Tetrameric assembly and conservation in the ATP-binding domain of rat branched-chain alpha-ketoacid dehydrogenase kinase

We showed previously that the rat branched-chain alpha-ketoacid dehydrogena se (BCKD) kinase is capable of autophosphorylation. However, despite its se quence similarity to bacterial histidine protein kinases, BCKD kinase does not function as a histidine protein kinase. In the present study, we report that the rat BCKD kinase exists as a homotetramer of M-r = 185,000, based on results of gel filtration and dynamic light scattering. This is in contr ast to the related mammalian pyruvate dehydrogenase kinase isozymes that oc cur as homodimers. The tetrameric assembly of BCKD kinase was confirmed by the presence of four 5' -adenylyl-imidodiphosphate-binding sites (K-D = 4.1 x 10(-6) M) per molecule of the kinase. Incubation of the BCKD kinase with increasing concentrations of urea resulted in dissociation of the tetramer to dimers and eventually to monomers as separated on a sucrose density gra dient. Both tetramers and dimers, but not the monomer, maintained the confo rmation capable of binding ATP and undergoing autophosphorylation. BCKD kin ase depends on a fully lipoylated transacylase for maximal activity, but th e interaction between the kinase and the transacylase is impeded in the pre sence of high salt concentrations. Alterations of conserved residues in the ATP-binding domain led to a marked reduction or complete loss in the catal ytic efficiency of the BCKD kinase. The results indicate that BCKD kinase, similar to pyruvate dehydrogenase kinase isozymes, belongs to the superfami ly of ATPase/kinase.