Information on EC 1.2.5.1 - pyruvate dehydrogenase (quinone)

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.2.5.1
-
RECOMMENDED NAME
GeneOntology No.
pyruvate dehydrogenase (quinone)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
pyruvate + ubiquinone + H2O = acetate + CO2 + ubiquinol
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
pyruvate to cytochrome bd terminal oxidase electron transfer
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pyruvate to cytochrome bo oxidase electron transfer
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acetate fermentation
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Pyruvate metabolism
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SYSTEMATIC NAME
IUBMB Comments
pyruvate:ubiquinone oxidoreductase
Flavoprotein (FAD) [1]. This bacterial enzyme is located on the inner surface of the cytoplasmic membrane and coupled to the respiratory chain via ubiquinone [2,3]. Does not accept menaquinone. Activity is greatly enhanced by lipids [4,5,6]. Requires thiamine diphosphate [7]. The enzyme can also form acetoin [8].
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
pyruvate + a quinone
acetate + CO2 + a quinol
show the reaction diagram
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-
-
-
?
pyruvate + ferricyanide
acetate + CO2 + ferrocyanide
show the reaction diagram
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activity assayed photometrically by monitoring the reduction of 2,6-dichloroindophenol
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-
?
pyruvate + ferricyanide + H2O
acetate + CO2 + ferrocyanide
show the reaction diagram
pyruvate + ferricytochrome b1 + H2O
acetate + CO2 + ferrocytochrome b1
show the reaction diagram
pyruvate + oxidized 2,6-dichloroindophenol
acetate + CO2 + reduced 2,6-dichloroindophenol
show the reaction diagram
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activity assayed photometrically by monitoring the reduction of 2,6-dichloroindophenol, pH 6.0, 40°C
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-
?
pyruvate + oxidized 2,6-dichloroindophenol + H2O
acetate + CO2 + reduced 2,6-dichloroindophenol
show the reaction diagram
-
-
-
-
?
pyruvate + ubiquinol-6 + H2O
acetate + CO2 + ubiquinol-6
show the reaction diagram
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the natural electron acceptor for the reduced enzyme is a cell-membrane-associated electron transport system including both ubiquinone-6 and cytochrome b1, with oxygen being the terminal electron acceptor
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-
?
pyruvate + ubiquinone + H2O
acetate + CO2 + ubiquinol
show the reaction diagram
pyruvate + ubiquinone-30 + H2O
acetate + CO2 + ubiquinol-30
show the reaction diagram
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ubiquinone-30 is rapidly reduced by pyruvate oxidase only in the presence of palmitic acid
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-
?
pyruvate + ubiquinone-6 + H2O
acetate + CO2 + ubiquinol-6
show the reaction diagram
-
-
-
-
?
pyruvate + ubiquinone-8
acetate + CO2 + ubiquinol-8
show the reaction diagram
pyruvate + ubiquinone-8 + H2O
acetate + CO2 + ubiquinol-8
show the reaction diagram
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
pyruvate + a quinone
acetate + CO2 + a quinol
show the reaction diagram
-
-
-
-
?
pyruvate + ferricytochrome b1 + H2O
acetate + CO2 + ferrocytochrome b1
show the reaction diagram
pyruvate + ubiquinol-6 + H2O
acetate + CO2 + ubiquinol-6
show the reaction diagram
-
the natural electron acceptor for the reduced enzyme is a cell-membrane-associated electron transport system including both ubiquinone-6 and cytochrome b1, with oxygen being the terminal electron acceptor
-
-
?
pyruvate + ubiquinone + H2O
acetate + CO2 + ubiquinol
show the reaction diagram
pyruvate + ubiquinone-6 + H2O
acetate + CO2 + ubiquinol-6
show the reaction diagram
-
-
-
-
?
pyruvate + ubiquinone-8
acetate + CO2 + ubiquinol-8
show the reaction diagram
pyruvate + ubiquinone-8 + H2O
acetate + CO2 + ubiquinol-8
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
thiamine diphosphate
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
alpha-chymotrypsin
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20 micromol/ml, wild-type enzyme and the mutant poxB4 are activated 15- and 70fold, respectively, and the mutant poxB3 is activated 14fold
Anionic detergents
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the enzyme activity is stimulated 20- to 50fold, if the enzyme is removed from the membrane particulate fraction of the cell by incubation with a wide variety of amphiphiles
Cationic detergents
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the enzyme activity is stimulated 20- to 50fold, if the enzyme is removed from the membrane particulate fraction of the cell by incubation with a wide variety of amphiphiles
Mg2+
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as cofactor
Mn2+
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divalent metal ion required
SDS
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20 microM, activates the wild-type and mutant enzymes, poxB3 and poxB4, 15-, 20-, and 70fold, respectively
Triton X-100
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2.2 mM, activates the wild-type and mutant enzymes, poxB3 and poxB4, 32-, 4-, and 6fold, respectively. With the addition of alpha-chymotrypsin the activation of the mutant enzymes poxB3 and poxB4 is increased 14- and 39fold, respectively
Zwitterionic detergents
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the enzyme activity is stimulated 20- to 50fold, if the enzyme is removed from the membrane particulate fraction of the cell by incubation with a wide variety of amphiphiles
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
cis-12-hydroxy-9-octadecenoic acid
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119% of the activitation with palmitic acid
elaidic acid
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122% of the activitation with palmitic acid
lauric acid
lecithin
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the hydrophobic moieties of lecithin activate pyruvate oxidase whereas the hydrophilic portions of the molecule have no stimulatory effect
linoleic acid
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116% of the activitation with palmitic acid
linolelaidic acid
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113% of the activitation with palmitic acid
linolenic acid
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126% of the activitation with palmitic acid
Lipids
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enzyme is activated by lipids, high affinity binding site
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lysophosphatidylethanolamine
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highest stimulating activity among the phospholipid extracted from cell membranes tested, if the phospholipids are added directly to the assay mixtures. When water-soluble micellar preparations are substituted for direct addition of the phospholipid to the assay, all the phosphatides demonstrate higher specific activities for stimulating pyruvate oxidase, and the differences in their stimulating capacity are minimized
myristic acid
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109% of the activitation with palmitic acid
n-nonanoic acid
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42% of the activitation with palmitic acid
oleic acid
palmitic acid
palmitoleic acid
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137% of the activitation with palmitic acid
trans-12-hydroxy-9-octadecenoic acid
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103% of the activitation with palmitic acid
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
60 - 883
ferricyanide
167
pyruvate
Escherichia coli
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pH and temperature not specified in the publication
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
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mutant poxB4 in presence of 20 microM SDS, pH and temperature not specified in the publication
90
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wild-type enzyme, pH and temperature not specified in the publication
270
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270 micromol of ferricyanide min-1 mg-1 of flavoprotein subunit, 25°C, pH not specified in the publication
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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the pyruvate oxidase system and the electron transport system are associated with the cell envelope-membrane fraction
Manually annotated by BRENDA team
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cell membrane-associated
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Manually annotated by BRENDA team
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inner surface of the cytoplasmic membrane and is coupled to the Escherichia coli aerobic respiratory chain
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Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Escherichia coli (strain K12)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60000
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the lauric acid-labeled enzyme is not digested neither by trypsin nor alpha-chymotrypsin in the presence of 0.1% SDS. Effective digestion is achieved by thermolysin, to a 45000 and a 15000 Da fragment
240000
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PAGE
265000
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determined by the Archibald method
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 62018, calculated
tetramer
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4 * 62000, SDS-PAGE
additional information
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when two poxB gene alleles coexist in cells either on a single plasmid or on two compatible plasmids, heterotetrameric species are formed in addition to homotetramers. The concentration of tetramer species varies according to the concentrations of the different subunit present. The distribution of each tetramer species seems virtually identical to those theoretically expected based on random mixing. The intrinsic activity of pyruvate oxidase is not affected by interactions among the four subunits. Each subunit of the tetramer catalyzes the oxidase reaction independently
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
the crystalline enzyme does not contain thiamine diphosphate but has an absolute thiamine diphosphate requirement for the reduction of the enzyme-bound FAD
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DEAE-Sephadex, ammonium sulfate precipitation, dialysis
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purified to homogeneity in the absence of detergents
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the enzyme is purified from an Escherichia coli strain CG3 harboring a plasmid carrying a plasmid th eoxidase gene
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the poxB4 mutant is purified from the strain CG3, carrying plasmid pYYC16, which overproduces the enzyme about 20fold. Purified on DEAE-cellulose, >90% pure
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli strain CG3
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mutant genes, poxB3 and posB4, are cloned on plasmid pBR322
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
systematical substitution of cysteine at 18 amino acid positions within the C-terminal region to obtain a panel of proteins each having a single residue changed to cysteine. In the absence of pyruvate, the cysteine residues of the modified PoxB proteins fail to form disulfide bonds, generally fail to react with a large and rigid hydrophilic sulfhydryl reagent, 4-acetamido-4'-[(iodoacetyl)amino]stilbene-2,2'-disulfonic acid (IASD), and in some cases react weakly with a smaller more hydrophobic reagent, N-ethylmaleimide. In this conformation, the C termini appear fixed in a rigid environment having limited exposure to solvent. In the presence of pyruvate, all of the C-terminal cysteine residues, except the two most distal from the C terminus, react with both sulfhydryl reagents and readily formed disulfide cross-linked species. In the presence of lipid activators, Triton X-100 or dipalmitoylphosphatidylglycerol, a subset of the cysteine-substituted proteins no longer reacts with the membrane-impermeable IASD reagent, indicating penetration of these protein segments into the lipid micelle
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A533T
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in native gel electrophoresis, mutant enzymes show differing electrophoretic mobilities. A533T mobility is similar to wild-type, and slower than Y549Term
A553V
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in native gel electrophoresis, mutant enzymes show differing electrophoretic mobilities. A553V mobility is similar to wild-type, and slower than Y549Term
E564P
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in native gel electrophoresis, mutant enzymes show differing electrophoretic mobilities. E564P has the slowest mobilityamong the mutants tested
R572E
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in native gel electrophoresis, mutant enzymes show differing electrophoretic mobilities. R572E has the fastest mobility among the mutants tested
R572G
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in native gel electrophoresis, mutant enzymes show differing electrophoretic mobilities. R572G shows a midway mobility
R572K
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in native gel electrophoresis, mutant enzymes show differing electrophoretic mobilities. R572K mobility is similar to wild-type, and slower than Y549Term
R572Term
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deletion of last amino acid. In native gel electrophoresis, mutant enzymes show differing electrophoretic mobilities. R572Term shows a midway mobility
W570Term
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deletion of last three amino acids. In native gel electrophoresis, mutant enzymes show differing electrophoretic mobilities.. W570Term shows a midway mobility
Y549Term
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deletion of last 24 amino acids. In native gel electrophoresis, mutant enzymes show differing electrophoretic mobilities.. Y549Term shows a midway mobility
A467T
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mutant poxB4 is deficient in lipid activation. Mutation is located in the C-terminal half of the gene. The difference between poxB3 and poxB4 is the binding of Triton detergents
S536P
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mutant poxB3 is deficient in lipid activation but retains full catlytic activity. Mutation is located in the C-terminal half of the gene. The difference between poxB3 and poxB4 is the binding of Triton detergents
additional information
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expression of a truncated gene lacking the last 24 amino acids of the C-terminus, thus being closely analogous to the activated species produced in vitro by limited chymotrypsin cleavage. The truncated protein is fully active in vitro in the absence of lipid, and its activity is not further increased by addition of lipid activators. The truncated enzyme fails to bind Triton X-114. Strains producing the truncated protein are devoid of oxidase activity in vivo
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
reconstitution of a minimal respiratory chain consisting of pyruvate oxidase and cytochrome d terminal oxidase plus ubiquinone8 incorporated in phospholipid vesicles. The catalytic velocity of the reconstituted liposome system is about 30% of that observed when the flavoprotein is reconstituted with Escherichia coli membranes
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reconstitution of enzyme with a supported lipidic structure. The activated enzyme can be efficiently regulated by the oxidation level of the quinone pool in natural membranes
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reconstitution of the native enzymatically active protein can be accomplished by incubating equimolar concentrations of apomonomers and FAD at pH 6.5. The second order reaction of apomonomers with FAD to form an initial monomer-FAD complex is fast. The rate-limiting step for enzymatic reactivation appears to be the folding of the polypeptide chain in the monomer-FAD complex to reconstitute the three-dimensional FAD binding site prior to subunit reassociation. The subsequent formation of native tetramers proceeds via an essentially irreversible dimer assembly pathway
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removal of the lipids from the membrane particles by extraction with aqueous acetone or hydrolysis of the phospholipids by treatment with Bacillus cereus phospholipase C results in a complete loss of electron transport activity. Practically all the neutral lipids and 65% of the phospholipids are removed by this treatment. Phospholipase treatment results in a loss of 75% of the membrane phospholipid phosphorus. The diglycerides and the neutral lipids produced by phospholipase hydrolysis remain associated with the particles. Addition of neutral lipid and detergent hepta-D,L-alanyl-dodecylamide to the acetone-extracted material results in a restoration of 37% of the original particle activity. Addition of neutral lipid and hepta-DL-alanyl dodecylamide to phospholipase-treated particles completely restores the original electron transport activity. Addition of ubiquinone from either yeast or Escherichia coli will restore pyruvate oxidase activity when the quinones are supplemented with photoinactivated neutral lipid. No restoration of activity to phospholipase-treated particles is noted upon the addition of either menaquinone 6 or menaquinone 8 to the reconstitution system
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