Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli strain BL21(DE3) pLysS | Candida albicans |
Protein Variants | Comment | Organism |
---|---|---|
additional information | expression of truncated enzyme variants as His-tagged proteins. Fragments encompassing residues 1-345 and 346-712 represent the functional glutamine amide-hydrolysing GAH and hexose phosphate-isomerizing domains ISOM, respectively. The native GAH domain is monomeric, whereas the native ISOM domain forms tetramers, as does the whole enzyme. The binding site for the feedback inhibitor, uridine 5'-diphospho-N-acetyl-D-glucosamine, is located in the ISOM domain. Inhibitor binding affects amidohydrolysing activity of the GAH domain and, as a consequence, the D-glucosamine-6-phosphate-synthetic activity of the whole enzyme. The fragment containing residues 218-283 is neither involved in ligand binding nor in protein oligomerization. An intramolecular channel connects the GAH and ISOM domains. The channel becomes leaky upon deletion of amino acids 709-712 and in the W97F and W97G mutants | Candida albicans |
V711F | mutation results in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities | Candida albicans |
V711F | reduction of the glucosamine 6-phosphate-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities | Candida albicans |
W97F | mutation results in an almost complete elimination of the GlcN-6-P synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities | Candida albicans |
W97F | reduction of the glucosamine 6-phosphate-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities. Residue W97 functions as a molecular gate, opening and closing the intramolecular channel that connects the glutamine amide-hydrolysing and hexose phosphate-isomerizing domains | Candida albicans |
W97G | mutation results in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities | Candida albicans |
W97G | almost complete elimination of the glucosamine 6-phosphate-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities. Residue W97 functions as a molecular gate, opening and closing the intramolecular channel that connects the glutamine amide-hydrolysing and hexose phosphate-isomerizing domains | Candida albicans |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.31 | - |
L-gamma-glutamyl-p-nitroanilide | residues 1-345, His-tagged glutamine amide-hydrolysing domain, pH 7.5, 25°C | Candida albicans | |
0.34 | - |
L-glutamate | reaction mixture contains 10 mM L-glutamate, 1 mM EDTA, 1 mM DTT and an appropriately diluted enzyme preparation in 20 mM HEPES buffer (pH 7.5) at 25°C | Candida albicans | |
0.34 | - |
L-glutamine | glutamine 6-phosphate-synthetic activity, wild-type, pH 7.5, 25°C | Candida albicans | |
0.64 | - |
L-gamma-glutamyl-p-nitroanilide | wild-type with C-terminal His-tag, pH 7.5, 25°C | Candida albicans | |
0.67 | - |
L-gamma-glutamyl-p-nitroanilide | wild-type, pH 7.5, 25°C | Candida albicans | |
0.67 | - |
L-gamma -glutamyl-p-nitroanilide | reaction mixture contains 1 mM L-gamma-glutamyl-p-nitroanilide, 1 mM EDTA, 1 mM DTT and the appropriately diluted enzyme preparation in 20 mM HEPES buffer (pH 7.5) at 25°C | Candida albicans | |
1.08 | - |
D-fructose 6-phosphate | hexose phosphate-isomerizing activity, wild-type with N-terminal His-tag, pH 7.5, 25°C | Candida albicans | |
1.2 | - |
D-fructose 6-phosphate | reaction mixture contains 2 mM D-fructose 6-phosphate, 0.5 mM NADP+, 1 mM EDTA, and appropriately diluted GlcN-6-P synthase preparation in 50 mM Tris/HCl buffer (pH 7.5) at 25°C | Candida albicans | |
1.2 | - |
D-fructose 6-phosphate | hexose phosphate-isomerizing activity, wild-type, pH 7.5, 25°C | Candida albicans | |
1.4 | - |
D-fructose 6-phosphate | hexose phosphate-isomerizing activity, residues 346-712, His-tagged hexose phosphate-isomerizing domain, pH 7.5, 25°C | Candida albicans | |
1.41 | - |
D-fructose 6-phosphate | glutamine 6-phosphate-synthetic activity, wild-type, pH 7.5, 25°C | Candida albicans | |
6.5 | - |
D-fructose 6-phosphate | hexose phosphate-isomerizing activity, wild-type with C-terminal His-tag, pH 7.5, 25°C | Candida albicans | |
12.2 | - |
L-gamma-glutamyl-p-nitroanilide | wild-type with N-terminal His-tag, pH 7.5, 25°C | Candida albicans |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
39500 | - |
4 * 39500, hexose phosphate-isomerizing domain, Superdex 200 HR 10/30 gel filtration | Candida albicans |
41000 | - |
glutamine amide-hydrolysing domain, SDS-PAGE | Candida albicans |
42000 | - |
1 * 42000, glutamine amide-hydrolysing domain, Superdex 200 HR 10/30 gel filtration | Candida albicans |
159000 | - |
hexose phosphate-isomerizing domain, Superdex 200 HR 10/30 gel filtration | Candida albicans |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Candida albicans | - |
- |
- |
Purification (Comment) | Organism |
---|---|
Ni2+-IDA agarose affinity chromatography and HiTrap desalting column chromatography | Candida albicans |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.021 | - |
glutamine 6-phosphate-synthetic activity, mutant V711F, pH 7.5, 25°C | Candida albicans |
0.05 | - |
glutamine 6-phosphate-synthetic activity, mutant W97G, pH 7.5, 25°C | Candida albicans |
0.095 | - |
amidohydrolysing activity of the mutant enzyme W97G using D-fructose 6-phosphate as substrate | Candida albicans |
0.256 | - |
amidohydrolysing activity of the mutant enzyme W97F using D-fructose 6-phosphate as substrate | Candida albicans |
0.89 | - |
amidohydrolysing activity of the wild type enzyme using D-fructose 6-phosphate as substrate | Candida albicans |
0.94 | - |
amidohydrolysing activity of the mutant enzyme V711F using D-fructose 6-phosphate as substrate | Candida albicans |
1.2 | - |
glutamine 6-phosphate-synthetic activity, mutant W97F, pH 7.5, 25°C | Candida albicans |
12 | - |
glutamine 6-phosphate-synthetic activity, wild-type, pH 7.5, 25°C | Candida albicans |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-fructose 6-phosphate | determination of hexose phosphate-isomerizing activity | Candida albicans | D-glucose 6-phosphate | - |
? | |
L-gamma-glutamyl-p-nitroanilide + D-fructose 6-phosphate | - |
Candida albicans | ? | - |
? | |
L-gamma-glutamyl-p-nitroanilide + H2O | determination of amidohydrolysing activity | Candida albicans | L-glutamine + p-nitroaniline | - |
? | |
L-glutamine + D-fructose 6-phosphate | - |
Candida albicans | L-glutamate + D-glucosamine 6-phosphate | - |
? |
Subunits | Comment | Organism |
---|---|---|
homotetramer | 4 * 39500, hexose phosphate-isomerizing domain, Superdex 200 HR 10/30 gel filtration | Candida albicans |
monomer | 1 * 42000, glutamine amide-hydrolysing domain, Superdex 200 HR 10/30 gel filtration | Candida albicans |
Synonyms | Comment | Organism |
---|---|---|
Gfa1p | - |
Candida albicans |
glucosamine-6-phosphate synthase | - |
Candida albicans |
L-glutamine:L-fructose-6-phosphate amidotransferase | - |
Candida albicans |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00092 | - |
L-gamma-glutamyl-p-nitroanilide | wild-type with N-terminal His-tag, pH 7.5, 25°C | Candida albicans | |
0.004 | - |
D-fructose 6-phosphate | hexose phosphate-isomerizing activity, wild-type with C-terminal His-tag, pH 7.5, 25°C | Candida albicans | |
0.0075 | - |
L-gamma-glutamyl-p-nitroanilide | residues 1-345, His-tagged glutamine amide-hydrolysing domain, pH 7.5, 25°C | Candida albicans | |
0.0092 | - |
D-fructose 6-phosphate | hexose phosphate-isomerizing activity, residues 346-712, His-tagged hexose phosphate-isomerizing domain, pH 7.5, 25°C | Candida albicans | |
0.012 | - |
L-gamma-glutamyl-p-nitroanilide | wild-type, pH 7.5, 25°C | Candida albicans | |
0.0123 | - |
L-gamma-glutamyl-p-nitroanilide | reaction mixture contains 1 mM L-gamma-glutamyl-p-nitroanilide, 1 mM EDTA, 1 mM DTT and the appropriately diluted enzyme preparation in 20 mM HEPES buffer (pH 7.5) at 25°C | Candida albicans | |
0.013 | - |
L-gamma-glutamyl-p-nitroanilide | wild-type with C-terminal His-tag, pH 7.5, 25°C | Candida albicans | |
0.021 | - |
D-fructose 6-phosphate | hexose phosphate-isomerizing activity, wild-type with N-terminal His-tag, pH 7.5, 25°C | Candida albicans | |
0.021 | - |
D-fructose 6-phosphate | hexose phosphate-isomerizing activity, wild-type, pH 7.5, 25°C | Candida albicans | |
0.0213 | - |
D-fructose 6-phosphate | reaction mixture contains 2 mM D-fructose 6-phosphate, 0.5 mM NADP+, 1 mM EDTA, and appropriately diluted GlcN-6-P synthase preparation in 50 mM Tris/HCl buffer (pH 7.5) at 25°C | Candida albicans | |
12 | - |
L-glutamate | reaction mixture contains 10 mM L-glutamate, 1 mM EDTA, 1 mM DTT and an appropriately diluted enzyme preparation in 20 mM HEPES buffer (pH 7.5) at 25°C | Candida albicans | |
12 | - |
L-glutamine | glutamine 6-phosphate-synthetic activity, wild-type, pH 7.5, 25°C | Candida albicans |