Pig heart CoA transferase exists as two oligomeric forms separated by a large kinetic barrier

Pig heart CoA transferase (EC 2.8.3.5) has been shown previously to adopt a homodimeric structure, in which each subunit has a molecular weight of 52 197 and consists of N- and C-domains linked by a hydrophilic linker or "hin ge", Here we identify and characterize a second oligomeric form constituent in purified enzyme preparations, albeit at low concentrations. Both specie s catalyze the transfer of CoA with similar values for k(cat) and K-M. This second form sediments more rapidly than the homodimer under the conditions of conventional sedimentation velocity and active enzyme centrifugation. A pparent molecular weight values determined by sedimentation equilibrium and gel filtration chromatography are 4-fold greater than the subunit molecula r weight, confirming that this form is a homotetramer. The subunits of both oligomeric forms are indistinguishable with respect to molecular mass, far -UV CD, intrinsic tryptophan fluorescence, and equilibrium unfolding. Disso ciation of the homotetramer to the homodimer occurs very slowly in benign s olutions containing high salt concentrations (0.25-2.0 M KCl). The homotetr amer is fully converted to homodimer during refolding from denaturant at lo w protein concentrations. Disruption of the hydrophilic linker between the N- and C-domains by mutagenesis or mild proteolysis causes a decrease in th e relative amount of the larger conformer. The homotetramer is stabilized b y interactions involving the helical hinge region, and a substantial kineti c barrier hinders interconversion of the two oligomeric species under nonde naturing conditions.