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Literature summary for 2.2.1.6 extracted from
- Characterization of acetohydroxyacid synthase from the hyperthermophilic bacterium (2015), Biochem. Biophys. Rep., 4, 89-97.
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expressed in Escherichia coli BL21 (DE3) Rosetta 2 cells | Thermotoga maritima |
| expression in Escherichia coli | Thermotoga maritima |
| recombinant expression of the catalytic subunit in Escherichia coli strain BL21 (DE3) Rosetta 2, subcloning in Escherichia coli strain DH5alpha, reconstitution of the catalytic and regulatory subunits leads to increased AHAS activity | Thermotoga maritima |
| recombinant expression of the regulatory subunit in Escherichia coli strain BL21 (DE3) Rosetta 2, subcloning in Escherichia coli strain DH5alpha, reconstitution of the catalytic and regulatory subunits leads to increased AHAS activity | Thermotoga maritima |
General Stability
| General Stability | Organism |
|---|---|
| stirring has detrimental effects on activity. When stirred, both aerobic and anaerobic aliquots lose major portions of their activity at almost the same rate resulting in a time required for the loss of its 50% activity of about 18 h | Thermotoga maritima |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | Michaelis-Menten steady-state kinetic analysis, overview | Thermotoga maritima | |
| additional information | - |
additional information | activity is dependent on the ionic strength of the buffer and diminishes considerably (approximately 80%) when assayed in buffers with less than 100 mM concentrations. At concentrations higher than 100 mM the activity levels are quite similar (tested up to 500 mM) | Thermotoga maritima | |
| 16.4 | - |
pyruvate | pH 7.0, 80°C | Thermotoga maritima |  |
| 16.4 | - |
pyruvate | pH 7.0, 85°C, recombinant catalytic subunit | Thermotoga maritima | |
| 16.4 | - |
pyruvate | at pH 7.0 and 80°C | Thermotoga maritima | |
Metals/Ions
| Metals/Ions | Comment | Organism | Structure |
|---|---|---|---|
| Mg2+ | required | Thermotoga maritima | |
| additional information | the AHAS activity is dependent on the ionic strength of the buffer and diminishes considerably (approximately 80%) when assayed in buffers with less than 100 mM concentrations. At concentrations higher than 100 mM the activity levels are quite similar (up to 500 mM is tested), suggesting it might be needed to maintain the functional oligomeric state of the enzyme | Thermotoga maritima |
Molecular Weight [Da]
| Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
|---|---|---|---|
| 20508 | - |
2 * 20508, regulatory subunit, sequence calculation, 2 * 22210, regulatory subunit, SDS-PAGE | Thermotoga maritima |
| 20508 | - |
2 * 65497 + 2 * 20508, calculated from amino acid sequence | Thermotoga maritima |
| 22200 | - |
1 * 65497, calculated, 1 * 66400, SDS-PAGE, plus 1 * 2058, calculated, 1 * 22200, SDS-PAGE, catalytic and regulatory subunit, respectively | Thermotoga maritima |
| 22200 | - |
2 * 66400 + 2 * 22200, SDS-PAGE | Thermotoga maritima |
| 22210 | - |
2 * 20508, regulatory subunit, sequence calculation, 2 * 22210, regulatory subunit, SDS-PAGE | Thermotoga maritima |
| 37700 | - |
recombinant catalytic subunit, gel filtration | Thermotoga maritima |
| 65497 | - |
2 * 65497, catalytic subunit, sequence calculation, 2 * 66398, catalytic subunit, SDS-PAGE | Thermotoga maritima |
| 65497 | - |
1 * 65497, calculated, 1 * 66400, SDS-PAGE, plus 1 * 2058, calculated, 1 * 22200, SDS-PAGE, catalytic and regulatory subunit, respectively | Thermotoga maritima |
| 65497 | - |
2 * 65497 + 2 * 20508, calculated from amino acid sequence | Thermotoga maritima |
| 66398 | - |
2 * 65497, catalytic subunit, sequence calculation, 2 * 66398, catalytic subunit, SDS-PAGE | Thermotoga maritima |
| 66400 | - |
1 * 65497, calculated, 1 * 66400, SDS-PAGE, plus 1 * 2058, calculated, 1 * 22200, SDS-PAGE, catalytic and regulatory subunit, respectively | Thermotoga maritima |
| 66400 | - |
2 * 66400 + 2 * 22200, SDS-PAGE | Thermotoga maritima |
| 156000 | - |
recombinant catalytic subunit, gel filtration | Thermotoga maritima |
| 156400 | - |
gel filtration | Thermotoga maritima |
| 156800 | - |
gel filtration | Thermotoga maritima |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 2 pyruvate | Thermotoga maritima | - |
2-acetolactate + CO2 | - |
? | |
| pyruvate + 2-oxobutyrate | Thermotoga maritima | - |
2-aceto-2-hydroxybutyrate + CO2 | - |
? | |
| pyruvate + pyruvate | Thermotoga maritima | - |
2-acetolactate + CO2 | - |
? | |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Thermotoga maritima | - |
- |
- |
| Thermotoga maritima | Q9WZ19 | small subunit | - |
| Thermotoga maritima | Q9WZ19 and Q9WZ18 | Q9WZ19 i.e. regulatory subunit, Q9WZ18 i.e. catalytic subunit | - |
| Thermotoga maritima DSM 3109 | Q9WZ19 and Q9WZ18 | Q9WZ19 i.e. regulatory subunit, Q9WZ18 i.e. catalytic subunit | - |
Oxidation Stability
| Oxidation Stability | Organism |
|---|---|
| enzyme is not oxygen sensitive | Thermotoga maritima |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
- |
Thermotoga maritima |
| DEAE-Sepharose column chromatography and hydroxyapatite column chromatography | Thermotoga maritima |
| recombinant catalytic and regulatory subunits from Escherichia coli strain BL21(DE3) Rosetta 2, the catalytic subunit is purified 30.2fold by heat treatement, and anion exchange and hydroxyapatite chromatography | Thermotoga maritima |
| recombinant regulatory subunit from Escherichia coli strain BL21(DE3) Rosetta 2, the regulatory subunit is purified to homogeneity by heat treatment at 80°C for 1h and nickel affinity chromatography | Thermotoga maritima |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 5.4 | - |
crude extract, at pH 7.0 and 80°C | Thermotoga maritima |
| 163 | - |
pH 7.0, 85°C, purified recombinant catalytic subunit | Thermotoga maritima |
| 163 | - |
after 30.2fold purification, at pH 7.0 and 80°C | Thermotoga maritima |
Storage Stability
| Storage Stability | Organism |
|---|---|
| 0°C, purified recombinant enzyme, 5 days, 35-40% loss of activity | Thermotoga maritima |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| 2 pyruvate | - |
Thermotoga maritima | 2-acetolactate + CO2 | - |
? | |
| 2 pyruvate | - |
Thermotoga maritima DSM 3109 | 2-acetolactate + CO2 | - |
? | |
| pyruvate + 2-oxobutyrate | - |
Thermotoga maritima | 2-aceto-2-hydroxybutyrate + CO2 | - |
? | |
| pyruvate + pyruvate | - |
Thermotoga maritima | 2-acetolactate + CO2 | - |
? | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| dimer | 2 * 20508, regulatory subunit, sequence calculation, 2 * 22210, regulatory subunit, SDS-PAGE | Thermotoga maritima |
| dimer | 2 * 65497, catalytic subunit, sequence calculation, 2 * 66398, catalytic subunit, SDS-PAGE | Thermotoga maritima |
| dimer | 1 * 65497, calculated, 1 * 66400, SDS-PAGE, plus 1 * 2058, calculated, 1 * 22200, SDS-PAGE, catalytic and regulatory subunit, respectively | Thermotoga maritima |
| heterotetramer | 2 * 65497 + 2 * 20508, calculated from amino acid sequence | Thermotoga maritima |
| heterotetramer | 2 * 66400 + 2 * 22200, SDS-PAGE | Thermotoga maritima |
| More | the enzyme consists of a catalytic and a regulatory subunit | Thermotoga maritima |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| acetohydroxy acid synthase | - |
Thermotoga maritima |
| acetohydroxyacid synthase | - |
Thermotoga maritima |
| AHAS | - |
Thermotoga maritima |
| AHASL | - |
Thermotoga maritima |
| AHASS | - |
Thermotoga maritima |
| TM_0548 | - |
Thermotoga maritima |
| TM_0549 | - |
Thermotoga maritima |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 85 | - |
- |
Thermotoga maritima |
Temperature Range [°C]
| Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
|---|---|---|---|
| additional information | - |
incubation at either 70 or 80°C results in enhanced activity with the highest activity achieved after 1 h of incubation, longer treatments result in reduced activity | Thermotoga maritima |
| 50 | 100 | activity range, overview | Thermotoga maritima |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 98 | - |
pyruvate | pH 7.0, 80°C | Thermotoga maritima | |
| 98 | - |
pyruvate | pH 7.0, 85°C, recombinant catalytic subunit | Thermotoga maritima | |
| 98 | - |
pyruvate | at pH 7.0 and 80°C | Thermotoga maritima | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7 | - |
- |
Thermotoga maritima |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 4.5 | 10.5 | activity range, overview | Thermotoga maritima |
| 5.5 | 8 | more than 50% between pH 5.5 and 8.0 | Thermotoga maritima |
Cofactor
| Cofactor | Comment | Organism | Structure |
|---|---|---|---|
| FAD | - |
Thermotoga maritima | |
| FAD | required | Thermotoga maritima | |
| FAD | apparent Km value 0.00015 mM | Thermotoga maritima | |
| thiamine diphosphate | - |
Thermotoga maritima | |
| thiamine diphosphate | dependent on | Thermotoga maritima | |
| thiamine diphosphate | apparent Km value 0.057 mM | Thermotoga maritima | |
General Information
| General Information | Comment | Organism |
|---|---|---|
| metabolism | acetohydroxyacid synthase is the key enzyme in branched chain amino acid biosynthesis pathway, overview | Thermotoga maritima |
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Release 2023.1 (January 2023)
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