Sizing of bovine heart and kidney pyruvate dehydrogenase complex and dihydrolipoyl transacetylase core by quasielastic light scattering

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 (K d Ӎ 175 M for 2 L2/PDK2) but much tighter with dimeric glutathione S-transferase (GST)-L2 (K d Ӎ 3 M), supporting the importance of bifunctional binding. Reduction of lipoyl groups resulted in ϳ8-fold tighter binding of PDK2 to GST-L2 red , which was ϳ300-fold tighter than binding of 2 L2 red and also much tighter than binding by GST-L1 red and GST-L3 red . The E2 60-mer bound ϳ18 PDK2 dimers with a K d similar to GST-L2. E2⅐E1 bound more PDK2 (ϳ27.6) than E2 with ϳ2-fold tighter affinity. Lipoate reduction fostered somewhat tighter binding at more sites by E2 and severalfold tighter binding at the majority of sites on E2⅐E1. ATP and ADP decreased the affinity of PDK2 for E2 by 3-5-fold and adenosine 5-(␤,␥-imino)triphosphate or phosphorylation of E1 similarly reduced PDK2 binding to E2⅐E1. 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.The mitochondrial pyruvate dehydrogenase complex (PDC) 1 catalyzes the irreversible conversion of pyruvate to acetyl-CoA along with the reduction of NAD ϩ . Mammalian PDC has a highly organized structure in which the dihydrolipoyl acetyltransferase (E2) component plays a central role in the organization, integrated chemical reactions, and regulation of the complex (1-4). The other components of the complex, required for the overall reaction, include the pyruvate dehydrogenase (E1) component, the dihydrolipoyl dehydrogenase (E3) component, and the E3-binding protein (E3BP). The PDC reaction is regulated by a phosphorylation-dephosphorylation cycle, which is carried out by dedicated kinase and phosphatase components. The desired control in different organs is achieved by the selective expression and the novel regulation of at least four pyruvate dehydrogenase kinase (PDK) isozymes and at least two pyruvate dehydrogenase phosphatase (PDP) isoforms (Refs. 3-11 and references therein). The kinases act to inactivate PDC and the phosphatases to reactivate PDC by phosphorylation and dephosphorylation of the E1 component.The PDKs and the related kinase regulating the branchedchain ␣-keto acid dehydrogenase complex are unrelated to cytoplasmic Ser/Thr/Tyr kin...

Section: Methodssupporting

confidence: 61%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

Hiromasa

Roche

2003

“…At its N terminus, t-E2 includes an 11-amino acid segment (964.5 Da) of the C-terminal end of linker region preceding the I domain of E2; this extra structure is presumably located outside the dodecahedron and contributes modestly to the hydrodynamic diameter of t-E2. The appreciably larger f/f 0 (and R s ) of the E2 60-mer than t-E2 60-mer is consistent with the linker connected binding and lipoyl domains creating significant drag due its propensity to extend out from the inner core (24,53,54).…”

Section: Fig 3 Sedimentation Profiles For E2 and E2⅐e3bp The G(s*)supporting

confidence: 54%

Organization of the Cores of the Mammalian Pyruvate Dehydrogenase Complex Formed by E2 and E2 Plus the E3-binding Protein and Their Capacities to Bind the E1 and E3 Components

Hiromasa

Fujisawa

Aso

et al. 2004

113

143

The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the Cterminal I domain of E2 at the vertices of a dodecahedron. Exterior to this inner core structure, E2 has a pyruvate dehydrogenase component (E1)-binding domain followed by two lipoyl domains, all connected by mobile linker regions. The assembled core structure of mammalian pyruvate dehydrogenase complex also includes the dihydrolipoyl dehydrogenase (E3)-binding protein (E3BP) that binds the I domain of E2 by its C-terminal I domain. E3BP similarly has linker regions connecting an E3-binding domain and a lipoyl domain. The composition of E2⅐E3BP was thought to be 60 E2 plus ϳ12 E3BP. We have prepared homogenous human components. E2 and E2⅐E3BP have s 20,w values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2⅐E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2⅐E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound ϳ60 E1 and maximally sedimented 64.4 ؎ 1.5 S faster than E2, whereas E1-saturated E2⅐E3BP maximally sedimented 49.5 ؎ 1.4 S faster than E2⅐E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (ϳ12) were bound by E2⅐E3BP than by E2. The findings of a smaller E2⅐E3BP mass and a lower capacity to bind E1 support the smaller E3BP substituting for E2 subunits rather than adding to the 60-mer. We describe a substitution model in which 12 I domains of E3BP replace 12 I domains of E2 by forming 6 dimer edges that are symmetrically located in the dodecahedron structure. Twelve E3 dimers were bound per E2 48 ⅐E3BP 12 mass, which is consistent with this model.The mitochondrial pyruvate dehydrogenase complex (PDC) 1 catalyzes the irreversible conversion of pyruvate to acetyl-CoA along with the reduction of NAD ϩ . PDCs from all known sources contain the pyruvate dehydrogenase (E1), the dihydrolipoyl acetyltransferase (E2), and the dihydrolipoyl dehydrogenase (E3) components. Mammalian PDC has a highly organized structure in which the E2 component plays a central role in the organization, integrated chemical reactions, and regulation of the complex (1-5). Besides those universal components, the mammalian and subsequently some other eukaryotic PDCs were shown to contain another component, called E3-binding protein (E3BP); this protein was originally designated protein X (6, 7). Mammalian E3BP was first characterized as a component with a reactive lipoyl group on a single lipoyl domain (7-11) that, alone, could support the overall reaction (12). E3BP was tightly bound to E2 (7) by its C-terminal region (10). The E3BP component was then shown to contribute to the organization of the complex by binding the E3 component (13)(14)(15)(16)(17)(18). This work provides new insights into the integration of E3BP into the central framework of the mammalian complex.Although first characterized in the bo...

“…2 Indeed, dissociation constants for E1-lipoyl domain interactions appear to be much higher than the K m of E1 for lipoyl domain substrates. A weak interaction is probably a desirable feature for sustaining high efficiency since the flexibly held lipoyl domains are concentrated in mammalian PDC at Ͼ1.0 mM (12,13) next to E1 and should, therefore, efficiently ferry lipoyl groups into the active site of E1.…”

mentioning

confidence: 99%

Specificity Determinants for the Pyruvate Dehydrogenase Component Reaction Mapped with Mutated and Prosthetic Group Modified Lipoyl Domains

Gong

Peng

Yakhnin

et al. 2000

Efficient catalysis in the second step of the pyruvate dehydrogenase (E1) component reaction requires a lipoyl group to be attached to a lipoyl domain that displays appropriately positioned specificity residues. As substrates, the human dihydrolipoyl acetyltransferase provides an N-terminal (L1) and an inner (L2) lipoyl domain. We evaluated the specificity requirements for the E1 reaction with 27 mutant L2 (including four substitutions for the lipoylated lysine, Lys 173 ), with three analogs substituted for the lipoyl group on Lys 173, and with selected L1 mutants. Besides Lys 173 mutants, only E170Q mutation prevented lipoylation. Based on analysis of the structural stability of mutants by differential scanning calorimetry, alanine substitutions of residues with aromatic side chains in terminal regions outside the folded portion of the L2 domain significantly decreased the stability of mutant L2, suggesting specific interactions of these terminal regions with the folded domain. E1 reaction rates were markedly reduced by the following substitutions in the L2 domain (equivalent site-L1): L140A, S141A (S14A-L1), T143A, E162A, D172N, and E179A (E52A-L1). These mutants gave diverse changes in kinetic parameters. These residues are spread over >24 Å on one side of the L2 structure, supporting extensive contact between E1 and L2 domain. The pyruvate dehydrogenase complex (PDC) 1 oxidatively decarboxylates pyruvate with the production of acetyl-CoA and NADH. The PDC reaction results from the sequential catalysis by the pyruvate dehydrogenase (E1), the dihydrolipoyl acetyltransferase (E2), and the dihydrolipoyl dehydrogenase (E3) components. In the intermediate steps, the lipoyl groups on mobile lipoyl domains of the E2 are reductively acetylated by E1, de-acetylated by E2, and re-oxidized by E3. Thus, lipoyl domains play a vital role in connecting the sequential series of reaction catalyzed by these three components. In mammalian PDC, two lipoyl domains, L1 and L2, and an E1-binding domain are provided in the outer structure of the E2 (1, 2). These flexibly held domains surround a pentagonal dodecahedron shaped inner core formed by association of 60 C-terminal catalytic domains of E2. The capacity of lipoyl domains to rapidly move between active sites is provided by highly flexible interdomain linker segments which are located between the globular domains of E2. In mammalian PDC-E2 these linker regions are 20 to 30 amino acids in length and are high in alanine and proline residues; prolines introduce stiffness in these interdomain segments (3). Mammalian PDC has a third lipoyl domain on an E3-binding protein (E3BP) which has a similar segmented structure (1).In the first step of the E1 reaction, the bound thiamin diphosphate (TPP) forms an adduct with pyruvate which is rapidly decarboxylated to generate 2-(1-hydroxyethylidene)-TPP (HE-TPP) (4). In the second step, reductive acetylation of a lipoyl group is achieved by linking the oxidation of the hydroxyethylidene group to the reduction of the disulfide in the dithiola...