Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli | Escherichia coli |
Protein Variants | Comment | Organism |
---|---|---|
additional information | an active chimera (CEC) consisting of the substrate-binding domain (domain I) of CsGDH and the coenzyme-binding domain (domain II) of Escherichia coli GDH is generated. Kinetic constants of chimeric protein: Km values for substrates L-glutamate, 2-oxoglutarate, NH4Cl highly increased compared to wild-type, Vmax values also highly increased compared to wild-type. The CEC chimera, like Escherichia coli GDH, has a marked preference for NADP(H) as coenzyme. selectivity for the phosphorylated coenzyme does indeed reside solely in domain II. Positive cooperativity toward L-glutamate, characteristic of wild-type CsGDH, retains with domain I. Although glutamate cooperativity occurs only at higher pH values in the wild-tpye CsGDH, the chimeric protein shows it over the full pH range explored. The chimera is capable of catalyzing severalfold higher reaction rates (Vmax) in both directions than either of the parent enzymes from which it is constructed | Escherichia coli |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.018 | - |
NADP+ | wild-type EcGDH, Vmax: 45.6 micromol/min/mg, pH 8.0, 25°C | Escherichia coli | |
0.06 | - |
NADPH | wild-type EcGDH, Vmax: 503 micromol/min/mg, pH 8.0, 25°C | Escherichia coli | |
0.163 | - |
NADP+ | chimeric protein CEC consisting of the substrate-binding domain of CsGDH and the coenzyme-binding domain of Escherichia coli GDH, Vmax: 80.8 micromol/min/mg, pH 8.0, 25°C | Escherichia coli | |
0.51 | - |
NADPH | chimeric protein CEC consisting of the substrate-binding domain of CsGDH and the coenzyme-binding domain of Escherichia coli GDH, Vmax: 2180 micromol/min/mg, pH 8.0, 25°C | Escherichia coli | |
0.68 | - |
2-oxoglutarate | wild-type EcGDH, Vmax: 464 micromol/min/mg, pH 8.0, 25°C | Escherichia coli | |
2.3 | - |
L-glutamate | wild-type EcGDH, Vmax: 37.9 micromol/min/mg, pH 8.0, 25°C | Escherichia coli | |
2.5 | 3 | NH4+ | wild-type EcGDH, Vmax: 298 micromol/min/mg, pH 8.0, 25°C | Escherichia coli | |
285 | - |
2-oxoglutarate | chimeric protein CEC consisting of the substrate-binding domain of CsGDH and the coenzyme-binding domain of Escherichia coli GDH, 781 mM NH4Cl, Vmax: 2260 micromol/min/mg, pH 8.0, 25°C | Escherichia coli | |
304 | - |
NH4+ | chimeric protein CEC consisting of the substrate-binding domain of CsGDH and the coenzyme-binding domain of Escherichia coli GDH, Vmax: 1960 micromol/min/mg, pH 8.0, 25°C | Escherichia coli | |
606 | - |
2-oxoglutarate | chimeric protein CEC consisting of the substrate-binding domain of CsGDH and the coenzyme-binding domain of Escherichia coli GDH, 1800 mM NH4Cl, Vmax: 200 micromol/min/mg, pH 8.0, 25°C | Escherichia coli | |
1349 | - |
L-glutamate | chimeric protein CEC consisting of the substrate-binding domain of CsGDH and the coenzyme-binding domain of Escherichia coli GDH, Vmax: 121.9 micromol/min/mg, pH 8.0, 25°C | Escherichia coli |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Escherichia coli | - |
- |
- |
Purification (Comment) | Organism |
---|---|
using dye-affintiy chromatography and anion-exchange chromatography | Escherichia coli |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.33 | - |
wild-type EcGDH using NAD+ as coenzyme | Escherichia coli |
0.49 | - |
chimeric protein CEC consisting of the substrate-binding domain of CsGDH and the coenzyme-binding domain of Escherichia coli GDH using NAD+ as coenzyme | Escherichia coli |
58 | - |
chimeric protein CEC consisting of the substrate-binding domain of CsGDH and the coenzyme-binding domain of Escherichia coli GDH using NADP+ as coenzyme | Escherichia coli |
63 | - |
wild-type EcGDH using NADP+ as coenzyme | Escherichia coli |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-oxoglutarate + NADPH + NH3 | - |
Escherichia coli | L-glutamate + NADP+ + H2O | - |
r | |
2-oxoglutarate + NH4+ + NADPH | - |
Escherichia coli | L-glutamate + NADP+ | - |
? | |
L-glutamate + H2O + NADP+ | - |
Escherichia coli | 2-oxoglutarate + NH3 + NADPH | - |
r |
Subunits | Comment | Organism |
---|---|---|
hexamer | native PAGE | Escherichia coli |
Synonyms | Comment | Organism |
---|---|---|
EcGDH | - |
Escherichia coli |
glutamate dehydrogenase | - |
Escherichia coli |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Escherichia coli |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Escherichia coli |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADPH | preferred coenzyme compared to NADH | Escherichia coli |