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 "hinge". Here we identify and characterize a second oligomeric form constituent in purified enzyme preparations, albeit at low concentrations. Both species 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. Apparent molecular weight values determined by sedimentation equilibrium and gel filtration chromatography are 4-fold greater than the subunit molecular 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. Dissociation of the homotetramer to the homodimer occurs very slowly in benign solutions containing high salt concentrations (0.25-2.0 M KCl). The homotetramer is fully converted to homodimer during refolding from denaturant at low protein concentrations. Disruption of the hydrophilic linker between the N- and C-domains by mutagenesis or mild proteolysis causes a decrease in the relative amount of the larger conformer. The homotetramer is stabilized by interactions involving the helical hinge region, and a substantial kinetic barrier hinders interconversion of the two oligomeric species under nondenaturing conditions.