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 | 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 | 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 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 | 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 Maximum [Da] | Comment | Organism |
---|---|---|---|
200000 | - |
about, recombinant wild-type and mutant enzymes, gel filtration | Sus scrofa |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | P09467 | - |
- |
Sus scrofa | P00636 | - |
- |
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 | Comment | Organism | Textmining |
---|---|---|---|
kidney | - |
Sus scrofa | - |
kidney | - |
Homo sapiens | - |
liver | - |
Sus scrofa | - |
liver | - |
Homo sapiens | - |
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 | 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 | 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 Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Sus scrofa |
37 | - |
assay at | Homo sapiens |
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Sus scrofa |
7.5 | - |
assay at | Homo sapiens |
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 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 | 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 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 |