Literature summary for 3.1.3.11 extracted from

Brown, G.; Singer, A.; Lunin, V.V.; Proudfoot, M.; Skarina, T.; Flick, R.; Kochinyan, S.; Sanishvili, R.; Joachimiak, A.; Edwards, A.M.; Savchenko, A.; Yakunin, A.F.
Structural and biochemical characterization of the type II D-fructose-1,6-bisphosphatase GlpX from Escherichia coli (2009), J. Biol. Chem., 284, 3784-3792.

Activating Compound

Activating Compound	Comment	Organism	Structure
DTT	increases activity by 40-50%	Escherichia coli
KCl	slightly increases activity by 20%	Escherichia coli

Cloned(Commentary)

Cloned (Comment)	Organism
expressed in Escherichia coli as a His-tagged fusion protein	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
crystal structure of GlpX is determined in a free state and in the complex with a D-fructose 1,6-bisphosphate or inhibitor (phosphate). The crystal structure of the ligand-free GlpX reveals a compact, globular shape with two alpha/beta-sandwich domains. The core fold of GlpX is structurally similar to that of Li+-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with D-fructose 1,6-bisphosphate reveals that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Phosphate (inhibitor) binds to the active site	Escherichia coli

Crystallization (Comment)

Organism

crystal structure of GlpX is determined in a free state and in the complex with a D-fructose 1,6-bisphosphate or inhibitor (phosphate). The crystal structure of the ligand-free GlpX reveals a compact, globular shape with two alpha/beta-sandwich domains. The core fold of GlpX is structurally similar to that of Li+-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with D-fructose 1,6-bisphosphate reveals that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Phosphate (inhibitor) binds to the active site

Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
D186A	Km (mM) (D-fructose 1,6-bisphosphate): 0.2 (wild-type: 0.07 mM), kcat (1/sec)(D-fructose 1,6-bisphosphate): 1.2 (wild-type: 5.7/sec)	Escherichia coli
E59A	Km (mM) (D-fructose 1,6-bisphosphate): 0.1 (wild-type: 0.07 mM), kcat (1/sec)(D-fructose 1,6-bisphosphate): 1.1 (wild-type: 5.7/sec)	Escherichia coli
K239A	Km (mM) (D-fructose 1,6-bisphosphate): 0.1 (wild-type: 0.07 mM), kcat (1/sec)(D-fructose 1,6-bisphosphate): 7.5 (wild-type: 5.7/sec)	Escherichia coli
K29A	Km (mM) (D-fructose 1,6-bisphosphate): 0.06 (wild-type: 0.07 mM), kcat (1/sec)(D-fructose 1,6-bisphosphate): 14 (wild-type: 5.7/sec)	Escherichia coli
R235A	Km (mM) (D-fructose 1,6-bisphosphate): 0.2 (wild-type: 0.07 mM), kcat (1/sec)(D-fructose 1,6-bisphosphate): 5.4 (wild-type: 5.7/sec)	Escherichia coli

Inhibitors

Inhibitors	Comment	Organism
ATP	1 mM, reduces activity by 40%	Escherichia coli
KCl	50 mM, residual activity 20%	Escherichia coli
LiCl	-	Escherichia coli
phosphate	-	Escherichia coli

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.06	-	D-fructose 1,6-bisphosphate	pH 9.0, 37°C, mutant K29A	Escherichia coli
0.07	-	D-fructose 1,6-bisphosphate	pH 9.0, 37°C, wild-type	Escherichia coli
0.1	-	fructose 1,6-bisphosphate	wild-type	Escherichia coli
0.1	-	D-fructose 1,6-bisphosphate	pH 9.0, 37°C, mutant E59A	Escherichia coli
0.1	-	D-fructose 1,6-bisphosphate	pH 9.0, 37°C, mutant K239A	Escherichia coli
0.2	-	D-fructose 1,6-bisphosphate	pH 9.0, 37°C, mutant D186A	Escherichia coli
0.2	-	D-fructose 1,6-bisphosphate	pH 9.0, 37°C, mutant R235A	Escherichia coli

Metals/Ions

Metals/Ions	Comment	Organism
KCl	50 mM, 20% stimulation	Escherichia coli
Mn2+	divalent metal cations are required for enzymatic activity. However, only Mn2+ supports activity of GlpX and YggF	Escherichia coli
Mn2+	divalent metal cations are required for enzymatic activity. Mn2+ supports activity of GlpX and YggF	Escherichia coli

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
34300	-	2 * 34300, calculated from sequence	Escherichia coli
36000	-	2 * 36000, calculated from sequence	Escherichia coli
76000	-	dimer, gel filtration	Escherichia coli

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P0A9C9	Escherichia coli has two class II D-fructose 1,6-bisphosphatase GlpX and YggF	-
Escherichia coli	P21437	uncharacterized protein YggF; Escherichia coli has two class II fructose 1,6-bisphosphatase GlpX and YggF	-

Purification (Commentary)

Purification (Comment)	Organism
using Ni-NTA chromatography	Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
D-fructose 1,6-bisphosphate + H2O	-	Escherichia coli	D-fructose 6-phosphate + phosphate	-	?
D-glucose 1,6-bisphosphate + H2O	-	Escherichia coli	D-glucose 6-phosphate + phosphate	-	?

Subunits

Subunits	Comment	Organism
homodimer	2 * 34300, calculated from sequence	Escherichia coli
homodimer	2 * 36000, calculated from sequence	Escherichia coli

Synonyms

Synonyms	Comment	Organism
D-fructose 1,6-bisphosphatase	-	Escherichia coli
fructose 1,6-bisphosphatase	-	Escherichia coli
GlpX	-	Escherichia coli
YggF	-	Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
1.1	-	D-fructose 1,6-bisphosphate	pH 9.0, 37°C, mutant E59A	Escherichia coli
1.2	-	D-fructose 1,6-bisphosphate	pH 9.0, 37°C, mutant D186A	Escherichia coli
2.5	-	fructose 1,6-bisphosphate	wild-type	Escherichia coli
5.4	-	D-fructose 1,6-bisphosphate	pH 9.0, 37°C, mutant R235A	Escherichia coli
5.7	-	D-fructose 1,6-bisphosphate	pH 9.0, 37°C, wild-type	Escherichia coli
7.5	-	D-fructose 1,6-bisphosphate	pH 9.0, 37°C, mutant K239A	Escherichia coli
14	-	D-fructose 1,6-bisphosphate	pH 9.0, 37°C, mutant K29A	Escherichia coli

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	pH optimum: 7.5-8.0	Escherichia coli

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor
1.2	-	-	Escherichia coli	phosphate
3	-	-	Escherichia coli	phosphate
15.8	-	-	Escherichia coli	LiCl
70	-	-	Escherichia coli	LiCl