Cloned (Comment) | Organism |
---|---|
expressed in Escherichia coli | [Clostridium] symbiosum |
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 | [Clostridium] symbiosum |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.055 | - |
NADH | wild-type CsGDH, Vmax: 285 micromol/min/mg, pH 8.0, 25°C | [Clostridium] symbiosum | |
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 | [Clostridium] symbiosum | |
0.168 | - |
NAD+ | wild-type CsGDH, Vmax: 40.6 micromol/min/mg, pH 8.0, 25°C | [Clostridium] symbiosum | |
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 | [Clostridium] symbiosum | |
1.39 | - |
L-glutamate | wild-type CsGDH, Vmax: 47.1 micromol/min/mg, pH 8.0, 25°C | [Clostridium] symbiosum | |
2.85 | - |
2-oxoglutarate | wild-type CsGDH, Vmax: 546 micromol/min/mg, pH 8.0, 25°C | [Clostridium] symbiosum | |
12.9 | - |
NH4+ | wild-type CsGDH, Vmax: 307 micromol/min/mg, pH 8.0, 25°C | [Clostridium] symbiosum | |
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 | [Clostridium] symbiosum | |
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 | [Clostridium] symbiosum | |
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 | [Clostridium] symbiosum | |
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 | [Clostridium] symbiosum |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
[Clostridium] symbiosum | - |
- |
- |
Purification (Comment) | Organism |
---|---|
using dye-affintiy chromatography and anion-exchange chromatography | [Clostridium] symbiosum |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
0.49 | - |
chimeric protein CEC consisting of the substrate-binding domain of CsGDH and the coenzyme-binding domain of Escherichia coli GDH using NAD+ | [Clostridium] symbiosum |
58 | - |
chimeric protein CEC consisting of the substrate-binding domain of CsGDH and the coenzyme-binding domain of Escherichia coli GDH using NADP+ | [Clostridium] symbiosum |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-oxoglutarate + NADH + NH3 | - |
[Clostridium] symbiosum | L-glutamate + NAD+ + H2O | - |
r | |
2-oxoglutarate + NADPH + NH3 | - |
[Clostridium] symbiosum | L-glutamate + NADP+ + H2O | - |
r | |
2-oxoglutarate + NH4+ + NADPH | - |
[Clostridium] symbiosum | L-glutamate + NADP+ | - |
? | |
L-glutamate + H2O + NAD+ | - |
[Clostridium] symbiosum | 2-oxoglutarate + NH3 + NADH | - |
r | |
L-glutamate + NADP+ + H2O | - |
[Clostridium] symbiosum | 2-oxoglutarate + NADPH + NH3 | - |
r |
Subunits | Comment | Organism |
---|---|---|
hexamer | native PAGE | [Clostridium] symbiosum |
Synonyms | Comment | Organism |
---|---|---|
CsGDH | - |
[Clostridium] symbiosum |
glutamate dehydrogenase | - |
[Clostridium] symbiosum |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | [Clostridium] symbiosum |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | [Clostridium] symbiosum |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NADH | preferred coenzyme compared to NADP+ | [Clostridium] symbiosum |