Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli RM448 cells | Geobacillus stearothermophilus |
Crystallization (Comment) | Organism |
---|---|
vapor diffusion method, using 80 mM sodium acetate, 20% (w/v) polyethylene glycol 4000, and 6.4% (v/v) isopropanol | Geobacillus stearothermophilus |
Protein Variants | Comment | Organism |
---|---|---|
W336A | the mutant shows about 28fold reduced catalytic efficiency compared to the wild type | Geobacillus stearothermophilus |
W336D | the mutant shows severely reduced catalytic efficiency compared to the wild type | Geobacillus stearothermophilus |
W336F | the mutant shows about 3fold increased catalytic efficiency compared to the wild type | Geobacillus stearothermophilus |
W336N | the mutant shows about 10fold reduced catalytic efficiency compared to the wild type | Geobacillus stearothermophilus |
W336S | the mutant shows about 100fold reduced catalytic efficiency compared to the wild type | Geobacillus stearothermophilus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.21 | - |
4-nitrophenyl alpha-D-galactopyranoside | wild type enzyme isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
0.24 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336F of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
0.44 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336A of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
0.83 | - |
4-nitrophenyl alpha-D-galactopyranoside | wild type enzyme isoform AgaB, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
0.84 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336N of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
1.33 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336D of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
3.8 | - |
raffinose | wild type enzyme isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
8.7 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336S of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
36.6 | - |
raffinose | mutant enzyme W336S of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
200 | - |
raffinose | wild type enzyme isoform AgaB, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
205 | - |
raffinose | mutant enzyme W336N of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
250 | - |
raffinose | mutant enzyme W336F of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
252 | - |
raffinose | mutant enzyme W336A of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Geobacillus stearothermophilus | Q9ALJ4 | - |
- |
Purification (Comment) | Organism |
---|---|
DMAE column chromatography, Mono Q column chromatography and Superdex 200 gel filtration | Geobacillus stearothermophilus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
4-nitrophenyl alpha-D-galactopyranoside + H2O | - |
Geobacillus stearothermophilus | 4-nitrophenol + alpha-D-galactopyranose | - |
? | |
raffinose + H2O | - |
Geobacillus stearothermophilus | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
AgaA | isoform | Geobacillus stearothermophilus |
AgaB | isoform | Geobacillus stearothermophilus |
Alpha-D-galactoside galactohydrolase | - |
Geobacillus stearothermophilus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.97 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336D of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
5.2 | - |
raffinose | mutant enzyme W336S of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
7.9 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336A of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
10.3 | - |
raffinose | mutant enzyme W336N of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
38.2 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336N of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
48 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336S of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
48.1 | - |
raffinose | mutant enzyme W336A of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
105.6 | - |
4-nitrophenyl alpha-D-galactopyranoside | wild type enzyme isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
180 | - |
raffinose | wild type enzyme isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
375 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336F of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
832 | - |
raffinose | mutant enzyme W336F of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
1205 | - |
raffinose | wild type enzyme isoform AgaB, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
2382 | - |
4-nitrophenyl alpha-D-galactopyranoside | wild type enzyme isoform AgaB, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.05 | - |
raffinose | mutant enzyme W336N of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
0.14 | - |
raffinose | mutant enzyme W336S of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
0.19 | - |
raffinose | mutant enzyme W336A of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
0.7 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336D of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
3.3 | - |
raffinose | mutant enzyme W336F of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
5.5 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336S of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
6 | - |
raffinose | wild type enzyme isoform AgaB, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
17.9 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336A of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
45.5 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336N of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
64.3 | - |
raffinose | wild type enzyme isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
502 | - |
4-nitrophenyl alpha-D-galactopyranoside | wild type enzyme isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
1562 | - |
4-nitrophenyl alpha-D-galactopyranoside | mutant enzyme W336F of isoform AgaA, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus | |
2870 | - |
4-nitrophenyl alpha-D-galactopyranoside | wild type enzyme isoform AgaB, in 100 mM potassium phosphate buffer, pH 6.5, at 25°C | Geobacillus stearothermophilus |