Ligand-induced effects on pyruvate dehydrogenase kinase isoform 2

Yasuaki Hiromasa, Liangyan Hu, Thomas E. Roche

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17 Citations (Scopus)

Abstract

Tryptophan fluorescence was used to analyze binding of ligands to human pyruvate dehydrogenase isoform 2 (PDHK2) and to demonstrate effects of ligand binding on distal structure of PDHK2 that is required for binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase. Ligand-altered binding of PDHK2 to L2 and effects of specific ligands on PDHK2 oligomeric state were characterized by analytical ultracentrifugation. ATP, ADP, and pyruvate markedly quenched the tryptophan fluorescence of PDHK2 and gave maximum quenching/ L0.5 estimates: ∼53%/3 μM for ATP; ∼49%/15 μM for ADP; and ∼71%/∼590 μM for pyruvate. The conversion of Trp-383 to phenylalanine completely removed ATP- and ADP-induced quenching and ≥80% of the absolute decrease in fluorescence due to pyruvate. The W383F-PDHK2 mutant retained high catalytic activity. Pyruvate, added after ADP, quenched Trp fluorescence with an L0.5 of 3.4 μM pyruvate, ≥150-fold lower concentration than needed with pyruvate alone. ADP-enhanced binding of pyruvate was maintained with W383F-PDHK2. Binding of PDHK2 dimer to L2 is enhanced when L2 are housed in oligomeric structures, including the glutathione S-transferase (GST)-L2 dimer, and further strengthened by reduction of the lipoyl groups (GST-L2red) (Hiromasa and Roche (2003) J. Biol. Chem. 278, 33681-33693). Binding of PDHK2 to GST-L2red was modestly hindered by 200 μM level of ATP or ADP or 5.0 mM pyruvate; a marked change to nearly complete prevention of binding was observed with ATP or ADP plus pyruvate at only 100 μM levels, and these conditions caused PDHK2 dimer to associate to a tetramer. These changes should make major contributions to synergistic inhibition of PDHK2 activity by ADP and pyruvate. Ligand-induced changes that interfere with PDHK2 binding to GST-L2red may involve release of an interdomain cross arm between PDHK2 subunits in which Trp-383 plays a critical anchoring role.

Original languageEnglish
Pages (from-to)12568-12579
Number of pages12
JournalJournal of Biological Chemistry
Volume281
Issue number18
DOIs
Publication statusPublished - May 5 2006
Externally publishedYes

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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