Facilitated Interaction between the Pyruvate Dehydrogenase Kinase Isoform 2 and the Dihydrolipoyl Acetyltransferase

Yasuaki Hiromasa, Thomas E. Roche

研究成果: ジャーナルへの寄稿学術誌査読

26 被引用数 (Scopus)


The dihydrolipoyl acetyltransferase (E2) has an enormous impact on pyruvate dehydrogenase kinase (PDK) phosphorylation of the pyruvate dehydrogenase (E1) component by acting as a mobile binding framework and in facilitating and mediating regulation of PDK activity. Analytical ultracentrifugation (AUC) studies established that the soluble PDK2 isoform is a stable dimer. The interaction of PDK2 with the lipoyl domains of E2 (L1, L2) and the E3-binding protein (L3) were characterized by AUC. PDK2 interacted very weakly with L2 (Kd ≃ 175 μM for 2 L2/PDK2) but much tighter with dimeric glutathione S-transferase (GST)-L2 (Kd ≃ 3 μM), supporting the importance of bifunctional binding. Reduction of lipoyl groups resulted in ∼8-fold tighter binding of PDK2 to GST-L2red, which was ∼300-fold tighter than binding of 2 L2red and also much tighter than binding by GST-L1red and GST-L3red. The E2 60-mer bound ∼18 PDK2 dimers with a Kd similar to GST-L2. E2∼E1 bound more PDK2 (∼27.6) than E2 with ∼2-fold tighter affinity. Lipoate reduction fostered somewhat tighter binding at more sites by E2 and severalfold tighter binding at the majority of sites on E2·E1. ATP and ADP decreased the affinity of PDK2 for E2 by 3-5-fold and adenosine 5′-(β,γ-imino)triphosphate or phosphorylation of E1 similarly reduced PDK2 binding to E2·-E1. Reversible bifunctional binding to L2 with the mandatory singly held transition fits the proposed " hand-over-hand" movement of a kinase dimer to access E1 without dissociating from the complex. The gain in binding interactions upon lipoate reduction likely aids reduction-engendered stimulation of PDK2 activity; loosening of binding as a result of adenine nucleotides and phosphorylation may instigate movement of lipoyl domain-held kinase to a new E1 substrate.

ジャーナルJournal of Biological Chemistry
出版ステータス出版済み - 9月 5 2003

!!!All Science Journal Classification (ASJC) codes

  • 生化学
  • 分子生物学
  • 細胞生物学


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