Critical Role of a Lipoyl Cofactor of the Dihydrolipoyl Acetyltransferase in the Binding and Enhanced Function of the Pyruvate Dehydrogenase Kinase

Radke, Gary A.; Ôno, Kazuo; Ravindran, Sundari; Roche, Thomas E.

doi:10.1006/bbrc.1993.1146

Cited by 40 publications

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“…Nevertheless, PDHK3 binds the L2 domain Ͼ100-fold tighter than PDHK2 (15). 6 There are major differences in the sequences of the C-terminal regions beyond the cross arm in these two isozymes. In the PDHK3⅐L2 structure (37), the final 17 residues at the C terminus of PDHK3 (beyond Trp-381) contribute to the binding of L2 by interactions with the lipoyl domain and lipoyl group.…”

Section: Discussionmentioning

confidence: 99%

Ligand-induced Effects on Pyruvate Dehydrogenase Kinase Isoform 2

Hiromasa

Roche

2006

Journal of Biological Chemistry

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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/L 0.5 estimates: ϳ53%/3 M for ATP; ϳ49%/15 M for ADP; and ϳ71%/ϳ590 M 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 L 0.5 of 3.4 M 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-L2 red ) (Hiromasa and Roche (2003) J. Biol. Chem. 278, 33681-33693). Binding of PDHK2 to GST-L2 red was modestly hindered by 200 M 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 M 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-L2 red may involve release of an interdomain cross arm between PDHK2 subunits in which Trp-383 plays a critical anchoring role.Decarboxylation of pyruvate by the mitochondrial pyruvate dehydrogenase complex (PDC) 2 commits glucose and glucose-linked substrates to being either fully oxidized or converted to fatty acids for storage.Because this results in animals in the irrecoverable reduction of body carbohydrate reserves, PDC activity is tightly regulated by dedicated regulatory enzymes (1, 2). The pyruvate dehydrogenase kinase isozymes (PDHK1, PDHK2, PDHK3, and PDHK4) and pyruvate dehydrogenase phosphatase isozymes (PDHP1 and PDHP2) control the fractional activity of PDC in a tissue-specific manner. Phosphorylation of the pyruvate dehydrogenase (E1) component by the PDHKs results in inactivation and dephosphorylation by the PDHPs results in reactivation of E1.The components required in the mammalian complex for the five-step PDC reaction are E1, the dihydrolipoyl acetyltransferase (E2), the dihydrolipoyl dehydrogenase (E3), and the E3-binding protein (E3BP) (1, 2). E2 and E3BP associate via their C-terminal domains to form the pentagonal-dodecahedron shaped core of the complex, a 60-subunit structure with a stoichiometry estimated to be E2 48 E3...

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Section: Discussionmentioning

confidence: 99%

Ligand-induced Effects on Pyruvate Dehydrogenase Kinase Isoform 2

Hiromasa

Roche

2006

Journal of Biological Chemistry

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“…It is generally believed that only pyruvate directly regulates the kinase activity through a dedicated allosteric site. The effects of NAD ϩ /NADH and CoA/acetyl-CoA are mediated by the oxidation, reduction, and acetylation state of the lipoyl group (4). Structurally, this domain is an 80-amino acid, free-folding domain in the N-terminal region of E2.…”

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confidence: 99%

Structure of Pyruvate Dehydrogenase Kinase

Steussy

Popov

Bowker-Kinley

et al. 2001

Journal of Biological Chemistry

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The structure of mitochondrial pyruvate dehydrogenase kinase isozyme 2 is of interest because it represents a family of serine-specific protein kinases that lack sequence similarity with all other eukaryotic protein kinases. Similarity exists instead with key motifs of prokaryotic histidine protein kinases and a family of eukaryotic ATPases. The 2.5-Å crystal structure reported here reveals that pyruvate dehydrogenase kinase isozyme 2 has two domains of about the same size. The N-terminal half is dominated by a bundle of four amphipathic ␣-helices, whereas the C-terminal half is folded into an ␣/␤ sandwich that contains the nucleotide-binding site. Analysis of the structure reveals this C-terminal domain to be very similar to the nucleotidebinding domain of bacterial histidine kinases, but the catalytic mechanism appears similar to that of the eukaryotic serine kinases and ATPases.

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“…The difference in specific activities is therefore attributed either to PDK properties or docking site(s) to the dihydrolipoamide acetyltransferase (E2) core complex. Reconstitutions of mammalian PDCs have shown a 3-5-fold decrease in PDK activity (inactivation of PDC) in the absence of the E2 core [35][36][37]. This observation was explained by a proximity model in which the independent binding of E1 and PDK by the E2 core couples E1α phosphorylation.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, monocots such as maize possess a single mitochondrial E2 that contains one lipoyl domain [21,38]. Because mammalian PDKs have been shown to bind to the E2 core via the lipoyl domains [16,36,37], perhaps the divergence between monocot and dicot E2 cores hinders AtPDK binding to maize PDC, resulting in a lower specific activity in itro. Steric hindrance by the bulky fusion partner (MBP) might also contribute to the decreased activity.…”

Section: Discussionmentioning

confidence: 99%

Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues

Thelen

Miernyk

Randall

2000

Biochemical Journal

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Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Although PDKs inactivate mitochondrial PDC by phosphorylating specific Ser residues, the primary amino acid sequence indicates that they are more closely related to prokaryotic His kinases than to eukaryotic Ser/Thr kinases. Unlike Ser/Thr kinases, His kinases use a conserved His residue for phosphotransfer to Asp residues. To understand these unique kinases better, a presumptive PDK from Arabidopsis thaliana was heterologously expressed and purified for this investigation. Purified, recombinant A. thaliana PDK could inactivate kinase-depleted maize mitochondrial PDC by phosphorylating Ser residues. Additionally, A. thaliana PDK was capable of autophosphorylating Ser residues near its N-terminus, although this reaction is not part of the phosphotransfer pathway. To elucidate the mechanism involved, we performed site-directed mutagenesis of the canonical His residue likely to be involved in phosphotransfer. When His-121 was mutated to Ala or Gln, Ser-autophosphorylation was decreased by 50% and transphosphorylation of PDC was decreased concomitantly. We postulate that either (1) His-121 is not the sole phosphotransfer His residue or (2) mutagenesis of His-121 exposes an additional otherwise cryptic phosphotransfer His residue. Thus His-121 is one residue involved in kinase function.

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Critical Role of a Lipoyl Cofactor of the Dihydrolipoyl Acetyltransferase in the Binding and Enhanced Function of the Pyruvate Dehydrogenase Kinase

Cited by 40 publications

References 0 publications

Ligand-induced Effects on Pyruvate Dehydrogenase Kinase Isoform 2

Ligand-induced Effects on Pyruvate Dehydrogenase Kinase Isoform 2

Structure of Pyruvate Dehydrogenase Kinase

Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues

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