Conformational Studies on Lipoamide Dehydrogenase from Pig Heart

No structural differences could be detected between the dissociable and non‐dissociable dimer of lipoamide dehydrogenase from pig heart. Optical rotatory dispersion and circular dichroism studies show that upon dissociation the protein undergoes conformational changes, very probably accompanied by changes in α‐helix content. The monomeric apoenzyme probably has a higher α‐helix content than the holoenzyme. Treatment of the non‐dissociable enzyme with dioxan leads to monomerisation, accompanied by similar changes in optical rotatory dispersion and circular dichroism parameters in the 260 to 300‐nm region as obtained by spontaneous dissociation. The perturbation spectrum shows changes around a tryptophyl residue. Dissociation leads to significant changes in fluorescence characteristics. The protein fluorescence emission increases upon dissociation, the energy‐transfer from tryptophan to FAD increases. It could be shown that one tryptophyl residue becomes more exposed. Both the dimer and the monomer undergo a series of temperature‐dependent conformational changes. Upon reconstitution the dimer from apoenzyme and FAD, conformational changes occur during more than 48 h.