Competitive Interaction of Component Enzymes with the Peripheral Subunit-Binding Domain of the Pyruvate Dehydrogenase Multienzyme Complex of <i>Bacillus stearothermophilus</i>:  Kinetic Analysis Using Surface Plasmon Resonance Detection

Lessard, Ivan A. D.; Fuller, Christopher; Perham, Richard N.

doi:10.1021/bi961683r

Cited by 36 publications

(56 citation statements)

References 40 publications

(81 reference statements)

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“…3, both PDH2 and PDH1 bind similarly to the PDHbinding domain of E2 as determined by surface plasmon resonance. Previously, a similar approach was employed to study the interaction of PDH with the subunit-binding domain of E2 from Bacillus stearothermophilus (27). In the B. stearothermophilus PDC, the subunit-binding domain of E2 binds both PDH and E3 components, whereas in the eukaryotic PDC only PDH is bound to this domain of E2, and E3 is bound to the E3-binding domain of BP.…”

Section: Discussionmentioning

confidence: 99%

“…L2S was immobilized on the CM5 chip through the lipoyl moieties by a surface thiol coupling method. For this purpose the following reagents were applied on the sensor chip surface at a flow rate of 5 l/min: 35 l of the 1:1 mixture of 100 mM N-hydroxysuccinimide and 100 mM N-ethyl-NЈ- [3-(dimethylamino)propyl]carbodiimide hydrochloride to activate the surface; 15 l of 40 mM cystamine in 100 mM sodium borate buffer, pH 8.5, to introduce disulfides; 20 l of 100 mM dithioerythritol in 100 mM sodium borate buffer, pH 8.5, to reduce disulfides to thiols; 60 l of L2S (50 g/ml) in 10 mM CH 3 COONa, pH 4.0, to couple the ligand (L2S); and 35 l of 20 mM 2-(2-pyridinyldithio)ethaneamine hydrochloride in 100 mM sodium borate buffer, pH 4.3, 1 M NaCl to deactivate excess thiol (27). PDH2 or PDH1 in the concentration range 0 -3000 nM was injected to interact with the sensor surface at 10 l/min in HBS buffer (10 mM HEPES, pH 7.4, 15 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20).…”

Section: Methodsmentioning

confidence: 99%

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Characterization of Testis-specific Isoenzyme of Human Pyruvate Dehydrogenase

Korotchkina

Sidhu

Patel

2006

Journal of Biological Chemistry

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Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the ␣ subunit of ␣ 2 ␤ 2 PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37°C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 ␣ subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1. Mammalian pyruvate dehydrogenase complex (PDC)2 plays a central role in glucose oxidation by catalyzing the oxidative decarboxylation of pyruvic acid with formation of carbon dioxide, acetyl-CoA, NADH, and H ϩ (for reviews see Refs. 1-3). PDC is composed of multiple copies of the following three catalytic components: (i) pyruvate dehydrogenase (PDH) catalyzing the decarboxylation of pyruvate and reductive acetylation of the lipoyl moieties of dihydrolipoamide acetyltransferase (E2); (ii) E2 transferring acetyl moiety to CoA; (iii) and dihydrolipoamide dehydrogenase (E3) reoxidizing the reduced lipoyl moieties of E2 with the reduction of NAD ϩ to NADH (1-3). Forty eight to 60 subunits of E2and 12 subunits of E3-binding protein (BP), a noncatalytic component of mammalian PDC, form the central core of the multienzyme PDC, which binds 20 -30 tetramers of PDH, 6 -12 dimers of E3 and two regulatory enzymes, 1-2 copies of pyruvate dehydrogenase kinase (PDK), and 2-3 copies of pyruvate dehydrogenase phosphatase (PDP) (4, 5). PDK and PDP provide the finely tuned regulation of activity of PDC through reversible phosphorylation-dephosphorylation of PDH (and hence PDC) depending on cellular demand for glucose oxidation. Mammalian PDH is a tetramer consisting of two ␣ and two ␤ subunits. The hum...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Characterization of Testis-specific Isoenzyme of Human Pyruvate Dehydrogenase

Korotchkina

Sidhu

Patel

2006

Journal of Biological Chemistry

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“…2C). We do not attribute the size difference to a variable extent of E 1 binding to the E 2 core because the binding interaction between E 1 and the E 1 -binding domain of E 2 is very tight (K d ϭ 0.3 nM) (17) and the E 1 -binding sites were saturated. Moreover, the averaged protein density in the outer shell of the smaller structure (Fig.…”

Section: Comparison Of the 3d Reconstructions Representative Of The Smentioning

confidence: 99%

3D electron microscopy reveals the variable deposition and protein dynamics of the peripheral pyruvate dehydrogenase component about the core

Gu¹,

Zhou²,

McCarthy³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Cryo-electron microscopy was exploited to reveal and study the influence of pyruvate dehydrogenase (E 1) occupancy on the conformational states of the Saccharomyces cerevisiae pyruvate dehydrogenase complex (PDC). Structures representative of PDC preparations with Ϸ40% and full E 1 occupancy were determined after the electron microscopy images from each preparation were classified according to their sizes. The reconstructions derived from two size groups showed that the deposition of the E 1 molecules associated with the larger complex is, unexpectedly, not icosahedrally arranged, whereas in the smaller complex the E 1 molecules have an arrangement and architecture similar to their more ordered deposition in the WT bovine kidney PDC. This study also shows that the linker of dihydrolipamide acetyltransferase (E 2) that tethers E1 to the E2 core increases in length from Ϸ50 to 75 Å, accounting largely for the size difference of the smaller and larger structures, respectively. Extensive E 1 occupancy of its 60 E2 binding sites favors the extended conformation of the linker associated with the larger complex and appears to be related to the loss of icosahedral symmetry of the E 1 molecules. However, the presence of a significant fraction of larger molecules also in the WT PDC preparation with low E1 occupancy indicates that the conformational variability of the linker contributes to the overall protein dynamics of the PDC and the variable deposition of E1. The flexibility of the complex may enhance the catalytic proficiency of this macromolecular machine by promoting the channeling of the intermediates of catalysis between the active sites.A central feature of eukaryotic pyruvate dehydrogenase complexes (PDCs) is a 60-mer core with the morphology of a pentagonal dodecahedron (1-4). The dihydrolipoamide acetyltransferase (E 2 ) component serves as a scaffold to which the pyruvate dehydrogenase (E 1 ) and dihydrolipoamide dehydrogenase (E 3 ) components are attached. The E 2 dodecahedron consists of sets of three tightly bound subunits at each of its 20 vertices. The trimers are interconnected by 30 tenuous and flexible bridges that enable the core to breathe as evidenced by its size variability of Ϸ17% (240-280 Å in diameter) at room temperature (5).3D reconstructions of subcomplexes of the Saccharomyces cerevisiae E 2 core have revealed that 12 E 3 components are attached by a binding protein (BP) inside the 12 pentagonal openings of the cage-like E 2 (6), whereas the E 1 components form a shell that surrounds the underlying E 2 core (7-9). The reconstructions show that the 60 E 2 subunits are organized in 20 trimers. Each E 2 subunit contains an Ϸ50-Å-long linker that anchors an E 1 tetramer. Three of these linkers emanate from the outside edges of the triangular-shaped base of the E 2 trimer and form a cage around its base that may shelter the lipoyl domains of E 2 and the E 1 and E 2 active sites. We proposed that the lipoyl domain and its tether (swinging arm) rotates about the E 1 -binding domain of E 2 , which...

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“…The ThDP-dependent E1 component of the PDH multienzyme complex from Bacillus stearothermophilus has been intensively studied in this context (10,(12)(13)(14)(15). We have now solved the crystal structure of the subcomplex formed between the heterotetrameric (" 2 $ 2 ) B. stearothermophilus E1 and the peripheral subunit-binding domain (PSBD) from the lipoate acetyltransferase (E2) chain of the PDH complex (16).…”

mentioning

confidence: 99%

A Molecular Switch and Proton Wire Synchronize the Active Sites in Thiamine Enzymes

Frank

Titman

Pratap

et al. 2004

Science

Self Cite

172

186

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Thiamine diphosphate (ThDP) is used as a cofactor in many key metabolic enzymes. We present evidence that the ThDPs in the two active sites of the E1 (EC 1.2.4.1) component of the pyruvate dehydrogenase complex communicate over a distance of 20 angstroms by reversibly shuttling a proton through an acidic tunnel in the protein. This "proton wire" permits the co-factors to serve reciprocally as general acid/base in catalysis and to switch the conformation of crucial active-site peptide loops. This synchronizes the progression of chemical events and can account for the oligomeric organization, conformational asymmetry, and "ping-pong" kinetic properties of E1 and other thiamine-dependent enzymes.

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Competitive Interaction of Component Enzymes with the Peripheral Subunit-Binding Domain of the Pyruvate Dehydrogenase Multienzyme Complex of Bacillus stearothermophilus: Kinetic Analysis Using Surface Plasmon Resonance Detection

Cited by 36 publications

References 40 publications

Characterization of Testis-specific Isoenzyme of Human Pyruvate Dehydrogenase

Characterization of Testis-specific Isoenzyme of Human Pyruvate Dehydrogenase

3D electron microscopy reveals the variable deposition and protein dynamics of the peripheral pyruvate dehydrogenase component about the core

A Molecular Switch and Proton Wire Synchronize the Active Sites in Thiamine Enzymes

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