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Literature summary for 1.4.1.4 extracted from

  • Sharkey, M.A.; Engel, P.C.
    Modular coenzyme specificity: a domain-swopped chimera of glutamate dehydrogenase (2009), Proteins, 77, 268-278.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli Escherichia coli

Protein Variants

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 [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

Organism UniProt Comment Textmining
Escherichia coli
-
-
-

Purification (Commentary)

Purification (Comment) Organism
using dye-affintiy chromatography and anion-exchange chromatography Escherichia coli

Specific Activity [micromol/min/mg]

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 and Products (Substrate)

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

Subunits Comment Organism
hexamer native PAGE Escherichia coli

Synonyms

Synonyms Comment Organism
EcGDH
-
Escherichia coli
glutamate dehydrogenase
-
Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
assay at Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Escherichia coli

Cofactor

Cofactor Comment Organism Structure
NADPH preferred coenzyme compared to NADH Escherichia coli