Information on EC 1.6.1.5 - proton-translocating NAD(P)+ transhydrogenase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
1.6.1.5
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RECOMMENDED NAME
GeneOntology No.
proton-translocating NAD(P)+ transhydrogenase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
NADPH + NAD+ + H+[side 1] = NADP+ + NADH + H+[side 2]
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
NAD phosphorylation and dephosphorylation
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NAD phosphorylation and transhydrogenation
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SYSTEMATIC NAME
IUBMB Comments
NADPH:NAD+ oxidoreductase (H+-transporting)
The enzyme is a membrane bound proton-translocating pyridine nucleotide transhydrogenase that couples the reversible reduction of NADP by NADH to an inward proton translocation across the membrane. In the bacterium Escherichia coli the enzyme provides a major source of cytosolic NADPH. Detoxification of reactive oxygen species in mitochondria by glutathione peroxidases depends on NADPH produced by this enzyme.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
formerly Rhodopseudomonas capsulata
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Manually annotated by BRENDA team
Thermus thermophilus HB27 / ATCC BAA-163 / DSM 7039
subunit beta
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
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transhydrogenase PntAB is a major source of NADPH that is required for biosynthesis in Escherichia coli
physiological function
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the enzyme couples electron movement between the NADP/NADPH and NAD/NADH pools to the protonmotive force across the mitochondrial inner membrane
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,N6-etheno-NADPH + NAD+ + H+[side 1]
1,N6-etheno-NADP+ + NADH + H+[side 2]
show the reaction diagram
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-
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r
1,N6-etheno-NADPH + oxidized acetyl pyridine adenine dinucleotide + H+[side 1]
1,N6-etheno-NADP+ + reduced acetyl pyridine adenine dinucleotide + H+[side 2]
show the reaction diagram
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-
-
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r
deamino-NADPH + NAD+ + H+[side 1]
deamino-NADP+ + NADH + H+[side 2]
show the reaction diagram
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-
-
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r
deamino-NADPH + oxidized acetyl pyridine adenine dinucleotide + H+[side 1]
deamino-NADP+ + reduced acetyl pyridine adenine dinucleotide + H+[side 2]
show the reaction diagram
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-
-
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r
H+/out + NADH + NADP+
H+/in + NAD+ + NADPH
show the reaction diagram
NADH + NADP+
NAD+ + NADPH
show the reaction diagram
NADH + thio-NADP+
NAD+ + thio-NADPH
show the reaction diagram
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-
-
r
NADP+ + NADH
NADPH + NAD+
show the reaction diagram
NADPH + 3-acetylpyridine-NAD+
3-acetylpyridine-NADH + NADP+
show the reaction diagram
NADPH + 3-acetylpyridine-NAD+ + H+[side 1]
NADP+ + 3-acetylpyridine-NADH + H+[side 2]
show the reaction diagram
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?
NADPH + NAD+ + H+/in
NADP+ + NADH + H+/out
show the reaction diagram
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-
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r
NADPH + NAD+ + H+[side 1]
NADP+ + NADH + H+[side 2]
show the reaction diagram
NADPH + oxidized 3-acetylpyridin-adenine dinucleotide + H+[side 1]
NADP+ + reduced 3-acetylpyridin-adenine dinucleotide + H+[side 2]
show the reaction diagram
NADPH + oxidized 3-acetylpyridine adenine dinucleotide
NADP+ + reduced 3-acetylpyridine adenine dinucleotide
show the reaction diagram
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-
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?
NADPH + oxidized 3-acetylpyridine adenine dinucleotide + H+[side 1]
NADP+ + reduced 3-acetylpyridine adenine dinucleotide + H+[side 2]
show the reaction diagram
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r
NADPH + oxidized acetyl pyridine adenine dinucleotide + H+/in
NADP+ + reduced acetyl pyridine adenine dinucleotide + H+/out
show the reaction diagram
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r
NADPH + oxidized acetyl pyridine adenine dinucleotide + H+[side 1]
NADP+ + reduced acetyl pyridine adenine dinucleotide + H+[side 2]
show the reaction diagram
NADPH + oxidized acetylpyridine adenine dinucleotide + H+[side 1]
NADP+ + reduced acetylpyridine adenine dinucleotide + H+[side 2]
show the reaction diagram
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?
thio-NADH + NADP+
thio-NAD+ + NADPH
show the reaction diagram
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r
thio-NADP+ + NADH + H+[side 2]
thio-NADPH + NAD+ + H+[side 1]
show the reaction diagram
thio-NADPH + NAD+ + H+[side 1]
thio-NADP+ + NADH + H+[side 2]
show the reaction diagram
additional information
?
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NADP(H) binding leads to perturbation of a deeply buried part of the polypeptide backbone and to protonation of a carboxylic acid residue
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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
H+/out + NADH + NADP+
H+/in + NAD+ + NADPH
show the reaction diagram
NADH + NADP+
NAD+ + NADPH
show the reaction diagram
NADPH + NAD+ + H+/in
NADP+ + NADH + H+/out
show the reaction diagram
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-
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r
NADPH + NAD+ + H+[side 1]
NADP+ + NADH + H+[side 2]
show the reaction diagram
NADPH + oxidized 3-acetylpyridine adenine dinucleotide
NADP+ + reduced 3-acetylpyridine adenine dinucleotide
show the reaction diagram
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?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NAD(P)+
NAD(P)H
NADP+
NADPH
additional information
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no flavin cofactor, differentiation from EC 1.6.1.1.
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Na+
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strong activation of forward reaction, i.e. the reduction of NADP+ by NADH, in the light at concentrations of approx. 50-80 mM, half-maximal activation at 10 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-(4-maleimidoanilino)-naphthalene-6-sulfonic acid
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0.004 mM, 2 h incubation, 75% inhibition of reverse reaction catalyzed by A348C mutant enzyme, 95% inhibition of A390C mutant enzyme after 1 h, 90% inhibition of K424C mutant enzyme after 1 h, 55% inhibition of R425C mutant enzyme after 1h
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3-Aminopyridine adenine dinucleotide phosphate
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competitive vs. NADP(H)
acetylpyridine adenine dinucleotide
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Butane-2,3-dione
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40 mM, almost complete inactivation of enzyme activity in chromatophores after 12 min, NAD+ and NADP+ partially protect, complete protection by a combination of NAD+ and NADP+
Cd2+
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low concentrations of Cd2+ strongly inhibit revers transhydrogenation
N,N'-dicyclohexylcarbodiimide
N,N'-Dicylclohexylcarbodiimide
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NADH protects from inhibition, NADP+ and to a lesser extent NADPH increase the rate of inhibition
N-cyclohexyl-N'-(4-dimethylaminonaphthyl)carbodiimide
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about 25% residual activity after 200 min at 0.5 mM
N-ethylmaleimide
NADH
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high concentrations of NADH lead to substrate inhibition
NADP+
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mixed product inhibition vs. acetylpyridine adenine dinucleotide, competitive vs. NADPH
oxidized 3-acetylpyridin-adenine dinucleotide
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high concentrations of oxidized 3-acetylpyridin-adenine dinucleotide lead to substrate inhibition
reduced acetylpyridine adenine dinucleotide
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competitive inhibition vs. oxidized acetylpyridine adenine dinucleotide, mixed inhibition vs. NADPH
valinomycin
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Zn2+
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low concentrations of Zn2+ strongly inhibit revers transhydrogenation
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
carbonylcyanide-4-trifluoromethoxyphenyl hydrazone
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0.002 mM stimulates the rate of the forward reaction by 8fold and of the reverse reaction by 10fold
light
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forward reaction in chromatophores is accelerated more than 20fold during illumination with photosynthetically active light
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lysophosphatidylcholine
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.033 - 0.065
acetylpyridine adenine dinucleotide
0.0017 - 0.066
NADH
0.0051 - 0.12
NADPH
0.02 - 0.026
oxidized acetylpyridine adenine dinucleotide
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0.008 - 0.055
thio-NADP+
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0667 - 81.7
reduced acetylpyridine adenine dinucleotide
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.59 - 2.03
2'-AMP
0.1
3-aminopyridine dinucleotide phosphate
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approximate value
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0.51 - 2.63
5'-AMP
0.116
acetylpyridine adenine dinucleotide
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0.011
NADPH
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0013
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unpurified native enzyme, at pH 7.5, temperature not specified in the publication
0.0052
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unpurified recombinant enzyme, at pH 7.5, temperature not specified in the publication
0.022
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purified native enzyme, at pH 7.5, temperature not specified in the publication
0.0299
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purified recombinant enzyme, at pH 7.5, temperature not specified in the publication
0.1
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membrane bound mutant enzyme with a direct linker between alpha and beta subunits; purified mutant enzyme with a direct linker between alpha and beta subunits, forward reaction
0.11
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with oxidized acetyl pyridine adenine dinucleotide and deamino-NADPH as substrates, at pH 6.0 and 25°C
0.2
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reduction of NADP+ by NADH driven by electron transport, cysteine-free enzyme reconstituted in membrane vesicles
0.3
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membrane bound mutant enzyme with a 18 residues long linker between alpha and beta subunits
0.4
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purified mutant enzyme with a 18 residues long linker between alpha and beta subunits, forward reaction
0.42
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reduction of NADP+ by NADH driven by electron transport, wild-type enzyme reconstituted in membrane vesicles
0.47
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with oxidized acetyl pyridine adenine dinucleotide and 1-N6-etheno-NADPH as substrates, at pH 6.0 and 25°C
0.6
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membrane bound mutant enzyme with a 32 residues long linker between alpha and beta subunits
0.7
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purified mutant enzyme with a 32 residues long linker between alpha and beta subunits, forward reaction
0.8
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purified mutant enzyme with a direct linker between alpha and beta subunits, reverse reaction
0.9
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purified enzyme, forward reaction
1.4
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membrane bound enzyme, reverse reaction
1.46
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with oxidized acetyl pyridine adenine dinucleotide and NADPH as substrates, at pH 6.0 and 25°C
1.9
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reduction of acetylpyridine adenine dinucleotide by NADPH, cysteine-free enzyme reconstituted in membrane vesicles
3
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reduction of acetylpyridine adenine dinucleotide by NADPH, wild-type enzyme reconstituted in membrane vesicles
3.9
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purified mutant enzyme with a 18 residues long linker between alpha and beta subunits, reverse reaction
5.5
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purified mutant enzyme with a 32 residues long linker between alpha and beta subunits, reverse reaction
6
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purified enzyme, at pH 7.0 and 20°C
7
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purified mutant enzyme with a direct linker between alpha and beta subunits, cyclic reaction
9.6
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purified enzyme, reverse reaction
22
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partially purified enzyme from strain W6
29.9
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purified enzyme from strain JM83
42
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purified mutant enzyme with a 32 residues long linker between alpha and beta subunits, cyclic reaction
46
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purified mutant enzyme with a 18 residues long linker between alpha and beta subunits, cyclic reaction
63
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purified enzyme, cyclic reaction
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5
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H91E mutant enzyme, optima for forward reaction below pH 5.5, 7% of wild-type enzyme activity at pH 6.0
6
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reverse reaction catalyzed by H91E mutant enzyme, 20% of wild-type enzyme activity
6.3
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pH optimum for the reduction of thio-NADP+ by NADH
7.1
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pH optimum for the reduction of oxidized acetyl pyridine adenine dinucleotide by NADPH
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 8.5
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additional information
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titration of Escherichia coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
14900
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1 * 47800 + 1 * 14900, SDS-PAGE
40000
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2 * 40000, SDS-PAGE
40300
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2 * 40300, calculated from amino acid sequence
47000
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1 * 50000 + 1 * 47000, SDS-PAGE
47800
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1 * 47800 + 1 * 14900, SDS-PAGE
49000
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1 * 54000 + 1 * 49000, SDS-PAGE
53000
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x * 53000 + x * 48000, SDS-PAGE
84000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 53000 + x * 48000, SDS-PAGE
heterodimer
heterotetramer
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2 * 54000 + 2 * 48000, SDS-PAGE
homodimer
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2 * 40000, SDS-PAGE
tetramer
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domain I, i.e. alpha1 to alpha404, and III i.e. beta260 to beta462, are exposed to the cytosol and contain the binding sites for NAD(H) and NAD(P)H, respectively, domain II, i.e. alpha405 to alpha 510, spans the membrane
trimer
additional information
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the protein has three components: dI binds NADH, dIII binds NADP+, and dII spans the membrane. Transhydrogenase is a dimer of two dI-dII-dIII monomers. The two catalytic sites alternate during turnover
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
dimers of dI domains using hanging-drop vapor diffusion method
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hanging-drop method, crystal structures of the NAD(H)-binding domain I of transhydrogenase in the absence as well as in the presence of oxidized and reduced substrate. The structures are determined at 1.9-2.0 A resolution; NAD(H)-binding domain I in the absence and in the presence of NAD+ or NADH, hanging drop vapor diffusion method, using 0.2 M ammonium acetate, 0.1 M tri-sodium citrate dihydrate (pH 5.6) and 30% (w/v) PEG 4000
dIII domain in the presence of NADPH
(dI.Q132N)2dIII trimer
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; dI2dIII complex with bound NAD+ and NADP+, NADH and NADPH, and ADP-ribose and NADPH
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crystal structure of domain I i.e. alpha1 subunit, with and without bound NADH, 1.8 A resolution without NADH, 1.9 A with NADH bound
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crystal structure of domainI/domain III complex
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dI2dIII1 complex with bound NAD+ and 1,4,5,6-tetrahydronicotinamide adenine dinucleotide phosphate the dI2dIII1 complex with bound ,4,5,6-tetrahydronicotinamide adenine dinucleotide and NADP+. dI is the NAD(H)-binding component of transhydrogenase. dIII is the NADP(H)-binding component
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domain dIII, domain dI dimer and dI2dIII complex in the presence of limiting NADH using the vapor diffusion method
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mutant enzymes are crystallized by the hanging drop vapor diffusion method, using in 16-24% (w/v) 8K-PEG, 20-150 mM (NH4)2SO4, 100mM MES, pH 6.0, and 10% (v/v) glycerol in the presence of 50 mM NAD+ and 5 mM NADP+
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solution structure solved by NMR
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using 30% (w/v) PEG 400 in 0.1 M MES pH 6.5 with 0.1-0.4 M magnesium nitrate and 1 to 2.5% benzamidine hydrochloride
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
-15
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the enzyme remains stable for more than 3 months at -15°C
4
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the enzyme remains stable for more than 2 days at 4°C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
fusion protein is substantially more stable than wild type enzyme upon storage at 4°C
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purified enzyme is inactivated at 4°C even in presence of dithiothreitol
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-15°C, 20 mM sodium tricine buffer, pH 7.6, 1 mM dithiothreitol, 0.2% Triton X-100, 30% glycerol, 4 weeks, no loss of activity
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4°C, 50 mM Tris-HCl, pH 7.8, 1 mM, EDTA, 1 mM, dithiothreitol, 1 week, 10% loss of activity
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4°C, fusion protein is substantially more stable than wild type enzyme upon storage
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; recombinant domain I and recombinant E155W and Y171W mutant domain III
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; recombinant protein
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; the recombinant enzyme is purified by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution. The wild type enzyme is purified by phenyl Sepharose column chromatography, DEAE-Bio-Gel A column chromatography, and NAD+ agarose column chromatography
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DEAE-Sepharose column chromatography and hydroxyapatite column chromatography
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fusion protein using His-tag and on calmodulin-sepharose
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His-tagged H91E mutant enzyme
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Ni-NTA resin column chromatography
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phenyl-Sepharose column chromatography and DEAE-Trisacryl M column chromatography
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purification of bacterially expressed, recombinant membrane protein fused with calmodulin-binding domains. This method allows isolation of the protein fusions in a single chromatography step using elution with the calcium chelating agent EDTA. Unlike purification of His-tagged proteins on nickel chelate, it is not sensitive to the presence of strong reducing agents (e.g., DTT). The protocol involves disruption of host bacteria by sonication, sedimentation of membranes by differential centrifugation, solubilization of membrane proteins and affinity chromatography on calmodulin-agarose. To achieve maximum purity and yield, the use of a combination of non-ionic and anionic detergents is suggested. Purification takes two working days, with an overnight wash of the column to increase the purity of the product
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Q-Sepharose column chromatography and Mono Q column chromatography
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Q-Sepharose column chromatography, and butyl Toyopearl column chromatography
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recombinant domain I
recombinant domains I and III
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recombinant protein
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recombinant proteins
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recombinant wild-type, T393C, R425C, G430C and A432C mutant domain III
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; expressed in Escherichia coli as cysteine free variant
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; expressed in Escherichia coli JM83 cells
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dI and dIII domains separately expressed in Escherichia coli
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dIII domain expressed in Escherichia coli
domain dI and His-tag fusion protein of dIII separately expressed in Escherichia coli
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domain dI expressed in Escherichia coli
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domains expressed separately in Escherichia coli
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expressed in Escherichia coli
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expressed in Escherichia coli BL21(DE3) cells
expressed in Escherichia coli C600 and BL21(DE3) cells
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expressed in Escherichia coli JM 109 cells
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expressed in Escherichia coli JM83 cells
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expressed in Escherichia coli MC4100TH-
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expressed in Escherichia coli strain JM109
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expressed in Escherichia coli; expression of domain I and of E155W and Y171W mutant domain III
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expressed in Saccharomyces cerevisiae strain TN24
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expression of cysteine mutants A348C, A390C, K424C, and R425C in Escherichia coli
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expression of domain I in Escherichia coli
expression of domains I and III in Escherichia coli
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expression of wild-type domain III, T393C, R425C, G430C and A432C mutant domain III in Escherichia coli
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fusion protein with calmodulin-binding peptide and His-tag expressed in Escherichia coli JM109
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His-tagged H91E mutant enzyme expressed in Escherichia coli
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the NADP(H)-binding enzyme portion is expressed in Escherichia coli BL21(DE3) cells
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wild type dI and E155W and E155W/G173C mutants of dIII expressed in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A246C
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reverse activity stronger affected than cyclic activity
A253C
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reverse activity stronger affected than cyclic activity
A348C
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mutation introduced into a cysteine-free mutant enzyme, mutant shows markedly reduced activity
A390C
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mutation introduced into a cysteine-free mutant enzyme
A398C
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the mutant with wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
C292T/C339T/C395S/C397T/C435S
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cysteine of the alpha subunits replaced, similar activity as wild-type
C292T/C339T/C395S/C397T/C435S/C147S/C260S
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all 7 cysteines of the enzyme, 5 localized in the alpha subunit and 2 in the beta subunit, are replaced, the cysteine-free mutant shows about 5fold more activity in the reduction of acetylpyridine adenine dinucleotide by NADH than wild-type, the cyclic reduction of acetylpyridine adenine dinucleotide by NADH via NADPH is 2-2.5fold more activ
D213K
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mutation in domain II
D213R
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mutation in domain II
D238C
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reverse activity stronger affected than cyclic activity
D392C
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the mutant shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
D401E
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mutation in beta subunit
D401G
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mutation in beta subunit
D401V
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mutation in beta subunit
E124C
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domian dII, strongly reduced reverse activity, no effect on cyclic activity
E413D
-
mutation in beta subunit
E413G
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mutation in beta subunit
E413V
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mutation in beta subunit
G132A
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in domain dII, no effect on wild type reverse activity
G138A
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in domain dII, 57% of wild type reverse activity
G226A
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in domain dII, 50% of wild type reverse activity
G233A
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in domain dII, 49% of wild type reverse activity
G245A
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in domain dII, no effect of wild type reverse activity
G245C
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24% of reverse activity
G245L
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52% of cyclic activity; the mutation leads to a general inhibition of all enzyme activities
G249A
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in domain dII, 79% wild type reverse activity
G249C
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40% of reverse activity
G249L
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48% of cyclic activity; 70% of reverse activity; the mutation leads to a general inhibition of all enzyme activities
G252A
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in domain dII, 2.6% of wild type reverse activity
G252L
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13% of cyclic activity; the mutation leads to a general inhibition of all enzyme activities
G252S
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in domain dII, 2.4% of wild type reverse activity
G252T
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in domain dII, 2.3% of wild type reverse activity
G252V
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in domain dII, 2.5% of wild type reverse activity
G408C
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the mutant with wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
G476C
-
domian dII, little effect on activity
G95A
-
in domain dII, 56% of wild type reverse activity
H91C
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the mutant of the beta subunit is unable to undergo the conformational change that occurs on binding of the substrates NADP+ or NADPH. The mutant retains 12% of the hydride transfer activity while proton translocation is reduced to 7% compared to the wild type enzyme
H91D
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the mutant of the beta subunit retains 15% of the hydride transfer activity while proton translocation is reduced to 9% compared to the wild type enzyme
H91E
-
mutation in beta subunit
H91N
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the mutant of the beta subunit retains 80% of the hydride transfer activity while proton translocation is reduced to 7% compared to the wild type enzyme
H91R
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mutation in domain II, leads to occlusion of NADP(H) at the NADP(H)-binding site of domain III
H91S
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the mutant of the beta subunit is unable to undergo the conformational change that occurs on binding of the substrates NADP+ or NADPH. The mutant retains 19% of the hydride transfer activity while proton translocation is reduced to 11% compared to the wild type enzyme
H91T
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the mutant of the beta subunit is unable to undergo the conformational change that occurs on binding of the substrates NADP+ or NADPH. The mutant retains 11% of the hydride transfer activity while proton translocation is reduced to 8% compared to the wild type enzyme
I258C
-
reverse activity stronger affected than cyclic activity
I406C
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the mutant with 450% of wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
K416G
-
mutation in beta subunit
K424C
-
mutation introduced into a cysteine-free mutant enzyme, mutant shows markedly reduced activity
K424G
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mutation in beta subunit
K424R
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mutation in beta subunit
K452D
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mutation in beta subunit
K452G
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mutation in beta subunit
L240C
-
reverse activity stronger affected than cyclic activity
L241C
-
reverse activity stronger affected than cyclic activity
L254C
-
reverse activity stronger affected than cyclic activity
L255C
-
reverse activity stronger affected than cyclic activity
M259C
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21% of reverse activity, 215% of cyclic activity
M409C
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the mutant with 75% of wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
N222K
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mutation in domain II, leads to occlusion of NADP(H) at the NADP(H)-binding site of domain III
N222R
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mutation in domain II, leads to occlusion of NADP(H) at the NADP(H)-binding site of domain III
N238C
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reverse activity stronger affected than cyclic activity
R425E
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mutation in beta subunit
R425G
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mutation in beta subunit
R425K
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mutation in beta subunit
S105C
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domian dII, significantly reduced activity
S183C
-
domian dII, significantly reduced activity
S237C
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domian dII, slightly reduced reverse activity, no efect on cyclic activity
S250C
-
strongly increased reverse and cyclic activity; the mutation leads to enhanced activities of all enzyme activities
S251C
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strongly increased reverse and cyclic activity; the mutation leads to enhanced activities of all enzyme activities
S256C
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the mutation leads to enhanced activities of all enzyme activities
S2C
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domian dII, no effect on activity
S404C
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the mutant with 75% of wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
T244C
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reverse activity stronger affected than cyclic activity
T54C
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domian dII, significantly reduced activity
V243C
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reverse activity stronger affected than cyclic activity
V248C
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reverse activity stronger affected than cyclic activity
V411C
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the mutant with 125% of wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
Y257C
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reverse activity stronger affected than cyclic activity
Y431C
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the mutant with 450% of wild type activity shows increased ratios between the rates of the forward and reverse reactions, thus approaching that of the wild type enzyme
D135N
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mutation has no effect in binding affinity of either NAD+ or NADH; the mutant shows reduced activity compared to the wild type enzyme
E155W/G173C
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dIII domain, catalytic properties are similar to the wild type dIII, increased rate of reverse reaction
E155W/R165A
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the mutations mutation do not significantly affect catalytic activity
M239F
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the Km for APAD+ during reverse transhydrogenation is 6fold greater compared to the wild type. Cyclic transhydrogenation (in membranes and the recombinant system) is substantially more inhibited (84%) than either forward or reverse transhydrogenation
M293I
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the Km for APAD+ during reverse transhydrogenation is 5fold greater compared to the wild type. Cyclic transhydrogenation (in membranes and the recombinant system) is substantially more inhibited (70%) than either forward or reverse transhydrogenation
R127A
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mutation strongly inhibits the rate of transhydrogenation and alters the nucleotide-binding properties of the dI protein. When dIR127A is reconstituted into the intact enzyme in membranes, transhydrogenation rates are negligible. dI is the NAD(H)-binding component of the transhydrogenase; the mutant shows reduced activity compared to the wild type enzyme
R127M
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mutation strongly inhibits the rate of transhydrogenation and alters the nucleotide-binding properties of the dI protein. When dIR127M is reconstituted into the intact enzyme in membranes, transhydrogenation rates are negligible. dI is the NAD(H)-binding component of the transhydrogenase; the mutant shows reduced activity compared to the wild type enzyme
R165A
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a higher concentration of the nucleotide is needed to achieve the half-maximal rate compared to with the wild type protein
S135A
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mutation has no effect in binding affinity of either NAD+ or NADH
S138A
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the mutant shows reduced activity compared to the wild type enzyme
W72F
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the mutant shows wild type activity
Y146A
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mutation in component dI that binds NADH. dI.Y146A more readily dissociates into monomers than wild-type dI. dI.Y146A monomers bind NADH much more weakly than dimers. dI.Y146A reconstitutes activity to dI-depleted membranes in its dimeric form but not in its monomeric form; the mutant binds NADH much more weakly than the wild type enzyme
Y146F
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mutation in component dI that binds NADH. Wild-type dI and dI.Y146F reconstituted activity to dI-depleted membranes with similar characteristics; the mutant shows wild type NADH binding ability
Y171W
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mutation in domain III, similar catalytic activities as wild-type, used for tryptophan fluorescence measurements; the mutant shows wild type activity
additional information
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properties of a variety of mutant enzymes containing modified conserved and semiconserved basic and acidic residues in the beta subunit