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Literature summary for 1.4.1.2 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
Search for more literature for 1.4.1.2

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli [Clostridium] symbiosum

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 [Clostridium] symbiosum

KM Value [mM]

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

Organism UniProt Comment Textmining
[Clostridium] symbiosum
-
-
-

Purification (Commentary)

Purification (Comment) Organism
using dye-affintiy chromatography and anion-exchange chromatography [Clostridium] symbiosum

Specific Activity [micromol/min/mg]

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 and Products (Substrate)

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

Subunits Comment Organism
hexamer native PAGE [Clostridium] symbiosum

Synonyms

Synonyms Comment Organism
CsGDH
-
 [Clostridium] symbiosum
glutamate dehydrogenase
-
 [Clostridium] symbiosum

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at [Clostridium] symbiosum

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
 assay at [Clostridium] symbiosum

Cofactor

Cofactor Comment Organism Structure
NADH preferred coenzyme compared to NADP+ [Clostridium] symbiosum