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

R. Max Wynn, Jacinta L. Chuang, Cynthia D. Cote, David T. Chuang

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Abstract

We showed previously that the rat branched-chain α-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.

Original languageEnglish (US)
Pages (from-to)30512-30519
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number39
StatePublished - Sep 29 2000

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3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)
Rats
Conservation
Phosphotransferases
Adenosine Triphosphate
Histidine
Dimers
Protein Kinases
Isoenzymes
Monomers
Adenylyl Imidodiphosphate
Bacterial Proteins
Dynamic light scattering
Gel Chromatography
Adenosine Triphosphatases
Sucrose
Conformations
Urea

ASJC Scopus subject areas

  • Biochemistry

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Tetrameric assembly and conservation in the ATP-binding domain of rat branched-chain α-ketoacid dehydrogenase kinase. / Wynn, R. Max; Chuang, Jacinta L.; Cote, Cynthia D.; Chuang, David T.

In: Journal of Biological Chemistry, Vol. 275, No. 39, 29.09.2000, p. 30512-30519.

Research output: Contribution to journalArticle

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