Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli BL21(DE3) cells | Thermus thermophilus |
Crystallization (Comment) | Organism |
---|---|
complexed with N-(5'-phosphopyridoxyl)-L-glutamate, vapor diffusion method, using 16% (w/v) PEG3350, 100 mM HEPES, pH 7.0, and 200 mM calcium acetate, at 20°C | Thermus thermophilus |
Protein Variants | Comment | Organism |
---|---|---|
R23A | the mutation causes 4.1fold increase in the Km value for L-2-aminoadipate, the Km value for L-Glu increases 546fold, the Km value for L-Leu is not greatly affected | Thermus thermophilus |
R23Q | the mutation causes 13.7fold increase in the Km value for L-2-aminoadipate, the Km value for L-Glu increases 134fold, the Km value for L-Leu is not greatly affected | Thermus thermophilus |
S20E | the mutant shows decreased kcat values compared to the wild type enzyme | Thermus thermophilus |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.048 | - |
2-oxoisocaproate | mutant enzyme R23A, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.05 | - |
2-oxoisocaproate | mutant enzyme S20E, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.091 | - |
2-oxoisocaproate | mutant enzyme R23Q, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.2 | - |
L-leucine | mutant enzyme S20E, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.27 | - |
2-oxoglutarate | wild type enzyme, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.3 | - |
2-oxoglutarate | wild type enzyme, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.39 | - |
L-leucine | mutant enzyme R23A, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.4 | - |
2-oxoglutarate | mutant enzyme S20E, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.41 | - |
2-oxoglutarate | mutant enzyme S20E, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.42 | - |
2-oxoglutarate | mutant enzyme R23Q, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.43 | - |
2-oxoisocaproate | wild type enzyme, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.46 | - |
L-glutamate | wild type enzyme, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.54 | - |
2-oxoglutarate | mutant enzyme R23A, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.55 | - |
2-oxoglutarate | mutant enzyme R23Q, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.61 | - |
2-oxoglutarate | mutant enzyme R23A, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.81 | - |
L-2-aminoadipate | wild type enzyme, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.88 | - |
L-leucine | mutant enzyme R23Q, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.95 | - |
L-leucine | wild type enzyme, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
3.3 | - |
L-2-aminoadipate | mutant enzyme R23A, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
7 | - |
L-2-aminoadipate | mutant enzyme S20E, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
11 | - |
L-2-aminoadipate | mutant enzyme R23Q, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
62 | - |
L-glutamate | mutant enzyme R23Q, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
82 | - |
L-glutamate | mutant enzyme S20E, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
250 | - |
L-glutamate | mutant enzyme R23A, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Thermus thermophilus | Q72LL6 | - |
- |
Purification (Comment) | Organism |
---|---|
ammonium sulfate precipitation and Superdex 200 gel filtration | Thermus thermophilus |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-oxoisocaproate + L-glutamate | - |
Thermus thermophilus | ? | - |
? | |
L-2-aminoadipate + 2-oxoglutarate | - |
Thermus thermophilus | 2-oxoadipate + L-glutamate | - |
? | |
L-leucine + 2-oxoglutarate | - |
Thermus thermophilus | 4-methyl-2-oxopentanoate + L-glutamate | - |
? | |
additional information | negligible activity is observed for L-Asp | Thermus thermophilus | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
AAA-AT | - |
Thermus thermophilus |
alpha-aminoadipate aminotransferase | - |
Thermus thermophilus |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.009 | - |
L-leucine | mutant enzyme R23A, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.011 | - |
L-2-aminoadipate | mutant enzyme R23A, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.023 | - |
L-leucine | mutant enzyme S20E, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.04 | - |
L-2-aminoadipate | mutant enzyme S20E, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.05 | - |
L-leucine | mutant enzyme R23Q, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.083 | - |
L-2-aminoadipate | mutant enzyme R23Q, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.23 | - |
L-glutamate | mutant enzyme R23A, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.5 | - |
L-glutamate | mutant enzyme R23Q, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
1.2 | - |
L-glutamate | mutant enzyme S20E, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
5.1 | - |
L-leucine | wild type enzyme, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
6.7 | - |
L-2-aminoadipate | wild type enzyme, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
16 | - |
L-glutamate | wild type enzyme, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
pyridoxal 5'-phosphate | - |
Thermus thermophilus |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.0009 | - |
L-glutamate | mutant enzyme R23A, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.0033 | - |
L-2-aminoadipate | mutant enzyme R23A, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.0057 | - |
L-2-aminoadipate | mutant enzyme S20E, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.0075 | - |
L-2-aminoadipate | mutant enzyme R23Q, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.0081 | - |
L-glutamate | mutant enzyme R23Q, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.014 | - |
L-glutamate | mutant enzyme S20E, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.015 | - |
2-oxoglutarate | mutant enzyme R23A, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.021 | - |
2-oxoglutarate | mutant enzyme R23A, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.023 | - |
L-leucine | mutant enzyme R23A, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.057 | - |
L-leucine | mutant enzyme R23Q, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.06 | - |
2-oxoglutarate | mutant enzyme S20E, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.091 | - |
2-oxoglutarate | mutant enzyme R23Q, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.097 | - |
2-oxoglutarate | mutant enzyme S20E, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.12 | - |
L-leucine | mutant enzyme S20E, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
0.2 | - |
2-oxoglutarate | mutant enzyme R23Q, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
4.8 | - |
2-oxoisocaproate | mutant enzyme R23A, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
5.4 | - |
L-leucine | wild type enzyme, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
5.5 | - |
2-oxoisocaproate | mutant enzyme R23Q, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
8.2 | - |
L-2-aminoadipate | wild type enzyme, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
19 | - |
2-oxoglutarate | wild type enzyme, using L-leucine as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
22 | - |
2-oxoglutarate | wild type enzyme, using L-2-aminoadipate as cosubstrate, in 50 mM HEPES-NaOH buffer, pH 8.0, 50 mM KCl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
24 | - |
2-oxoisocaproate | mutant enzyme S20E, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
34 | - |
2-oxoisocaproate | wild type enzyme, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus | |
37 | - |
L-glutamate | wild type enzyme, using 2-oxoisocaproate as cosubstrate, in 50 mM HEPESNaOH buffer, pH 8.0, containing 50 mM NH4Cl, 0.15 mM pyridoxal 5'-phosphate, at 45°C | Thermus thermophilus |