Information on EC 1.2.1.13 - glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating)

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

EC NUMBER
COMMENTARY hide
1.2.1.13
-
RECOMMENDED NAME
GeneOntology No.
glyceraldehyde-3-phosphate dehydrogenase (NADP+) (phosphorylating)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
D-glyceraldehyde 3-phosphate + phosphate + NADP+ = 3-phospho-D-glyceroyl phosphate + NADPH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
redox reaction
-
-
-
-
reduction
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Calvin-Benson-Bassham cycle
-
-
Carbon fixation in photosynthetic organisms
-
-
Metabolic pathways
-
-
Microbial metabolism in diverse environments
-
-
photosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
D-glyceraldehyde-3-phosphate:NADP+ oxidoreductase (phosphorylating)
-
CAS REGISTRY NUMBER
COMMENTARY hide
37250-87-6
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Ankistrodesmus braunii
-
-
-
Manually annotated by BRENDA team
strain P511, formerly Spirulina platensis
-
-
Manually annotated by BRENDA team
strain P511, formerly Spirulina platensis
-
-
Manually annotated by BRENDA team
isoform GapB
UniProt
Manually annotated by BRENDA team
isoform GapB
UniProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Methanothermobacter thermautotrophicum
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
no activity in Rhodopseudomonas capsulata
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Synechococcus PCC7942
-
-
Manually annotated by BRENDA team
Synechococcus PCC7942
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
-
a deletion mutant strain does not exhibit any growth under gluconeogenic conditions
metabolism
-
the enzyme plays a major role only in gluconeogenesis
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
3-phospho-D-glyceroyl phosphate + NADH
D-glyceraldehyde 3-phosphate + phosphate + NAD+
show the reaction diagram
3-phospho-D-glyceroyl phosphate + NADH + H+
D-glyceraldehyde 3-phosphate + phosphate + NAD+
show the reaction diagram
-
-
-
-
r
3-phospho-D-glyceroyl phosphate + NADPH
D-glyceraldehyde 3-phosphate + phosphate + NADP+
show the reaction diagram
3-phospho-D-glyceroyl phosphate + NADPH + H+
D-glyceraldehyde 3-phosphate + phosphate + NADP+
show the reaction diagram
3-phospho-D-glyceroyl phosphate + NADPH + H+
DL-glyceraldehyde 3-phosphate + phosphate + NADP+
show the reaction diagram
-
-
-
-
r
D-glyceraldehyde + phosphate + NADP+
D-glyceroylphosphate + NADPH
show the reaction diagram
-
with 10fold lower efficiency than D-glyceraldehyde 3-phosphate
-
-
?
D-glyceraldehyde 3-phosphate + arsenate + NADP+
3-phospho-D-glyceroyl arsenate + NADPH
show the reaction diagram
D-glyceraldehyde 3-phosphate + phosphate + NAD+
3-phospho-D-glyceroyl phosphate + NADH
show the reaction diagram
D-glyceraldehyde 3-phosphate + phosphate + NAD+
3-phospho-D-glyceroyl phosphate + NADH + H+
show the reaction diagram
-
Vmax/Km for NADP+ is 33fold higher compared to Vmax/Km for NAD+
-
-
?
D-glyceraldehyde 3-phosphate + phosphate + NADP+
3-phospho-D-glyceroyl phosphate + NADPH
show the reaction diagram
D-glyceraldehyde 3-phosphate + phosphate + NADP+
3-phospho-D-glyceroyl phosphate + NADPH + H+
show the reaction diagram
DL-glyceraldehyde 3-phosphate + phosphate + NAD+
3-phospho-D-glyceroyl phosphate + NADH + H+
show the reaction diagram
-
-
-
-
r
DL-glyceraldehyde 3-phosphate + phosphate + NADP+
3-phospho-D-glyceroyl phosphate + NADPH + H+
show the reaction diagram
-
-
-
-
r
additional information
?
-
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
3-phospho-D-glyceroyl phosphate + NADPH
D-glyceraldehyde 3-phosphate + phosphate + NADP+
show the reaction diagram
3-phospho-D-glyceroyl phosphate + NADPH + H+
D-glyceraldehyde 3-phosphate + phosphate + NADP+
show the reaction diagram
3-phospho-D-glyceroyl phosphate + NADPH + H+
DL-glyceraldehyde 3-phosphate + phosphate + NADP+
show the reaction diagram
-
-
-
-
r
D-glyceraldehyde 3-phosphate + phosphate + NADP+
3-phospho-D-glyceroyl phosphate + NADPH + H+
show the reaction diagram
DL-glyceraldehyde 3-phosphate + phosphate + NAD+
3-phospho-D-glyceroyl phosphate + NADH + H+
show the reaction diagram
-
-
-
-
r
DL-glyceraldehyde 3-phosphate + phosphate + NADP+
3-phospho-D-glyceroyl phosphate + NADPH + H+
show the reaction diagram
-
-
-
-
r
additional information
?
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-phospho-D-glyceroyl phosphate
-
-
6-Hydroxydopamine
-
-
chloroplast protein CP12
-
the chloroplast protein CP12 behaves as a negative regulator of GAPDH activity
-
Cu2+
-
inhibition is reversed by EDTA
cystamine
-
inhibitory when the enzyme is activated by effectors
DL-glyceraldehyde 3-phosphate
-
-
GAPDH segregator
H2O2
-
rapid reversible deactivation of untreated and GSH-treated enzyme preparation, GSH reverses the inhibition
iodoacetamide
iodoacetate
-
preincubation with glyceraldehyde-phosphate, 10fold molar excess over iodoacetate, prevents inactivation
NADH
-
strong inhibition with excess co-substrate s observed and no activity is observed with over 2 mM NADH
NADPH
-
strong inhibition with excess co-substrate s observed and no activity is observed with over 2 mM NADPH
sulfite
-
non-competitive inhibitor with respect to both NADPH and 3-phosphoglycerate
thioredoxin
-
a regulatory disulfide between Cys359 and Cys358 of the C-terminal extension of GapB does form in presence of oxidized thioredoxin. This covalent modification is required for the NAD+-dependent association into higher oligomers and inhibition of the NADPH-dependent activity
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,3-diphosphoglycerate
2-mercaptoethanol
-
activation
Br-
-
0.6 M, maximal stimulation
cholate
-
activation
Cl-
-
1.0 M, maximal stimulation
Cl3COO2-
-
0.24 M, maximal stimulation
-
ClO4-
-
0.08 M, maximal stimulation
cysteine
-
required for maximal activity
dithiothreitol
Ferredoxin
-
enzyme is present throughout the life cycle of the plants, inactive enzyme is converted during greening to an active state by light either via enzyme effectors or via the ferredoxin-thioredoxin system
-
glutathione
-
required for maximal activity
glycerate 1,3-bisphosphate
-
most effective on a molar basis in stimulating NADPH-activity of dark chloroplast extracts and purified enzyme
GSH
-
activates in the physiological concentration range 0.1-2 mM
I-
-
0.35 M, maximal stimulation
phosphate
-
stimulates
SCN-
-
0.05 M, maximal stimulation
thioredoxin
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00041 - 5.8
3-phospho-D-glyceroyl phosphate
30 - 120
arsenate
0.75 - 0.838
D-glyceraldehyde 3-phosphate
0.21 - 0.77
DL-glyceraldehyde 3-phosphate
0.2 - 16.8
NAD+
0.01 - 0.872
NADH
0.003 - 0.4
NADP+
0.002 - 0.3
NADPH
10.7 - 409
phosphate
additional information
additional information
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
40 - 419
3-phospho-D-glyceroyl phosphate
8.4 - 137
NADH
32 - 430
NADPH
additional information
additional information
Chlamydomonas reinhardtii
-
catalytic rate constant 238 s-1, control; catalytic rate constant 289 s-1, 3 microM 3-phospho-D-glyceroyl phosphate, NADPH-dependent activity of GAPDH in the GAPDH/CP12 complex; catalytic rate constant 316 s-1, 160 microM 3-phospho-D-glyceroyl phosphate, NADPH-dependent activity of GAPDH in the GAPDH/CP12 complex; catalytic rate constant 330 s-1, 10 microM thioredoxin, NADPH-dependent activity of GAPDH in the GAPDH/CP12 complex; catalytic rate constant 390 s-1, 10 microM thioredoxin, 3 microM 3-phospho-D-glyceroyl phosphate, NADPH-dependent activity of GAPDH in the GAPDH/CP12 complex; catalytic rate constant 462 s-1, 10 microM thioredoxin, 160 microM 3-phospho-D-glyceroyl phosphate, NADPH-dependent activity of GAPDH in the GAPDH/CP12 complex
-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
49.1
3-phospho-D-glyceroyl phosphate
-
wild type enzyme, in 50 mM EPPS/NaOH (pH 8.0), 20 mM potassium phosphate, at 50C
2.8 - 13.1
DL-glyceraldehyde 3-phosphate
10.7
NAD+
-
wild type enzyme, in 50 mM EPPS/NaOH (pH 8.0), 20 mM potassium phosphate, at 60C
0.081
NADH
-
wild type enzyme, in 50 mM EPPS/NaOH (pH 8.0), 20 mM potassium phosphate, at 50C
0.75
NADP+
-
wild type enzyme, in 50 mM EPPS/NaOH (pH 8.0), 20 mM potassium phosphate, at 60C
0.035
NADPH
-
wild type enzyme, in 50 mM EPPS/NaOH (pH 8.0), 20 mM potassium phosphate, at 50C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.008
6-Hydroxydopamine
Oryctolagus cuniculus
-
-
0.015
GAPDH segregator
Oryctolagus cuniculus
-
GAPDS inhibts the glycolytic enzymatic function of GAPDH
-
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.026
-
pH not specified in the publication, 37C, activity in undialyzed cell extracts
0.0602
-
effect of water stress on the activity, stress, 1 day, control, 39.6% of control value, Lutescens 758, seedlings
0.0759
-
effect of water stress on the activity, stress, 1 day, Kartolin-4, 50.5% of control value, Lutescens 758, seedlings
0.076
-
effect of water stress on the activity, rehydration, control, 50.1% of control value, Lutescens 758, seedlings
0.0843
-
effect of water stress on the activity, stress, 1 day, control, 25.4% of control value, Mironovskaya 808, seedlings
0.0916
-
effect of water stress on the activity, rehydration, Kartolin-4, 61.0% of control value, Lutescens 758, seedlings
0.1103
-
effect of water stress on the activity, stress, 1 day, BAP, 33.2% of control value, Mironovskaya 808, seedlings
0.1169
-
effect of water stress on the activity, stress, 1 day, TDZ, 35.2% of control value, Mironovskaya 808, seedlings
0.123
-
effect of water stress on the activity, stress, 2 weeks, control, 51.0% of control value, Lutescens 758, leaves
0.1265
-
effect of water stress on the activity, stress, 1 day, Kartolin-2, 38.1% of control value, Mironovskaya 808, seedlings
0.1457
-
effect of water stress on the activity, rehydration, control, 60.4% of control value, Lutescens 758, leaves
0.1502
-
effect of water stress on the activity, original Kartolin-4, 100% of control value, Lutescens 758, seedlings
0.152
-
effect of water stress on the activity, original control, 100% of control value, Lutescens 758, seedlings
0.2002
-
effect of water stress on the activity, stress, 2 weeks, Kartolin-4, 79.9% of control value, Lutescens 758, leaves
0.2153
-
effect of water stress on the activity, rehydration, Kartolin-4, 86.0% of control value, Lutescens 758, leaves
0.2412
-
effect of water stress on the activity, original, control, 100% of control value, Lutescens 758, leaves
0.2503
-
effect of water stress on the activity, original, Kartolin-4, 100% of control value, Lutescens 758, leaves
0.3321
-
effect of water stress on the activity, original, control, 100% of control value, Mironovskaya 808, seedlings
0.7602
-
effect of water stress on the activity, stress, 2 weeks, control, 62.2% of control value, Mironovskaya 808, leaves
0.8568
-
effect of water stress on the activity, stress, 2 weeks, TDZ, 69.9% of control value, Mironovskaya 808, leaves
0.86
-
effect of water stress on the activity, stress, 2 weeks, BAP, 70.1% of control value, Mironovskaya 808, leaves
0.9806
-
effect of water stress on the activity, stress, 1 day, control, 80.1% of control value, Mironovskaya 808, leaves
0.9878
-
effect of water stress on the activity, stress, 2 weeks, Kartolin-2, 80.7% of control value, Mironovskaya 808, leaves
0.9989
-
effect of water stress on the activity, stress, 1 day, TDZ, 80.8% of control value, Mironovskaya 808, leaves
1.101
-
effect of water stress on the activity, rehydration, control, 89.9% of control value, Mironovskaya 808, leaves; effect of water stress on the activity, stress, 1 day, BAP, 89.9% of control value, Mironovskaya 808, leaves
1.11
-
effect of water stress on the activity, stress, 1 day, Kartolin-2, 90.1% of control value, Mironovskaya 808, leaves
1.138
-
effect of water stress on the activity, rehydration, TDZ, 94.0% of control value, Mironovskaya 808, leaves
1.145
-
effect of water stress on the activity, rehydration, BAP, 94.5% of control value, Mironovskaya 808, leaves
1.187
-
effect of water stress on the activity, rehydration, Kartolin-2, 97.8% of control value, Mironovskaya 808, leaves
1.224
-
effect of water stress on the activity, original, control, 100% of control value, Mironovskaya 808, leaves
4.97
-
pH 7.5, 50C
13.92
-
NAD+-dependent activity
15.4
-
NADP+-dependent activity
740
-
NADH-enzyme activity
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
-
oxidation of D-glyceraldehyde-3-phosphate
7.7
-
native enzyme, reaction with NADPH
7.8
-
native and recombinant enzyme, reaction with NADH
8 - 8.8
-
both directions
8
-
reduction of 3-phospho-D-glyceroyl phosphate
8.3
-
activity assay
8.4
Methanothermobacter thermautotrophicum
-
-
8.5 - 8.8
-
-
8.5
-
both directions
10.5
-
at 60C
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9.1
-
pH 7.0: about 40% of maximal activity, pH 9.1: about 90% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
activity assay
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
very low activity
Manually annotated by BRENDA team
expression of isoforms GapA-1, GapB and phosphoribulokinase and peptide Cp12-2 is co-ordinately regulated with the same organ specificity, all four genes being mostly expressed in leaf and flower stalk, less expressed in flower, and little or not expressed in roots and siliques. Expression in leaf is terminated during prolonged darkness or following sucrose treatment, and their transcripts decay with similar kinetics; tissue with highest activity
Manually annotated by BRENDA team
-
peritoneal
Manually annotated by BRENDA team
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
15000
-
GAPDH III, gel filtration
132000
-
GapA, estimated by gel filtration
148000
-
gel filtration
148300
-
sedimentation analysis
151000
-
B(minCTE) mutant, estimated by gel filtration
152000
-
native A4 glyceraldehyde 3-phosphate dehydrogenase in complex with the small protein CP12, gel filtration
155000
189000
-
tetrameric conformation of GAPDH-A2B2 stabilized by NADPH, estimated by gel filtration
200000
-
gel filtration
210000
-
gel filtration
232000
-
GapB mutant B(E326Q) in the presence of NADPH, estimated by gel filtration
243000
-
wild-type GapB, tetramer in the presence of NADPH, estimated by gel filtration
247000
-
GapB mutant B(S188A) in the presence of NADPH, estimated by gel filtration
270000
-
A(plusCTE) mutant, tetramer in the presence of NADPH, estimated by gel filtration
278000
-
GapB mutant B(R77A) in the presence of NADPH, estimated by gel filtration
300000
-
GAPDH II, gel filtration
491000
-
wild-type GapB in the presence of NADH, compatible with octameric structure, estimated by gel filtration
520000
-
GapB mutant B(E326Q) in the presence of NADH, compatible with octameric structure, estimated by gel filtration; GapB mutant B(R77A) in the presence of NADH, compatible with octameric structure, estimated by gel filtration
553000
-
GapB mutant B(S188A) in the presence of NADH, compatible with octameric structure, estimated by gel filtration
600000
760000
-
oligomer of GAPDH in the presence of NADH, suggesting A8B8 stoichiometry, estimated by gel filtration
820000
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heteromer
-
AB isoform
heterotetramer
-
fully active enzyme
homotetramer
monomer
-
1 * 42000, SDS-PAGE
tetramer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in presence and absence of NADP+, to 2.6 A and 1.74 A resolution, respectively
-
crystal structure of the non-regulatory A(4) isoform of glyceraldehyde-3-phosphate dehydrogenase complexed with NADP+
-
hanging-drop vapour-diffusion method is used to grow crystals of recombinant A4-GAPDH, T33A and S188A A4-GAPDH mutants complexed with NADP+
-
molecular docking of ferredoxin-NADP-reductase EC 1.18.1.2 and GAPD. enzymes are able to form at least two different complexes, one involving a single GAPD monomer and an ferredoxin-NADP-reductase monomer or dimer. The amino acid residues located at the putative interface are highly conserved on the chloroplastic forms of both enzymes. The other potential complex involves the GAPD A2B2 tetramer and an FNR monomer or dimer. Ferredoxin is able to interact with FNR in either complex
-
sitting drop vapour diffusion method with 2.0-2.5 M or 1.5-1.2 M ammonium sulfate and 0.1 M potassium phosphate (pH 7.0-8.0)
-
two crystal forms of the A4-GAPDH isoform are used to solve the structure of the apo form to a resolution of 3.0 A
-
crystallized from ammonium sulfate to produce crystals that diffract to 2.4 A with a space group of P4(3)2(1)2 or P4(1)2(1)2
-
determination of the crystal structure of the apoenzyme by multiple isomorphous replacement at 2.05 A resolution, hanging drop technique. The crystals belong to space group P4(1)2(1)2 or its enantiomorph with cell dimensions a = b = 102.3 A, c = 181.6 A, which contract upon cryocooling at 100 K to a = b = 101.6 A, c = 179.9 A. The asymmetric unit contains two subunits with a molecular mass of 37611 Da
-
the structure of NADP-dependent GAPDH in complex with NADP is solved by molecular replacement and refined to an R factor of 19.1% and a free R factor of 24% at 2.5 A resolution
-
the crystal structure of apo-glyceraldehyde-3-phosphate dehydrogenase is solved by molecular replacement and refined to an R of 21.7% and Rfree of 27.5% at 2.9 A resolution
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
Methanothermobacter thermautotrophicum
-
inactivation above
70
-
40 min, stable
75
-
stable up to
87
-
half-life: 45 min
90
-
stabilized by NADP+ and at high ionic strength up to 90C
95
-
30 min, complete inactivation
96
-
half-life: 130 min
97
-
half-life: 65 min
104
-
half-life: 9 min without stabilizer, stable for 30 min in presence of 250 mM potassium citrate
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
the enzyme is stabilized by glycerol, several salts, especially sodium or potassium phosphate
-
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
oxidation typically leads to about 50% inhibition of the NADPH-dependent activity
-
689747
very sensitive to modification by H2O2, NAD+ promotes inactivation
-
654427
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, GAPDH I is stable for several months, GAPDH II and GAPDH III spontaneously lose activity
-
4C, pH 8.5, protein concentration 2.5 mg/ml, apoprotein, complete loss of activity after 48 h
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
2 isoenzymes: 1 and 2
3 enzyme forms: GAPDH I, GAPDH II, GAPDH III
-
by Ni2+ chelate chromatography and pH-shift under denaturating conditions
-
cell-free preparations for measuring the enzyme activities are obtained
-
DEAE Trisacryl column chromatography
-
heat treatment at 80C is an effective first step in the purification of these recombinant enzymes from extracts of the Escherichia coli host
-
HisTrap column chromatography and Superdex 200 gel filtration
-
native and recombinant enzyme
-
purified from spinach leaves
-
recombinant and native GAPDH are purified to apparent homogeneity from Escherichia coli cells and Chlamydomonas reinhardtii, respectively
-
using a Q-Sepharose HP and a MonoQ anion-exchange column
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned in vector pJF118EH and expressed in Escherichia coli
-
expressed in Escherichia coli
expressed in Escherichia coli BL21(DE3) cells
-
expressed in Escherichia coli C43 (DE3) cells
-
expressed in Saccharomyces cerevisiae mutant AG2 with deletion of NAD+-dependent glycerol-3-phosphate dehydrogenase gene GPD2
-
expression in Escherichia coli
expression in Escherichia coli BL21
-
expression in Escherichia coli. The phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase genes from Sulfolobus solfataricus overlap by 8-bp
-
into the pET29 vector for expression in Escherichia coli BL21DE3 cells
-
into the pJC45Flag vector for expression in the Escherichia coli strain PAPlaclQ DE3, into the vaccination vector pcDNA3.1+ for transformation of DH5alpha cells
-
overexpression in Escherichia coli
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
a slight increase in enzyme activity is observed in the presence of D-fructose 6-phosphate and coenzyme A (1.1fold)
-
ATP, ADP, AMP, D-glucose 6-phosphate and galactose 1-phosphate exhibit no effect on enzyme activity
-
expression of isoforms GapA-1, GapB and phosphoribulokinase and peptide Cp12-2 is co-ordinately regulated with the same organ specificity, all four genes being mostly expressed in leaf and flower stalk, less expressed in flower, and little or not expressed in roots and siliques. Expression in leaf is terminated during prolonged darkness or following sucrose treatment, and their transcripts decay with similar kinetics
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K128A
-
Km-value for NADPH is nearly identical to wild-type value, turnover-number for NADPH is decreased about 2fold. Km-value for NADH is increased 2.1fold compared to wild-type enzyme, turnover-number for NADH is decreased 2.6fold
K128E
-
Km-value for NADPH is about 80% of the wild-type value, turnover-number for NADPH is decreased 2.7fold. Km-value for NADH is increased 2.3fold compared to wild-type enzyme, turnover-number for NADH is decreased 1.9fold
R190A
-
the catalytic constant, kcat, of the mutant in the presence of NADH decreases 10fold while the Km for NADH decreases 12fold. The mutant shows no activity with NADPH
R197A
-
Km-value for NADPH is identical to wild-type value, turnover-number for NADPH is about 90% of the wild-type value. Km-value for NADH is about 80% of the wild-type value, turnover-number for NADH is nearly identical to wild-type value
R197E
-
Km-value for NADPH is increased 1.3fold compared to wild-type enzyme, turnover-number for NADPH is decreased 2fold. Km-value for NADH is about 90% of the wild-type value, turnover-number for NADH is increased 1.3fold
R82A
-
the mutation leads to a 10fold increase in the Km for NADPH but does not affect the kinetics of NADH
R82D
-
the mutation leads to a 10fold increase in the Km for NADPH but does not affect the kinetics of NADH
S195A
-
the mutation has no effect on the affinity of the enzyme for NADPH and its affinity for NADH and for BPGA in the presence of NADH is reduced
K225A
-
K225 is critical for binding of GAPDH to Siah1, an ubiquitin-E3-ligase, eliciting the translocation of GAPDH to the nucleus
Y123W
-
increase of temperature of irreversible inactivation by 1.3C
Y323S
-
decrease of temperature of irreversible inactivation by 4.5C
A(plusCTE)
-
chimeric mutant for testing the regulatory function of CTE
B(E326Q)
-
site specific mutant of the GAPDH B-subunit
B(minCTE)
-
deletion mutant for testing the regulatory function of CTE
B(R77A)
-
site specific mutant of the GAPDH B-subunit
B(S188A)
-
site specific mutant of the GAPDH B-subunit
C18S
-
mutant of GapB subunit still shows stron redox regulation
C274S
-
mutant of GapB subunit still shows stron redox regulation
C285S
-
mutant of GapB subunit still shows stron redox regulation
C349S
-
mutant of GapB subunit is less redox-sensitive than GapB. Active tetramer, unable to aggregate to higher oligomers in presence of NAD+
C349S/C358S
-
mutant of GapB subunit is less redox-sensitive than GapB. Active tetramer, unable to aggregate to higher oligomers in presence of NAD+
c358S
-
mutant of GapB subunit is less redox-sensitive than GapB. Active tetramer, unable to aggregate to higher oligomers in presence of NAD+
D351N
-
the mutation only marginally affects the redox sensitivity
E356Q
-
the mutation only marginally affects the redox sensitivity
E356Q/E357Q
-
complete redox insensitivity is achieved in the double mutant
E357Q
-
the mutation only marginally affects the redox sensitivity
S188A
-
affinity for NADPH is significantly decreased, decrease in the ratio of turnover number to Km-value in the NADPH-dependent reaction, significant expansion of the A4-tetramer
T33A
-
affinity for NADPH is significantly decreased, turnover-number for NADPH is lowered
T33A/S188A
-
affinity for NADPH is significantly decreased, turnover-number for NADPH is lowered
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine