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Conservation of Protein Domains Over Different Proteins

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A Guide to Production, Crystallization, and Structure Determination of Human IKK1/α
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Tetrameric assembly and conservation in the ATP-binding domain of rat branched-chain alpha-ketoacid dehydrogenase

R M Wynn1, J L Chuang, C D Cote

  • 1Departments of Internal Medicine and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. wynn@utsw.swmed.edu

The Journal of Biological Chemistry
|July 21, 2000
PubMed
Summary
This summary is machine-generated.

Rat branched-chain alpha-ketoacid dehydrogenase (BCKD) kinase forms a tetramer, unlike related enzymes. This tetrameric structure is crucial for its ATP binding and autophosphorylation activity.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Structure

Background:

  • Rat branched-chain alpha-ketoacid dehydrogenase (BCKD) kinase can autophosphorylate.
  • BCKD kinase shares sequence similarity with bacterial histidine protein kinases but does not function as one.

Purpose of the Study:

  • To investigate the quaternary structure of rat BCKD kinase.
  • To determine the functional significance of its oligomeric state.
  • To elucidate the relationship between BCKD kinase and the ATPase/kinase superfamily.

Main Methods:

  • Gel filtration and dynamic light scattering to determine molecular weight and oligomeric state.
  • Sucrose density gradient centrifugation to analyze dissociation.
  • Analysis of ATP-binding sites and effects of mutations in the ATP-binding domain.

Main Results:

  • Rat BCKD kinase exists as a homotetramer (185,000 M(r)), contrasting with homodimeric mammalian pyruvate dehydrogenase kinase isozymes.
  • The tetramer possesses four 5'-adenylyl-imidodiphosphate-binding sites.
  • Urea treatment dissociates the tetramer into dimers and monomers; only tetramers and dimers retain ATP-binding and autophosphorylation capabilities.
  • Interaction with lipoylated transacylase is salt-sensitive.
  • Mutations in the ATP-binding domain significantly impair catalytic efficiency.

Conclusions:

  • Rat BCKD kinase functions as a tetramer, essential for its catalytic activity.
  • The enzyme belongs to the ATPase/kinase superfamily, similar to pyruvate dehydrogenase kinase isozymes.