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Literature summary extracted from

  • List, F.; Bocola, M.; Haeger, M.C.; Sterner, R.
    Constitutively active glutaminase variants provide insights into the activation mechanism of anthranilate synthase (2012), Biochemistry, 51, 2812-2818.
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
3.5.1.2 additional information strong stimulation of glutaminase subunit TrpG activity by the associated synthase subunit TrpE within the glutamine amidotransferase, GATase, enzyme complex Thermotoga maritima
4.1.3.27 additional information activation mechanism of anthranilate synthase, overview Thermotoga maritima

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.1.2 gene trpG, expression of His-tagged wild-type TrpG and mutant TrpG variants T129F and T129A in Escherichia coli strain BL21(DE3)Rosetta Thermotoga maritima
4.1.3.27 expression of gene trpE in Escherichia coli strain BL21(DE3)RIPL and of wild-type and mutant genes trpGD and trpG in Escherichia coli strain BL21(DE3)Rosetta, all as N- and C-terminally His6-tagged proteins Thermotoga maritima

Protein Variants

EC Number Protein Variants Comment Organism
3.5.1.2 L126G site-directed mutagenesis, the mutant shows constitutive activity unlike the wild-type enzyme and does not require presence of TrpE Thermotoga maritima
3.5.1.2 L126G/V127Y/T129Y/Y131V site-directed mutagenesis, the mutant shows constitutive activity unlike the wild-type enzyme and does not require presence of TrpE Thermotoga maritima
3.5.1.2 T129Y site-directed mutagenesis, the mutant shows constitutive activity unlike the wild-type enzyme and does not require presence of TrpE Thermotoga maritima
3.5.1.2 V127Y site-directed mutagenesis, the mutant shows no constitutive activity like the wild-type enzyme and requires presence of TrpE Thermotoga maritima
3.5.1.2 Y131V site-directed mutagenesis, the mutant shows no constitutive activity like the wild-type enzyme and requires presence of TrpE Thermotoga maritima
4.1.3.27 L126G site-directed mutagenesis of a residue of the glutaminase subunit TrpG from anthranilate synthase, the mutant constitutively hydrolyzes glutamine in the absence of TrpE in contrast to the wild-type enzyme Thermotoga maritima
4.1.3.27 L126G/V127Y/T129Y/Y131V site-directed mutagenesis of anthranilate synthase glutaminase subunit TrpG residues, the mutant constitutively hydrolyzes glutamine in the absence of TrpE in contrast to the wild-type enzyme Thermotoga maritima
4.1.3.27 additional information in contrast to wild-type TrpG, two TrpG variants with single exchanges constitutively hydrolyze glutamine in the absence of TrpE. The introduced amino acid exchanges result in a distance reduction between the active site Cys-His pair, which facilitates the deprotonation of the sulfhydryl group of the catalytic cysteine and thus enables its nucleophilic attack onto the carboxamide group of the glutamine side chain, molecular dynamics simulations, overview Thermotoga maritima
4.1.3.27 T129A site-directed mutagenesis of a residue of the glutaminase subunit TrpG from anthranilate synthase, the mutant shows no activity with glutamine in absence of TrpE like the wild-type enzyme Thermotoga maritima
4.1.3.27 T129F site-directed mutagenesis of a residue of the glutaminase subunit TrpG from anthranilate synthase, the mutant constitutively hydrolyzes glutamine in the absence of TrpE in contrast to the wild-type enzyme Thermotoga maritima
4.1.3.27 T129Y site-directed mutagenesis of a residue of the glutaminase subunit TrpG from anthranilate synthase, the mutant constitutively hydrolyzes glutamine in the absence of TrpE in contrast to the wild-type enzyme Thermotoga maritima
4.1.3.27 V127Y site-directed mutagenesis of a residue of the glutaminase subunit TrpG from anthranilate synthase, the mutant shows no activity with glutamine in absence of TrpE like the wild-type enzyme Thermotoga maritima
4.1.3.27 Y131V site-directed mutagenesis of a residue of the glutaminase subunit TrpG from anthranilate synthase, the mutant shows no activity with glutamine in absence of TrpE like the wild-type enzyme Thermotoga maritima

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.1.2 additional information
-
 additional information glutaminase activity steady-state enzyme kinetics of TrpG and the AS complex, generated by mixing the recombinant TrpE and TrpGD subunits Thermotoga maritima
4.1.3.27 additional information
-
 additional information steady-state enzyme kinetics, overview Thermotoga maritima

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
3.5.1.2 L-glutamine + H2O Thermotoga maritima
-
 L-glutamate + NH3
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.2 Thermotoga maritima
-
 gene trpG
-
4.1.3.27 Thermotoga maritima
-
 genes trpE, trpGD, and trpG
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.1.2 recombinant His-tagged TrpG, and mutant TrpG variants T129F and T129A from Escherichia coli strain BL21(DE3)Rosetta by heat treatment for 20 min at 60°C, nickel affinity chromatography, and anion exchange chromatography Thermotoga maritima
4.1.3.27 recombinant wild-type and mutant His-tagged enzymes from Escherichia coli by nickel affinity and anion exchange chromatography Thermotoga maritima

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.1.2 L-glutamine + H2O
-
 Thermotoga maritima L-glutamate + NH3
-
 ?

Subunits

EC Number Subunits Comment Organism
3.5.1.2 More the glutaminase subunit TrpG, which adopts the triad G-type amidotransferase fold, and the synthase subunit TrpE, which adopts a complicated alpha/beta folding pattern, assemble to heterotetrameric (TrpE/TrpG)2-complexes. The sequence stretch L126-V127-A128-T129-R130-Y131 is localized between the catalytic triad Cys83-His175-Glu177 and the associated TrpE subunit, homology modeling based on the structure of Sulfolobus solfataricus TrpG Thermotoga maritima
4.1.3.27 More synthase subunit TrpE and glutaminase subunit TrpG Thermotoga maritima

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.2 25
-
 assay at Thermotoga maritima
4.1.3.27 25
-
 assay at Thermotoga maritima

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.1.2 8
-
 assay at Thermotoga maritima
4.1.3.27 8
-
 assay at Thermotoga maritima

Expression

EC Number Organism Comment Expression
3.5.1.2 Thermotoga maritima wild-type TrpG shows no constitutive activity and needs to be induced by TrpE up

General Information

EC Number General Information Comment Organism
4.1.3.27 additional information the enzyme shows a mechanism of this tight activity regulation, catalytic Cys-His-Glu triad, molecular dynamics simulations, overview Thermotoga maritima