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Literature summary for 3.1.3.11 extracted from

  • Topaz, G.R.; Epiter-Smith, V.; Robolo, C.; Emad, M.; Ford, V.; Daley, J.; Byron, J.; Stieglitz, K.A.
    Characterization of recombinant fructose 1,6-bisphosphatase (FBPase) gene mutations evidence of inhibition/activation of FBPase protein by gene mutation (2019), Biosci. Rep., 39, BSR20180960.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene FBP1, sequence comparisons of human and porcine enzymes, recombinant overexpression of C-terminally His6-tagged wild-type and mutant pig kidney FBPases in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain XL-1 Blue supercompetent cells Sus scrofa
gene FBP1, sequence comparisons of human and porcine kidney enzymes, recombinant overexpression of C-terminally His6-tagged human kidney FBPase in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain XL-1 Blue supercompetent cells Homo sapiens

Protein Variants

Protein Variants Comment Organism
K112A site-directed mutagenesis, mutation in the AMP-binding site to eliminate AMP hydrogen bonding to amino acids in the binding pocket Sus scrofa
L56A site-directed mutagenesis, interfacial mutant, that displays an about 5fold increased Ki for D-fructose 2,6-bisphosphate compared to wild-type Sus scrofa
L73A site-directed mutagenesis, interfacial mutant, that displays an about 5fold increased Ki for D-fructose 2,6-bisphosphate compared to wild-type Sus scrofa
M177A site-directed mutagenesis, the mutant data correlates with clinical data Sus scrofa
M248D site-directed mutagenesis, active site mutant, that displays an about 7fold increase in Ki for D-fructose 2,6-bisphosphate, a 4fold decrease in its apparent Km, and a 6fold increase in catalytic efficiency as compared to wild-type. The M248 residue is mutated to aspartic acid in an attempt to activate the enzyme as a means to enhance its binding affinity to the activating metals manganese and magnesium Sus scrofa
Y113A site-directed mutagenesis, mutation in the AMP-binding site to eliminate AMP hydrogen bonding to amino acids in the binding pocket Sus scrofa
Y164A site-directed mutagenesis, the mutant data correlates with clinical data Sus scrofa

Inhibitors

Inhibitors Comment Organism Structure
(4-[3-(6,7-diethoxy-quinazolin-4-ylamino)-phenyl]-thiazol-2-yl)-methanol i.e. PFE, allosteric inhibitor Homo sapiens
(4-[3-(6,7-diethoxy-quinazolin-4-ylamino)-phenyl]-thiazol-2-yl)-methanol i.e. PFE, allosteric inhibitor, residue L56 coordinates the (4-[3-(6,7-diethoxy-quinazolin-4-ylamino)-phenyl]-thiazol-2-yl)-methanol (PFE) inhibitor ligand, as does residue L73, both of which exhibit hydrophobic interactions with the ligand in the PFE-binding site. In addition, L73 and L56 are part of a network that leads from the allosteric binding site to the active site of the enzyme. This hydrophobic network, also involving residues V48 and L120 may stabilize previously described hydrogen bonding networks including residues R49, S169, and D127, shown in the network, leading to the active site where the metal binds D121, D118, and E280. The M177 and Y164 interfacial residues are positioned between the AMP-binding site and active sites Sus scrofa
AMP allosteric inhibitor Homo sapiens
AMP allosteric inhibitor Sus scrofa
D-fructose 2,6-bisphosphate a natural heterotropic inhibitor Homo sapiens
D-fructose 2,6-bisphosphate a natural heterotropic inhibitor Sus scrofa
additional information the FBPase pig kidney tetramer overlay of human and pig kidney (PDB IDs 1FTA and 1KZ8, respectively) show nearly identical orientation and conformation in the active site, AMP allosteric binding site, and inhibitor (4-[3-(6,7-diethoxy-quinazolin-4-ylamino)-phenyl]-thiazol-2-yl)-methanol allosteric binding site architecture Homo sapiens
additional information the FBPase pig kidney tetramer overlay of human and pig kidney (PDB IDs 1FTA and 1KZ8, respectively) show nearly identical orientation and conformation in the active site, AMP allosteric binding site, and inhibitor (4-[3-(6,7-diethoxy-quinazolin-4-ylamino)-phenyl]-thiazol-2-yl)-methanol allosteric binding site architecture Sus scrofa

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information Michaelis-Menten kinetics Sus scrofa
additional information
-
additional information Michaelis-Menten kinetics Homo sapiens
0.00125
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant M248D Sus scrofa
0.0025
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant Y113A Sus scrofa
0.00325
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant K112A Sus scrofa
0.0042 0.0048 D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant M177A Sus scrofa
0.0048
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant wild-type enzyme Sus scrofa
0.0051
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant L73A Sus scrofa
0.0052
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant L56A Sus scrofa
0.0053
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant Y164A Sus scrofa

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ activates, required for activity Homo sapiens
Mg2+ activates, required for activity, Ka values for wild-type and mutant enzymes, kinetics, overview Sus scrofa
Mn2+ activates Sus scrofa
Mn2+ activates Homo sapiens

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
200000
-
about, recombinant wild-type and mutant enzymes, gel filtration Sus scrofa

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
D-fructose 1,6-bisphosphate + H2O Sus scrofa
-
D-fructose 6-phosphate + phosphate
-
?
D-fructose 1,6-bisphosphate + H2O Homo sapiens
-
D-fructose 6-phosphate + phosphate
-
?

Organism

Organism UniProt Comment Textmining
Homo sapiens P09467
-
-
Sus scrofa P00636
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged kidney FBPase from Escherichia coli strain BL21(DE3) by crude cell extract by dialysis and nickel affinity chromatography, cleavage of the His-tag causing low solubility of the human enzyme Homo sapiens
recombinant His6-tagged wild-type and mutant pig kidney FBPases from Escherichia coli strain BL21(DE3) crude cell extract by dialysis and nickel affinity chromatography, cleavage of the His-tag, and gel filtration Sus scrofa

Source Tissue

Source Tissue Comment Organism Textmining
kidney
-
Sus scrofa
-
kidney
-
Homo sapiens
-
liver
-
Sus scrofa
-
liver
-
Homo sapiens
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
D-fructose 1,6-bisphosphate + H2O
-
Sus scrofa D-fructose 6-phosphate + phosphate
-
?
D-fructose 1,6-bisphosphate + H2O
-
Homo sapiens D-fructose 6-phosphate + phosphate
-
?

Subunits

Subunits Comment Organism
homotetramer
-
Sus scrofa
homotetramer
-
Homo sapiens
More structure comparisons of human and porcine enzymes, overview. The FBPase pig kidney tetramer overlay of human and pig kidney (PDB IDs 1FTA and 1KZ8, respectively) show nearly identical orientation and conformation in the active site, AMP allosteric binding site, and inhibitor (4-[3-(6,7-diethoxy-quinazolin-4-ylamino)-phenyl]-thiazol-2-yl)-methanol allosteric binding site architecture Homo sapiens
More structure comparisons of human and porcine kidney enzymes, overview. The FBPase pig kidney tetramer overlay of human and pig kidney (PDB IDs 1FTA and 1KZ8, respectively) show nearly identical orientation and conformation in the active site, AMP allosteric binding site, and inhibitor (4-[3-(6,7-diethoxy-quinazolin-4-ylamino)-phenyl]-thiazol-2-yl)-methanol allosteric binding site architecture Sus scrofa

Synonyms

Synonyms Comment Organism
FBP1
-
Sus scrofa
FBP1
-
Homo sapiens
FBPase
-
Sus scrofa
FBPase
-
Homo sapiens
fructose 1,6-bisphosphatase
-
Sus scrofa
fructose 1,6-bisphosphatase
-
Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
assay at Sus scrofa
37
-
assay at Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
11.5 12.8 D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant M177A Sus scrofa
13.5
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant Y164A Sus scrofa
20.5
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant wild-type enzyme Sus scrofa
22.5
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant L73A Sus scrofa
25.6
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant L56A Sus scrofa
26.8
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant K112A Sus scrofa
27.2
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant Y113A Sus scrofa
30.5
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant M248D Sus scrofa

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Sus scrofa
7.5
-
assay at Homo sapiens

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.00015
-
D-fructose 2,6-bisphosphate pH 7.5, 37°C, recombinant mutant Y164A Sus scrofa
0.00017
-
D-fructose 2,6-bisphosphate pH 7.5, 37°C, recombinant mutant M177A Sus scrofa
0.00024
-
D-fructose 2,6-bisphosphate pH 7.5, 37°C, recombinant mutant K112A Sus scrofa
0.00025
-
D-fructose 2,6-bisphosphate pH 7.5, 37°C, recombinant wild-type enzyme Sus scrofa
0.00025
-
D-fructose 2,6-bisphosphate pH 7.5, 37°C, recombinant mutant Y113A Sus scrofa
0.00125
-
D-fructose 2,6-bisphosphate pH 7.5, 37°C, recombinant mutant L56A Sus scrofa
0.00135
-
D-fructose 2,6-bisphosphate pH 7.5, 37°C, recombinant mutant L73A Sus scrofa
0.0015
-
D-fructose 2,6-bisphosphate pH 7.5, 37°C, recombinant mutant M248D Sus scrofa

IC50 Value

IC50 Value IC50 Value Maximum Comment Organism Inhibitor Structure
0.00062 0.00075 pH 7.5, 37°C, recombinant mutant M177A Sus scrofa D-fructose 2,6-bisphosphate
0.00095
-
pH 7.5, 37°C, recombinant mutant Y164A Sus scrofa D-fructose 2,6-bisphosphate
0.0015
-
pH 7.5, 37°C, recombinant wild-type enzyme Sus scrofa D-fructose 2,6-bisphosphate
0.0015
-
pH 7.5, 37°C, recombinant mutant K112A Sus scrofa D-fructose 2,6-bisphosphate
0.0017 0.0018 pH 7.5, 37°C, recombinant mutant Y113A Sus scrofa D-fructose 2,6-bisphosphate
0.0025 0.0035 pH 7.5, 37°C, recombinant mutant Y164A Sus scrofa AMP
0.0035 0.0039 pH 7.5, 37°C, recombinant mutant M177A Sus scrofa AMP
0.00695
-
pH 7.5, 37°C, recombinant mutant L73A Sus scrofa AMP
0.0072
-
pH 7.5, 37°C, recombinant mutant M248D Sus scrofa AMP
0.00725
-
pH 7.5, 37°C, recombinant mutant L73A Sus scrofa D-fructose 2,6-bisphosphate
0.00735
-
pH 7.5, 37°C, recombinant mutant L56A Sus scrofa AMP
0.0075
-
pH 7.5, 37°C, recombinant wild-type enzyme Sus scrofa AMP
0.0075
-
pH 7.5, 37°C, recombinant mutant L56A Sus scrofa D-fructose 2,6-bisphosphate
0.0105
-
pH 7.5, 37°C, recombinant mutant M248D Sus scrofa D-fructose 2,6-bisphosphate
0.0486 0.0553 pH 7.5, 37°C, recombinant mutant Y113A Sus scrofa AMP
0.0523 0.0685 pH 7.5, 37°C, recombinant mutant K112A Sus scrofa AMP

General Information

General Information Comment Organism
malfunction two significant mutations in the coding region of the FBPase gene in patients with hypoglycemia link the AMP-binding site to the active site of the enzyme. Individuals with FBPase deficiency exhibit hypoglycemia and metabolic acidosis due to impaired gluconeogenesis. In rare cases, hypoglycemia, an autosomal recessive disorder characterized by insufficient blood glucose levels, is genetically linked to FBPase deficiency in clinical studies. The M177 and Y164 interfacial residues are positioned between the AMP-binding site and active sites and are mutated in humans with hypoglycemia Homo sapiens
metabolism enzyme FBPase is a key rate-controlling enzyme in the gluconeogenic pathway Sus scrofa
metabolism enzyme FBPase is a key rate-controlling enzyme in the gluconeogenic pathway Homo sapiens
additional information sequence comparisons of human and porcine FBPase 1, overview. Residue L56 coordinates the (4-[3-(6,7-diethoxy-quinazolin-4-ylamino)-phenyl]-thiazol-2-yl)-methanol (PFE) inhibitor ligand, as does residue L73, both of which exhibit hydrophobic interactions with the ligand in the PFE-binding site. In addition, L73 and L56 are part of a network that leads from the allosteric binding site to the active site of the enzyme. Residue M248 is positioned near the triad of acidic residues co-ordinating manganese and is found in the active site to co-ordinate D-fructose 6-phosphate Sus scrofa
additional information sequence comparisons of human and porcine FBPase 1, overview. The M177 and Y164 interfacial residues are positioned between the AMP-binding site and active sites Homo sapiens
physiological function enzyme FBPase is a key rate-controlling enzyme in the gluconeogenic pathway. FBPase activity is regulated synergistically by the allosteric inhibitors AMP and fructose-2,6-bisphosphate (F2,6-BP). FBPase functions in the degradation of fructose-1,6-bisphosphate (FBP), which hydrolyzes to fructose-6-phosphate (F6P) and phosphate Sus scrofa
physiological function enzyme FBPase is a key rate-controlling enzyme in the gluconeogenic pathway. FBPase activity is regulated synergistically by the allosteric inhibitors AMP and fructose-2,6-bisphosphate. FBPase functions in the degradation of fructose-1,6-bisphosphate, which hydrolyzes to fructose-6-phosphate and phosphate Homo sapiens

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2547
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant Y164A Sus scrofa
2667
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant M177A Sus scrofa
4271
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant wild-type enzyme Sus scrofa
4412
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant L73A Sus scrofa
4923
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant L56A Sus scrofa
8246
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant K112A Sus scrofa
10880
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant Y113A Sus scrofa
24400
-
D-fructose 1,6-bisphosphate pH 7.5, 37°C, recombinant mutant M248D Sus scrofa