BRENDA - Enzyme Database

Characterization of the ubiquinone binding site in the alternative NADH-quinone oxidoreductase of Saccharomyces cerevisiae by photoaffinity labeling

Murai, M.; Yamashita, T.; Senoh, M.; Mashimo, Y.; Kataoka, M.; Kosaka, H.; Matsuno-Yagi, A.; Yagi, T.; Miyoshi, H.; Biochemistry 49, 2973-2980 (2010)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
1.6.5.9
expressed in Escherichia coli membranes
Saccharomyces cerevisiae
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
1.6.5.2
benzoquinol
shows noncompetitive inhibition in all conditions, it can bind to multiple enzyme forms or sites, even when either of the substrates (NADH or benzoquinone) is present at high concentrations
Saccharomyces cerevisiae
1.6.5.2
additional information
the product inhibition pattern expected if WrbA follows a ping pong mechanism is that the pairs NAD/BQ and NADH/BQH2 display competitive inhibition, whereas the pairs NAD/NADH and BQ/BQH2 display non-competitive inhibition
Saccharomyces cerevisiae
1.6.5.2
NAD+
noncompetitive inhibition with either substrate when the other substrate is at low concentrations. At high NADH concentrations, NAD and benzoquinone are competitive, indicating they bind to a common site or sites
Saccharomyces cerevisiae
1.6.5.9
aurachin analogue AC0-10
specific inhibitor
Saccharomyces cerevisiae
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.6.5.2
additional information
-
additional information
WrbA steady-state kinetics, overview. Initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depends on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process
Saccharomyces cerevisiae
1.6.5.2
14.9
-
benzoquinone
at 0.02 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
16.2
-
NADH
at 0.02 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
19
-
benzoquinone
at 0.01 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
19
-
NADH
at 0.01 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
19.3
-
benzoquinone
at 0.05 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
20.7
-
NADH
at 0.05 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
28.2
-
NADH
at 0.1 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
36.3
-
benzoquinone
at 0.1 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.9
0.0079
-
ubiquinone-2
in 50 mM sodium phosphate buffer (pH 6.0) and 1 mM EDTA, temperature not specified in the publication
Saccharomyces cerevisiae
1.6.5.9
0.0152
-
ubiquinone-1
in 50 mM sodium phosphate buffer (pH 6.0) and 1 mM EDTA, temperature not specified in the publication
Saccharomyces cerevisiae
1.6.5.9
0.0181
-
N-(6-[[(4E,8E)-10-(4-azido-5-methoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]amino]-6-oxohexyl)-6-([5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl]amino)hexanamide
in 50 mM sodium phosphate buffer (pH 6.0) and 1 mM EDTA, temperature not specified in the publication
Saccharomyces cerevisiae
1.6.5.9
0.022
-
N-[(4E,8E)-10-(4-azido-5-methoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]-6-([5-[(3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl]amino)hexanamide
in 50 mM sodium phosphate buffer (pH 6.0) and 1 mM EDTA, temperature not specified in the publication
Saccharomyces cerevisiae
1.6.5.9
0.033
-
N-[2-[2-(2-[[(4E,8E)-10-(4-azido-5-methoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]oxy]ethoxy)ethoxy]ethyl]-5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamide
in 50 mM sodium phosphate buffer (pH 6.0) and 1 mM EDTA, temperature not specified in the publication
Saccharomyces cerevisiae
Localization
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
1.6.5.9
mitochondrial membrane
-
Saccharomyces cerevisiae
31966
-
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.6.5.9
53000
-
estimated from amino acid sequence
Saccharomyces cerevisiae
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.6.5.2
NADH + H+ + a quinone
Saccharomyces cerevisiae
-
NAD+ + a hydroquinone
-
-
?
1.6.5.2
NADPH + H+ + a quinone
Saccharomyces cerevisiae
-
NADP+ + a hydroquinone
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
1.6.5.2
Saccharomyces cerevisiae
-
-
-
1.6.5.9
Saccharomyces cerevisiae
P32340
-
-
Purification (Commentary)
EC Number
Commentary
Organism
1.6.5.9
streptavidin-agarose chromatography
Saccharomyces cerevisiae
Reaction
EC Number
Reaction
Commentary
Organism
1.6.5.2
NAD(P)H + H+ + a quinone = NAD(P)+ + a hydroquinone
ping-pong reaction mechanism
Saccharomyces cerevisiae
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.6.5.2
NADH + H+ + a quinone
-
714189
Saccharomyces cerevisiae
NAD+ + a hydroquinone
-
-
-
?
1.6.5.2
NADH + H+ + benzoquinone
-
714189
Saccharomyces cerevisiae
NAD+ + benzoquinol
-
-
-
?
1.6.5.2
NADPH + H+ + a quinone
-
714189
Saccharomyces cerevisiae
NADP+ + a hydroquinone
-
-
-
?
1.6.5.2
NADPH + H+ + benzoquinone
-
714189
Saccharomyces cerevisiae
NADP+ + benzoquinol
-
-
-
?
1.6.5.9
NADH + H+ + 2-azido-5-geranyl-3-methoxy-6-methyl-1,4-benzoquinone
-
714189
Saccharomyces cerevisiae
NAD+ + 2-azido-5-geranyl-3-methoxy-6-methyl-1,4-benzoquinol
-
-
-
?
1.6.5.9
NADH + H+ + N-(6-[[(4E,8E)-10-(4-azido-5-methoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]amino]-6-oxohexyl)-6-([5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl]amino)hexanamide
-
714189
Saccharomyces cerevisiae
NAD+ + N-(6-[[(4E,8E)-10-(4-azido-3,6-dihydroxy-5-methoxy-2-methylcyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]amino]-6-oxohexyl)-6-([5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl]amino)hexanamide
-
-
-
?
1.6.5.9
NADH + H+ + N-[(4E,8E)-10-(4-azido-5-methoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]-6-([5-[(3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl]amino)hexanamide
-
714189
Saccharomyces cerevisiae
NAD+ + N-[(4E,8E)-10-(4-azido-3,6-dihydroxy-5-methoxy-2-methylcyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]-6-([5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl]amino)hexanamide
-
-
-
?
1.6.5.9
NADH + H+ + N-[2-[2-(2-[[(4E,8E)-10-(4-azido-5-methoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]oxy]ethoxy)ethoxy]ethyl]-5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamide
-
714189
Saccharomyces cerevisiae
NAD+ + N-[2-[2-(2-[[(4E,8E)-10-(4-azido-3,6-dihydroxy-5-methoxy-2-methylcyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]oxy]ethoxy)ethoxy]ethyl]-5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamide
-
-
-
?
1.6.5.9
NADH + H+ + ubiquinone-1
-
714189
Saccharomyces cerevisiae
NAD+ + ubiquinol-1
-
-
-
?
1.6.5.9
NADH + H+ + ubiquinone-2
-
714189
Saccharomyces cerevisiae
NAD+ + ubiquinol-2
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
1.6.5.2
More
dimer-tetramer assembly equilibrium documented for apoWrbA by analytical ultracentrifugation, large effect of temperature on the subunit assembly state of both apo- and holoWrbA, overview
Saccharomyces cerevisiae
1.6.5.2
tetramer
three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds
Saccharomyces cerevisiae
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.6.5.2
23
-
assay at room temperature
Saccharomyces cerevisiae
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.6.5.2
6
-
assay at
Saccharomyces cerevisiae
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
1.6.5.2
FMN
-
Saccharomyces cerevisiae
1.6.5.2
NADH
-
Saccharomyces cerevisiae
1.6.5.2
NADPH
-
Saccharomyces cerevisiae
1.6.5.9
FAD
Ndi1 contains noncovalently bound FAD
Saccharomyces cerevisiae
1.6.5.9
additional information
Ndi1 contains no iron-sulfur cluster
Saccharomyces cerevisiae
1.6.5.9
NADH
-
Saccharomyces cerevisiae
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
1.6.5.9
expressed in Escherichia coli membranes
Saccharomyces cerevisiae
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
1.6.5.2
FMN
-
Saccharomyces cerevisiae
1.6.5.2
NADH
-
Saccharomyces cerevisiae
1.6.5.2
NADPH
-
Saccharomyces cerevisiae
1.6.5.9
FAD
Ndi1 contains noncovalently bound FAD
Saccharomyces cerevisiae
1.6.5.9
additional information
Ndi1 contains no iron-sulfur cluster
Saccharomyces cerevisiae
1.6.5.9
NADH
-
Saccharomyces cerevisiae
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
1.6.5.2
benzoquinol
shows noncompetitive inhibition in all conditions, it can bind to multiple enzyme forms or sites, even when either of the substrates (NADH or benzoquinone) is present at high concentrations
Saccharomyces cerevisiae
1.6.5.2
additional information
the product inhibition pattern expected if WrbA follows a ping pong mechanism is that the pairs NAD/BQ and NADH/BQH2 display competitive inhibition, whereas the pairs NAD/NADH and BQ/BQH2 display non-competitive inhibition
Saccharomyces cerevisiae
1.6.5.2
NAD+
noncompetitive inhibition with either substrate when the other substrate is at low concentrations. At high NADH concentrations, NAD and benzoquinone are competitive, indicating they bind to a common site or sites
Saccharomyces cerevisiae
1.6.5.9
aurachin analogue AC0-10
specific inhibitor
Saccharomyces cerevisiae
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.6.5.2
additional information
-
additional information
WrbA steady-state kinetics, overview. Initial velocity as a function of either NADH or benzoquinone concentration present one or two Michaelis-Menten phases depends on the temperature at which the enzyme is held prior to assay. The effect of temperature is reversible, suggesting an intramolecular conformational process
Saccharomyces cerevisiae
1.6.5.2
14.9
-
benzoquinone
at 0.02 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
16.2
-
NADH
at 0.02 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
19
-
benzoquinone
at 0.01 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
19
-
NADH
at 0.01 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
19.3
-
benzoquinone
at 0.05 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
20.7
-
NADH
at 0.05 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
28.2
-
NADH
at 0.1 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.2
36.3
-
benzoquinone
at 0.1 mM, pH 6.0, 23°C
Saccharomyces cerevisiae
1.6.5.9
0.0079
-
ubiquinone-2
in 50 mM sodium phosphate buffer (pH 6.0) and 1 mM EDTA, temperature not specified in the publication
Saccharomyces cerevisiae
1.6.5.9
0.0152
-
ubiquinone-1
in 50 mM sodium phosphate buffer (pH 6.0) and 1 mM EDTA, temperature not specified in the publication
Saccharomyces cerevisiae
1.6.5.9
0.0181
-
N-(6-[[(4E,8E)-10-(4-azido-5-methoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]amino]-6-oxohexyl)-6-([5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl]amino)hexanamide
in 50 mM sodium phosphate buffer (pH 6.0) and 1 mM EDTA, temperature not specified in the publication
Saccharomyces cerevisiae
1.6.5.9
0.022
-
N-[(4E,8E)-10-(4-azido-5-methoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]-6-([5-[(3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl]amino)hexanamide
in 50 mM sodium phosphate buffer (pH 6.0) and 1 mM EDTA, temperature not specified in the publication
Saccharomyces cerevisiae
1.6.5.9
0.033
-
N-[2-[2-(2-[[(4E,8E)-10-(4-azido-5-methoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]oxy]ethoxy)ethoxy]ethyl]-5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamide
in 50 mM sodium phosphate buffer (pH 6.0) and 1 mM EDTA, temperature not specified in the publication
Saccharomyces cerevisiae
Localization (protein specific)
EC Number
Localization
Commentary
Organism
GeneOntology No.
Textmining
1.6.5.9
mitochondrial membrane
-
Saccharomyces cerevisiae
31966
-
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
1.6.5.9
53000
-
estimated from amino acid sequence
Saccharomyces cerevisiae
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
1.6.5.2
NADH + H+ + a quinone
Saccharomyces cerevisiae
-
NAD+ + a hydroquinone
-
-
?
1.6.5.2
NADPH + H+ + a quinone
Saccharomyces cerevisiae
-
NADP+ + a hydroquinone
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
1.6.5.9
streptavidin-agarose chromatography
Saccharomyces cerevisiae
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
1.6.5.2
NADH + H+ + a quinone
-
714189
Saccharomyces cerevisiae
NAD+ + a hydroquinone
-
-
-
?
1.6.5.2
NADH + H+ + benzoquinone
-
714189
Saccharomyces cerevisiae
NAD+ + benzoquinol
-
-
-
?
1.6.5.2
NADPH + H+ + a quinone
-
714189
Saccharomyces cerevisiae
NADP+ + a hydroquinone
-
-
-
?
1.6.5.2
NADPH + H+ + benzoquinone
-
714189
Saccharomyces cerevisiae
NADP+ + benzoquinol
-
-
-
?
1.6.5.9
NADH + H+ + 2-azido-5-geranyl-3-methoxy-6-methyl-1,4-benzoquinone
-
714189
Saccharomyces cerevisiae
NAD+ + 2-azido-5-geranyl-3-methoxy-6-methyl-1,4-benzoquinol
-
-
-
?
1.6.5.9
NADH + H+ + N-(6-[[(4E,8E)-10-(4-azido-5-methoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]amino]-6-oxohexyl)-6-([5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl]amino)hexanamide
-
714189
Saccharomyces cerevisiae
NAD+ + N-(6-[[(4E,8E)-10-(4-azido-3,6-dihydroxy-5-methoxy-2-methylcyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]amino]-6-oxohexyl)-6-([5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl]amino)hexanamide
-
-
-
?
1.6.5.9
NADH + H+ + N-[(4E,8E)-10-(4-azido-5-methoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]-6-([5-[(3aS,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl]amino)hexanamide
-
714189
Saccharomyces cerevisiae
NAD+ + N-[(4E,8E)-10-(4-azido-3,6-dihydroxy-5-methoxy-2-methylcyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]-6-([5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoyl]amino)hexanamide
-
-
-
?
1.6.5.9
NADH + H+ + N-[2-[2-(2-[[(4E,8E)-10-(4-azido-5-methoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]oxy]ethoxy)ethoxy]ethyl]-5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamide
-
714189
Saccharomyces cerevisiae
NAD+ + N-[2-[2-(2-[[(4E,8E)-10-(4-azido-3,6-dihydroxy-5-methoxy-2-methylcyclohexa-1,4-dien-1-yl)-4,8-dimethyldeca-4,8-dien-1-yl]oxy]ethoxy)ethoxy]ethyl]-5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamide
-
-
-
?
1.6.5.9
NADH + H+ + ubiquinone-1
-
714189
Saccharomyces cerevisiae
NAD+ + ubiquinol-1
-
-
-
?
1.6.5.9
NADH + H+ + ubiquinone-2
-
714189
Saccharomyces cerevisiae
NAD+ + ubiquinol-2
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
1.6.5.2
More
dimer-tetramer assembly equilibrium documented for apoWrbA by analytical ultracentrifugation, large effect of temperature on the subunit assembly state of both apo- and holoWrbA, overview
Saccharomyces cerevisiae
1.6.5.2
tetramer
three subunits of the tetrameric enzyme contribute to each of four identical, cavernous active sites that appear to accommodate NAD(P)H or various quinones, but not simultaneously, suggesting an obligate tetramer with a ping-pong mechanism in which NAD departs before oxidized quinone binds
Saccharomyces cerevisiae
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
1.6.5.2
23
-
assay at room temperature
Saccharomyces cerevisiae
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
1.6.5.2
6
-
assay at
Saccharomyces cerevisiae