Information on EC 2.7.1.146 - ADP-specific phosphofructokinase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.7.1.146
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RECOMMENDED NAME
GeneOntology No.
ADP-specific phosphofructokinase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ADP + D-fructose 6-phosphate = AMP + D-fructose 1,6-bisphosphate
show the reaction diagram
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phospho group transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
-
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Biosynthesis of secondary metabolites
-
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gluconeogenesis II (Methanobacterium thermoautotrophicum)
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Glycolysis / Gluconeogenesis
-
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glycolysis V (Pyrococcus)
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Metabolic pathways
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Methane metabolism
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Microbial metabolism in diverse environments
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Pentose phosphate pathway
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glycolysis
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SYSTEMATIC NAME
IUBMB Comments
ADP:D-fructose-6-phosphate 1-phosphotransferase
ADP can be replaced by GDP, ATP and GTP, to a limited extent. Divalent cations are necessary for activity, with Mg2+ followed by Co2+ being the most effective.
CAS REGISTRY NUMBER
COMMENTARY hide
237739-62-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
; bifunctional ADP-dependent phosphofructokinase/glucokinase, reactions of EC 2.7.1.147 and EC 2.7.1.146, respectively
UniProt
Manually annotated by BRENDA team
strain OT3
UniProt
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
Embden-Meyerhof glycolytic pathway, early step
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
acetyl-phosphate + D-fructose 6-phosphate
acetate + D-fructose 1,6-bisphosphate
show the reaction diagram
ADP + D-fructose 6-phosphate
AMP + D-fructose 1,6-bisphosphate
show the reaction diagram
ADP + D-glucose
AMP + D-glucose 6-phosphate
show the reaction diagram
AMP + D-fructose 1,6-bisphosphate
ADP + D-fructose 6-phosphate
show the reaction diagram
-
-
-
r
CDP + beta-D-fructose 6-phosphate
CMP + D-fructose 1,6-bisphosphate
show the reaction diagram
about 15% compared to the activity with ADP
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ir
D-fructose 6-phosphate + ADP
D-fructose 1,6-bisphosphate + AMP
show the reaction diagram
D-fructose 6-phosphate + CDP
D-fructose 1,6-bisphosphate + CMP
show the reaction diagram
D-fructose 6-phosphate + GDP
D-fructose 1,6-bisphosphate + GMP
show the reaction diagram
D-fructose 6-phosphate + IDP
D-fructose 1,6-bisphosphate + IMP
show the reaction diagram
D-fructose 6-phosphate + UDP
D-fructose 1,6-bisphosphate + UMP
show the reaction diagram
UDP + D-glucose
UMP + D-glucose 6-phosphate
show the reaction diagram
the enzyme phosphorylates both D-glucose and D-fructose 6-phosphate. Activity with UDP and D-glucose is about 20% compared to the activity with ADP and D-glucose
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r
additional information
?
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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
ADP + D-fructose 6-phosphate
AMP + D-fructose 1,6-bisphosphate
show the reaction diagram
ADP + D-glucose
AMP + D-glucose 6-phosphate
show the reaction diagram
Q6LXQ3
the enzyme phosphorylates both D-glucose and D-fructose 6-phosphate. Binding of both substrates to the same active site. At a sugar concentration of 10 mM the acctivity with D-fructose 6-phosphate is about 75% compared to the activity with D-glucose. No activity in presence of ATP. kcat/KM for the phosphorylation of D-fructose 6-phosphate is 440fold higher than the kcat/Km for the phosphorylation of glucose. Analysis of the kcat/Km ratios shows that the glucose dephosphorylation is 2fold more effective than the phosphorylation
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-
r
D-fructose 6-phosphate + ADP
D-fructose 1,6-bisphosphate + AMP
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CaCl2
2 mM ADP and 2 mM D-fructose 6-phosphate, CaCl2 show the lowest activity with 75% of the activity measured in the presence of MgCl2, activity does not change with the cation concentration in the range of 2.5 to 7 mM
Fe2+
-
16% of activity with Mg2+
MgSO4
2 mM ADP and 2 mM D-fructose 6-phosphate, magnesium is tested with 2 counter ions to discard any effect of the anion
MnCl2
2 mM ADP and 2 mM D-fructose 6-phosphate, increase in enzyme activity with the increase in MnCl2 concentration from 2.5 to 7 mM is observed
NiSO4
2 mM ADP and 2 mM D-fructose 6-phosphate, highest PhPFK activity is obtained with NiSO4 (97 units/mg), decrease in enzyme activity with the increase in MnCl2 concentration from 2.5 to 7 mM is observed
Pb2+
-
40% of activity
ZnCl2
2 mM ADP and 2 mM D-fructose 6-phosphate, no significant activity
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ADP
; strong inhibition effect in the reverse glucokinase reaction
Ca2+
about 50% activity at 2.5 mM
D-fructose 6-phosphate
; strong inhibition of glucokinase activity, competitive inhibion versus D-glucose as variable substrate
D-glucose
; substrate inhibition above 200 mM. At 10 mM 20% inhibition of phosphofructokinase activity
EDTA
50 mM, complete inhibition; complete loss of activity; completely abolishes activity
KCl
500 mM, 16% inhibition
Mg2+
about 60% activity at 2.5 mM
Mn2+
about 70% activity at 2.5 mM
NaCl
500 mM, 12% inhibition
Zn2+
no activity detected in presence of Zn2+
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
D-glucosamine
10 mM, 40% activation of glucokinase activity
additional information
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no allosterical regulation by ADP, AMP, phosphoenolpyruvate, or citrate
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
11.9
acetyl-phosphate
pH 6.5, 50°C
0.00747 - 8
ADP
1.4
AMP
-
pH 6.6, 80°C
0.1 - 5.7
D-fructose 1,6-bisphosphate
0.00395 - 65
D-fructose 6-phosphate
40
D-glucose
pH 6.5, 30°C
0.1 - 2
D-glucose 6-phosphate
pH 6.5, 30°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
30 - 98
ADP
0.45
D-fructose 1,6-bisphosphate
Methanococcus maripaludis
Q6LXQ3
pH 6.5, 30°C; pH 7.0, 30°C
0.0048 - 151
D-fructose 6-phosphate
23
D-glucose
Methanococcus maripaludis
Q6LXQ3
pH 6.5, 30°C
0.45
D-glucose 6-phosphate
Methanococcus maripaludis
Q6LXQ3
pH 6.5, 30°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.345 - 8400
ADP
13
3.75
D-fructose 1,6-bisphosphate
Methanococcus maripaludis
Q6LXQ3
pH 6.5, 30°C; pH 7.0, 30°C
106
0.0876 - 2990
D-fructose 6-phosphate
87
0.57
D-glucose
Methanococcus maripaludis
Q6LXQ3
pH 6.5, 30°C
35
1.09
D-glucose 6-phosphate
Methanococcus maripaludis
Q6LXQ3
pH 6.5, 30°C
105
additional information
ADP
Pyrococcus horikoshii
O59355
the calculated value for UDP is 40times higher than that of ADP, indicating a strong preference for ADP over UDP as the phosphoryl group donor
13
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
54
ADP
pH 6.5, 30°C; pH 7.0, 30°C
0.4
AMP
pH 7.5, 30°C, reverse glucokinase reaction
0.226
D-fructose 6-phosphate
pH 7.5, 30°C, glucokinase reaction
895
D-glucose
pH 7.5, 30°C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.08
ADP
Methanococcus maripaludis
Q6LXQ3
pH 7.5, 30°C, reverse glucokinase reaction
40
D-fructose
Methanococcus maripaludis
Q6LXQ3
pH 7.5, 30°C, phosphopfructokinase reaction
0.22
D-fructose 6-phosphate
Methanococcus maripaludis
Q6LXQ3
pH 6.5, 30°C; pH 7.5, 30°C, glucokinase reaction
40
D-glucose
Methanococcus maripaludis
Q6LXQ3
pH 6.5, 30°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.01
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substrate: D-fructose 6-phosphate, 50°C, pH not specified in the publication, enzyme from lactate-grown cells
0.11
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substrate: D-fructose 6-phosphate, 50°C, pH not specified in the publication, enzyme from starch-grown cells
8.2
; pH 6.5, 50°C, direction of phosphorylation
12
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purified enzyme, determined at pH 6.6, 80°C, reverse reaction
157
purified recombinant enzyme
392
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purified enzyme, determined at pH 6.6, 50°C, forward reaction
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
; glucokinase activity
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.9 - 9
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50% of maximal activity at pH 6.1 and pH 7.9
5 - 7
pH 5.0: about 50% of maximal activity, pH 7.0: about 65% of maximal activity; pH 5.0: about 50% of maximal phosphofructokinase activity, pH 7.0: about 65% of maximal phosphofructokinase activity
5.5 - 7.5
pH 5.5: about 50% of maximal phosphofructokinase activity, pH 7.5: about 75% of maximal phosphofructokinase activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
specific activity is 11fold higher in starch-grown cells than in lactate-grown cells
Manually annotated by BRENDA team
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specific activity is 11fold higher in starch-grown cells than in lactate-grown cells
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
42200
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x * 42200, SDS-PAGE
50000
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2 * 50000, SDS-PAGE; 4 * 50000, SDS-PAGE
50100
native PAGE; non-denaturing PAGE
52700
calculation from sequence
53000
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1 * 53000, SDS-PAGE
53362
1 * 53362, calculated from sequence
53900
4 * 53900, SDS-PAGE
55000
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high pressure liquid chromatography
66000
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gel filtration
100000
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dimeric enzyme form, gel filtration at higher protein concentration
125000
recombinant enzyme, gel filtration
180000
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gel filtration
200000
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 42200, SDS-PAGE
monomer
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
molecular modeling of structure. for binding of ADP, residues M347, I431 and L441 create a hydrophobic pocket around the adenine group. R194 makes a hydrogen bond with alpha and beta phosphates, carbonyl and NH groups from V432 peptide bond make a hydrogen bond with the NH2 group of C6 and the N1 atom of adenine
hanging drop vapor diffusion method, the apo-enzyme is crystallized using 22% PEG 4000, 0.1 M Tris-HCl, pH 8.5, and 0.2 M LiSO4, at 21°C. Crystals of the PFK complex with AMP are obtained by crystallization of the PFK D17A protein in the presence of 20% PEG 3350, 0.2 M lithium citrate, 10 mM D-fructose 6-phosphate, and 5 mM ADP; through hanging drop vapor diffusion at 21°C by mixing protein solution with solution consisting of 22% PEG 4000, 0.1 M Tris-HCl, pH 8.5, and 0.2 M Li2SO4, crystals of the ADP-dependent 6-phosphofructokinase complex with AMP are obtained by crystallization of the mutant protein D17A protein in the presence of 20% PEG 3350, 0.2 M lithium citrate, 10 mM fructose 6-phosphate, and 5 mM ADPcrystal structure of ADP-dependent 6-phosphofructokinase in both apo- and AMP-bound forms determined to 2.0 A and 1.9 A resolution
purified recombinant enzyme, sitting drop vapour diffusion method, 0.005 ml of 10 mg/ml protein in 150 mM NaCl, 5 mM Tris-HCl, pH 7.5, is mixed with an equal volume of reservoir solution containing 4 M NaCl, 100 mM Tris-HCl, pH 7.5, 1 week at 25°C, derivatization by soaking for 2 days in 2.85 mM K2PtCl4, 2.8 mM HAuCl4, and 2 mM 4-hydroxymercuribenzoic acid or 0.4 mM 4-hydroxymercuribenzenesulfonic acid, cryoprotection by 20% glycerol in mother liquor, X-ray diffraction structure determination and analysis at 2.6 A resolution
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80
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120 min, completely stable
100
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120 min, completely loss of activity after 60 min, in presence of 1 M NaCl the enzyme retains 30% activity after 90 min, in presence of 1 M KCl the enzyme retains 50% activity after 90 min
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, purified native enzyme, quite stable
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
1818fold to homogeneity by ultracentrifugation, anion exchange, ADP affinity, and cation exchange chromatography, and gel filtration
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by using nickel-nitrilotriacetic acid affinity chromatography and gel filtration; Ni-NTA column chromatography
recombinant enzyme from Escherichia coli strain JM109 by ammonium sulfate fractionation, adsorption and ion exchange chromatography, and gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, expression in Escherichia coli strain JM109
expressed in Escherichia coli BL21(DE3) and DL41(DE3) cells; recombinant wild type and mutant native and selenomethionine-labeled proteins are expressed in BL21(DE3) and DL41(DE3) Escherichia coli cells
expression in Escherichia coli
expression in Escherichia coli; expression in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A71E
production by site-directed mutagenesis; the mutant shows slightly decreased kcat compared to the wild type enzyme
D17A
production by site-directed mutagenesis; the mutant shows severely decreased kcat compared to the wild type enzyme
D433A
production by site-directed mutagenesis, loss of PFK activity; the mutant shows no acitivity
K158A
production by site-directed mutagenesis; the mutant shows slightly decreased kcat compared to the wild type enzyme
N15A
production by site-directed mutagenesis; the mutant shows increased kcat compared to the wild type enzyme
N160A
production by site-directed mutagenesis; the mutant shows strongly increased kcat compared to the wild type enzyme
Q224A
production by site-directed mutagenesis; the mutant shows increased kcat compared to the wild type enzyme
Q97A
production by site-directed mutagenesis; the mutant shows decreased kcat compared to the wild type enzyme
R185A
production by site-directed mutagenesis; the mutant shows no acitivity
R191A
production by site-directed mutagenesis; the mutant shows decreased kcat compared to the wild type enzyme
R191E
production by site-directed mutagenesis; the mutant shows almost wild type enzyme activity
S183A
the mutant shows strongly increased kcat compared to the wild type enzyme
S183E
production by site-directed mutagenesis, the S183E mutant displays significantly less activity when the phosphoryl donor is replaced with IDP, UDP, GDP, or CDP compared with the wild type; the mutant displays significantly less activity when the phosphoryl donor ADP is replaced with IDP, UDP, GDP, or CDP, compared with the wild type
S189A
production by site-directed mutagenesis; the mutant shows decreased kcat compared to the wild type enzyme
S263A
production by site-directed mutagenesis; the mutant shows increased kcat compared to the wild type enzyme
D17A
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the mutant shows severely decreased kcat compared to the wild type enzyme
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K158A
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the mutant shows slightly decreased kcat compared to the wild type enzyme
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N160A
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the mutant shows strongly increased kcat compared to the wild type enzyme
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R191A
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the mutant shows decreased kcat compared to the wild type enzyme
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S183A
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the mutant shows strongly increased kcat compared to the wild type enzyme
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