Relations between conformations and activities of lipoamide dehydrogenase: III. Protein association—dissociation and the influence on catalytic properties

Abstract

1. 1. The molecular weights of lipoamide dehydrogenase holoenzyme and its Cu 2+-modified form have been determined by gel filtration and ultracentrifugation to be twice those of the apoenzyme and the DCIP-active enzyme obtained by binding FAD to the apoenzyme at 0–5°, i.e., 104 000 and 52 000, respectively. 2. 2. Upon anaerobic reduction with excess NADH in 8 M urea, a protein could be isolated that was able to reconstitute the activity with oxidized lipoate. 3. 3. The DCIP-active enzyme obtained upon freezing of the oxidized enzyme shows, under the present conditions, the original molecular weight, while upon anaerobic reduction with NADH, a DCIP-active enzyme is formed which is a monomer. 4. 4. Association—dissociation phenomena are involved in the reversible conversion of activity with oxidized lipoate into DCIP activity. The evidence that association—dissociation of the protein occurs will be discussed in connection with proposed models for the structure of this enzyme.