Publications

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

  Wynn R.M., Chuang J.L., Cote C.D., Chuang D.T.

  We showed previously that the rat branched-chain alpha-ketoacid dehydrogenase (BCKD) kinase is capable of autophosphorylation. However, despite its sequence similarity to bacterial histidine protein kinases, BCKD kinase does not function as a histidine protein kinase. In the present study, we repo ... >> More

  We showed previously that the rat branched-chain alpha-ketoacid dehydrogenase (BCKD) kinase is capable of autophosphorylation. However, despite its sequence 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 contrast to the related mammalian pyruvate dehydrogenase kinase isozymes that occur 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 gradient. Both tetramers and dimers, but not the monomer, maintained the conformation capable of binding ATP and undergoing autophosphorylation. BCKD kinase depends on a fully lipoylated transacylase for maximal activity, but the interaction between the kinase and the transacylase is impeded in the presence of high salt concentrations. Alterations of conserved residues in the ATP-binding domain led to a marked reduction or complete loss in the catalytic efficiency of the BCKD kinase. The results indicate that BCKD kinase, similar to pyruvate dehydrogenase kinase isozymes, belongs to the superfamily of ATPase/kinase. << Less

  J Biol Chem 275:30512-30519(2000) [PubMed] [EuropePMC]

• The C-terminal hinge region of lipoic acid-bearing domain of E2b is essential for domain interaction with branched-chain alpha-keto acid dehydrogenase kinase.

  Chuang J.L., Wynn R.M., Chuang D.T.

  The branched-chain alpha-keto acid dehydrogenase (BCKD) kinase (abbreviated as BCK) down-regulates activity of the mammalian mitochondrial BCKD complex by reversible phosphorylation of the decarboxylase (E1b) component of the complex. The binding of BCK to the holotransacylase (E2b) core of the BC ... >> More

  The branched-chain alpha-keto acid dehydrogenase (BCKD) kinase (abbreviated as BCK) down-regulates activity of the mammalian mitochondrial BCKD complex by reversible phosphorylation of the decarboxylase (E1b) component of the complex. The binding of BCK to the holotransacylase (E2b) core of the BCKD complex results in the stimulation of BCK activity. Here we show that the lipoylated lipoic acid-bearing domain (lip-LBD) (residues 1-84) of E2b alone does not interact with BCK. However, lip-LBD constructs containing various lengths of the C-terminal hinge region of LBD are able to bind to BCK as measured by a newly developed solubility-based binding assay. Isothermal titration calorimetry measurements produced a dissociation constant of 8.06 x 10(-6) m and binding enthalpy of -3.68 kcal/mol for the interaction of BCK with a construct containing lip-LBD and the Glu-Glu-Asp-Xaa-Xaa-Glu sequence of the C-terminal hinge region of LBD. These thermodynamic parameters are similar to those obtained for binding of BCK to a lipoylated di-domain construct, which harbors LBD, the entire hinge region, and the downstream subunit-binding domain of E2b. Our data establish that the C-terminal hinge region of LBD containing the above negatively charged residues is essential for the interaction between the lip-LBD construct and BCK. << Less

  J Biol Chem 277:36905-36908(2002) [PubMed] [EuropePMC]

• Isolation of rabbit liver branched chain alpha-ketoacid dehydrogenase and regulation by phosphorylation.

  Paxton R., Harris R.A.

  J Biol Chem 257:14433-14439(1982) [PubMed] [EuropePMC]

• Mitochondrial alpha-ketoacid dehydrogenase kinases: a new family of protein kinases.

  Popov K.M., Hawes J.W., Harris R.A.

  Four mitochondrial protein kinases have been cloned. These proteins represent a new family of protein kinases, related by sequence to the bacterial protein kinases but by function to the eukaryotic serine protein kinases. Arg288 is required for recognition by BCKDK of the phosphorylation site on t ... >> More

  Four mitochondrial protein kinases have been cloned. These proteins represent a new family of protein kinases, related by sequence to the bacterial protein kinases but by function to the eukaryotic serine protein kinases. Arg288 is required for recognition by BCKDK of the phosphorylation site on the E1alpha subunit of the BCKDH complex. BCKDK inhibits the dehydrogenase activity of the BCKDH complex by introducing a negative charge into the active-site pocket of the E1 component. Protein starvation of rats induces an increase in the amount of BCKDK bound to the BCKDH complex. This causes inactivation of the BCKDH complex and conserves branched-chain amino acids for protein synthesis in the protein-starved state. Expression of the different PDK isoenzymes is tissue specific, and the different PDK isoenzymes are unique with respect to kinetic parameters for ATP and ADP and sensitivity to allosteric effectors (NADH, NAD+, coenzyme A, acetyl-CoA, pyruvate, and dichloroacetate). Preliminary experiments indicate that an increased amount of PDK2 protein partly explains the increase in PDK activity that occurs in rat liver in response to chemically induced diabetes. << Less

  Adv Second Messenger Phosphoprotein Res 31:105-111(1997) [PubMed] [EuropePMC]