase-deficient mutant enzymes (beta defect) always exhibit dramatically increased beta-ketoacyl synthase (alpha domain) activities. 4. The incorporation of FMN in vitro into wild-type fatty acid synthetase apo-enzyme from which the flavin had been removed leads to a fully active enzyme complex only when the cofactor is present during the subunit assembly process. However, addition of FMN to a fully associated apo-enzyme restores only 50% of its original overall specific activity. If FMN was replaced by FAD in either of these experiments, reactivation occurred only to an extent of 50-70% of that observed with FMN.

• PMID: 7018906
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Reference Type

Comparative Study | Journal Article | Research Support, Non-U.S. Gov't

Authors

Werkmeister K, Johnston RB, Schweizer E

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