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Literature summary for 6.3.5.4 extracted from

  • Meyer, M.E.; Gutierrez, J.A.; Raushel, F.M.; Richards, N.G.
    A conserved glutamate controls the commitment to acyl-adenylate formation in asparagine synthetase (2010), Biochemistry, 49, 9391-9401.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 6.3.5.4

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli as a His-tagged fusion protein Escherichia coli

Protein Variants

Protein Variants Comment Organism
E348A mutant exhibits similar glutaminase activity to the wild-type enzyme in the absence of ATP, but is not capable of catalyzing asparagine formation when glutamine is employed as a nitrogen source Escherichia coli
E348A mutant shows similar glutaminase activity to the wild-type enzyme in the absence of ATP, mutant is not capable of catalyzing asparagine formation when glutamine is employed as a nitrogen source, mutant shows no synthetase activity, ATP-dependent stimulation of glutaminase activity is less than that of wild-type enzyme Escherichia coli
E348D mutant exhibits similar glutaminase activity to the wild-type enzyme in the absence of ATP and is capable of catalyzing asparagine formation when glutamine is employed as a nitrogen source. Formation of the beta-aspartyl-AMP intermediate, and therefore the eventual production of asparagine, is dependent on the presence of a carboxylate side chain at this position in the synthetase active site. In addition, E348 may also play a role in mediating the conformational changes needed to coordinate, albeit weakly, the glutaminase and synthetase activities of the enzyme and to establish the structural integrity of the intramolecular tunnel along which ammonia is translocated Escherichia coli
E348D mutant shows similar glutaminase activity to the wild-type enzyme in the absence of ATP, mutant is capable of catalyzing asparagine formation when glutamine is employed as a nitrogen source, glutaminase activity of the E348D mutant is stimulated more than 6fold relative to wild-type by the presence of 5 mM ATP, a 5fold decrease in the Km value for aspartate is observed for both the glutamine- and ammonia-dependent synthetase reactions, together with a kcat that is half of that observed for the wildtype enzyme Escherichia coli
E348Q mutant exhibits similar glutaminase activity to the wild-type enzyme in the absence of ATP, but is not capable of catalyzing asparagine formation when glutamine is employed as a nitrogen source Escherichia coli
E348Q mutant shows similar glutaminase activity to the wild-type enzyme in the absence of ATP, mutant is not capable of catalyzing asparagine formation when glutamine is employed as a nitrogen source, mutant shows no synthetase activity, ATP-dependent stimulation of glutaminase activity is less than that of wild-type enzyme Escherichia coli

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.013
-
 ATP mutant E348D, glutamine-dependent activity, pH 8.0, 37°C Escherichia coli
0.013
-
 ATP mutant E348D, synthetase activity, glutamine-dependent activity, 20 mM L-Gln, pH 8.0, 37°C Escherichia coli
0.03
-
 ATP mutant E348D, ammonia-dependent activity, pH 8.0, 37°C Escherichia coli
0.03
-
 ATP mutant E348D, synthetase activity, ammonia-dependent activity, 100 mM NH4Cl, pH 8.0, 37°C Escherichia coli
0.1
-
 ATP wild-type, glutamine-dependent activity, pH 8.0, 37°C Escherichia coli
0.1
-
 ATP wild-type, synthetase activity, glutamine-dependent activity, 20 mM L-Gln, pH 8.0, 37°C Escherichia coli
0.11
-
 ATP wild-type, ammonia-dependent activity, pH 8.0, 37°C Escherichia coli
0.11
-
 ATP wild-type, synthetase activity, ammonia-dependent activity, 100 mM NH4Cl, pH 8.0, 37°C Escherichia coli
0.13
-
 L-Asp mutant E348D, glutamine-dependent activity, pH 8.0, 37°C Escherichia coli
0.13
-
 L-Asp mutant E348D, synthetase activity, glutamine-dependent activity, 20 mM L-Gln, pH 8.0, 37°C Escherichia coli
0.23
-
 L-Asp mutant E348D, ammonia-dependent activity, pH 8.0, 37°C Escherichia coli
0.23
-
 L-Asp mutant E348D, synthetase activity, ammonia-dependent activity, 100 mM NH4Cl, pH 8.0, 37°C Escherichia coli
0.58
-
 L-Asp wild-type, glutamine-dependent activity, pH 8.0, 37°C Escherichia coli
0.58
-
 L-Asp wild-type, synthetase activity, glutamine-dependent activity, 20 mM L-Gln, pH 8.0, 37°C Escherichia coli
1.1
-
 L-Gln mutant E348D, glutaminase activity, pH 8.0, 37°C, 5 mM ATP present Escherichia coli
1.2
-
 L-Asp wild-type, ammonia-dependent activity, pH 8.0, 37°C Escherichia coli
1.2
-
 L-Asp wild-type, synthetase activity, ammonia-dependent activity, 100 mM NH4Cl, pH 8.0, 37°C Escherichia coli
1.7
-
 L-Gln wild-type, glutaminase activity, pH 8.0, 37°C, 5 mM ATP present Escherichia coli
2.7
-
 L-Gln mutant E348D, glutaminase activity, pH 8.0, 37°C, ATP absent Escherichia coli
3.5
-
 L-Gln mutant E348A, glutaminase activity, pH 8.0, 37°C, 5 mM ATP present Escherichia coli
3.9
-
 L-Gln mutant E348Q, glutaminase activity, pH 8.0, 37°C, 5 mM ATP present Escherichia coli
5
-
 L-Gln wild-type, glutaminase activity, pH 8.0, 37°C, ATP absent Escherichia coli
5.8
-
 L-Gln mutant E348A, glutaminase activity, pH 8.0, 37°C, ATP absent Escherichia coli
9
-
 L-Gln mutant E348Q, glutaminase activity, pH 8.0, 37°C, ATP absent Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+
-
 Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli
-
-
-

Purification (Commentary)

Purification (Comment) Organism
using Ni-NTA chromatography Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-Asp + L-Gln
-
 Escherichia coli AMP + diphosphate + L-Asn + L-Glu
-
 ?
ATP + L-Asp + L-Gln + H2O
-
 Escherichia coli AMP + diphosphate + L-Asn + L-Glu
-
 ?
ATP + L-Asp + NH3
-
 Escherichia coli AMP + diphosphate + L-Asn
-
 ?

Synonyms

Synonyms Comment Organism
AS-B
-
 Escherichia coli
Asparagine synthetase
-
 Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.3
-
 L-Asp mutant E348D, ammonia-dependent activity, pH 8.0, 37°C Escherichia coli
0.3
-
 L-Asp mutant E348D, synthetase activity, ammonia-dependent activity, 100 mM NH4Cl, pH 8.0, 37°C Escherichia coli
0.43
-
 ATP mutant E348D, ammonia-dependent activity, pH 8.0, 37°C Escherichia coli
0.43
-
 ATP mutant E348D, synthetase activity, ammonia-dependent activity, 100 mM NH4Cl, pH 8.0, 37°C Escherichia coli
0.45
-
 L-Asp mutant E348D, glutamine-dependent activity, pH 8.0, 37°C Escherichia coli
0.45
-
 L-Asp mutant E348D, synthetase activity, glutamine-dependent activity, 20 mM L-Gln, pH 8.0, 37°C Escherichia coli
0.51
-
 ATP mutant E348D, glutamine-dependent activity, pH 8.0, 37°C Escherichia coli
0.51
-
 ATP mutant E348D, synthetase activity, glutamine-dependent activity, 20 mM L-Gln, pH 8.0, 37°C Escherichia coli
0.67
-
 L-Asp wild-type, glutamine-dependent activity, pH 8.0, 37°C Escherichia coli
0.67
-
 L-Asp wild-type, synthetase activity, glutamine-dependent activity, 20 mM L-Gln, pH 8.0, 37°C Escherichia coli
0.75
-
 L-Asp wild-type, ammonia-dependent activity, pH 8.0, 37°C Escherichia coli
0.75
-
 L-Asp wild-type, synthetase activity, ammonia-dependent activity, 100 mM NH4Cl, pH 8.0, 37°C Escherichia coli
0.9
-
 ATP wild-type, glutamine-dependent activity, pH 8.0, 37°C Escherichia coli
0.9
-
 ATP wild-type, synthetase activity, glutamine-dependent activity, 20 mM L-Gln, pH 8.0, 37°C Escherichia coli
0.96
-
 ATP wild-type, ammonia-dependent activity, pH 8.0, 37°C Escherichia coli
0.96
-
 ATP wild-type, synthetase activity, ammonia-dependent activity, 100 mM NH4Cl, pH 8.0, 37°C Escherichia coli
4
-
 L-Gln mutant E348Q, glutaminase activity, pH 8.0, 37°C, ATP absent Escherichia coli
4.1
-
 L-Gln mutant E348D, glutaminase activity, pH 8.0, 37°C, ATP absent Escherichia coli
4.45
-
 L-Gln mutant E348Q, glutaminase activity, pH 8.0, 37°C, 5 mM ATP present Escherichia coli
5.8
-
 L-Gln mutant E348A, glutaminase activity, pH 8.0, 37°C, 5 mM ATP present Escherichia coli
6.2
-
 L-Gln wild-type, glutaminase activity, pH 8.0, 37°C, ATP absent Escherichia coli
6.37
-
 L-Gln mutant E348A, glutaminase activity, pH 8.0, 37°C, ATP absent Escherichia coli
6.6
-
 L-Gln wild-type, glutaminase activity, pH 8.0, 37°C, 5 mM ATP present Escherichia coli
10.02
-
 L-Gln mutant E348D, glutaminase activity, pH 8.0, 37°C, 5 mM ATP present Escherichia coli

pH Optimum

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

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.45
-
 L-Gln mutant E348Q, glutaminase activity, pH 8.0, 37°C, ATP absent Escherichia coli
0.63
-
 L-Asp wild-type, ammonia-dependent activity, pH 8.0, 37°C Escherichia coli
0.63
-
 L-Asp wild-type, synthetase activity, ammonia-dependent activity, 100 mM NH4Cl, pH 8.0, 37°C Escherichia coli
1.1
-
 L-Gln mutant E348A, glutaminase activity, pH 8.0, 37°C, ATP absent Escherichia coli
1.14
-
 L-Gln mutant E348Q, glutaminase activity, pH 8.0, 37°C, 5 mM ATP present Escherichia coli
1.19
-
 L-Gln wild-type, glutaminase activity, pH 8.0, 37°C, ATP absent Escherichia coli
1.2
-
 L-Asp wild-type, glutamine-dependent activity, pH 8.0, 37°C Escherichia coli
1.2
-
 L-Asp wild-type, synthetase activity, glutamine-dependent activity, 20 mM L-Gln, pH 8.0, 37°C Escherichia coli
1.3
-
 L-Asp mutant E348D, ammonia-dependent activity, pH 8.0, 37°C Escherichia coli
1.3
-
 L-Asp mutant E348D, synthetase activity, ammonia-dependent activity, 100 mM NH4Cl, pH 8.0, 37°C Escherichia coli
1.51
-
 L-Gln mutant E348D, glutaminase activity, pH 8.0, 37°C, ATP absent Escherichia coli
1.66
-
 L-Gln mutant E348A, glutaminase activity, pH 8.0, 37°C, 5 mM ATP present Escherichia coli
3.4
-
 L-Asp mutant E348D, glutamine-dependent activity, pH 8.0, 37°C Escherichia coli
3.45
-
 L-Asp mutant E348D, synthetase activity, glutamine-dependent activity, 20 mM L-Gln, pH 8.0, 37°C Escherichia coli
3.88
-
 L-Gln wild-type, glutaminase activity, pH 8.0, 37°C, 5 mM ATP present Escherichia coli
8.7
-
 ATP wild-type, ammonia-dependent activity, pH 8.0, 37°C Escherichia coli
8.7
-
 ATP wild-type, synthetase activity, ammonia-dependent activity, 100 mM NH4Cl, pH 8.0, 37°C Escherichia coli
9
-
 ATP wild-type, glutamine-dependent activity, pH 8.0, 37°C Escherichia coli
9
-
 ATP wild-type, synthetase activity, glutamine-dependent activity, 20 mM L-Gln, pH 8.0, 37°C Escherichia coli
9.1
-
 L-Gln mutant E348D, glutaminase activity, pH 8.0, 37°C, 5 mM ATP present Escherichia coli
14.3
-
 ATP mutant E348D, ammonia-dependent activity, pH 8.0, 37°C Escherichia coli
14.3
-
 ATP mutant E348D, synthetase activity, ammonia-dependent activity, 100 mM NH4Cl, pH 8.0, 37°C Escherichia coli
39.2
-
 ATP mutant E348D, glutamine-dependent activity, pH 8.0, 37°C Escherichia coli
39.2
-
 ATP mutant E348D, synthetase activity, glutamine-dependent activity, 20 mM L-Gln, pH 8.0, 37°C Escherichia coli