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Literature summary for 1.8.1.9 extracted from

  • Eckenroth, B.E.; Lacey, B.M.; Lothrop, A.P.; Harris, K.M.; Hondal, R.J.
    Investigation of the C-terminal redox center of high-Mr thioredoxin reductase by protein engineering and semisynthesis (2007), Biochemistry, 46, 9472-9483.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 1.8.1.9

Cloned(Commentary)

Cloned (Comment) Organism
-
 Caenorhabditis elegans
the full-length Sec489Cys mutant as well as the truncated mTR3 missing the C-terminal CUG tripeptide sequence is expressed as a TR-intein-chitin binding domain fusion protein in Escherichia coli Mus musculus
wild-type and C-terminal variants Drosophila melanogaster

Protein Variants

Protein Variants Comment Organism
additional information the mutant in the which Sec and Cys residues are switched (TR-GUCG), shows activity similar to that of the Sec489Cys mutant (TR-GCCG). Replacement of the Cys-Sec dyad with a Sec-Sec dyad (TR-GUUG) results in a mutant enzyme with very low catalytic activity. Even if the kcat values are normalized for selenium content, the TR-GUCG and TR-GUUG mutants have catalytic activity 90fold and 185fold lower, respectively, than that of the wild-type enzyme. The mutants in which alanine residues are inserted to increase the ring size (TR-GCAUG and TR-GCAAUG) show only a modest decrease in catalytic activity, 6fold and 4fold, respectively Mus musculus
additional information When the C-terminus of DmTR is changed from a carboxylate to either a thiocarboxylate or a hydroxamic acid, the result is a mutant enzyme with an about 1.7fold increase in activity with thioredoxin. Alanine insertion mutants (DmTR-SCACS and DmTR-SCAACS) show activity with thioredoxin that is greatly reduced compared to that of wild-type DmTR. Increasing the ring size of the Cys-Cys dyad results in a 150-300fold loss in kcat, while the Km is affected little. The 5,5'-dithiobis(2-nitrobenzoic acid) reductase activity of DmTR is also increased when the negative charge at the C-terminus is either neutralized by converting the carboxylate to a neutral hydroxamic acid or modulated by conversion to a thiocarboxylate. Similar to the Sec-containing mammalian enzyme, the truncated DmTR mutant also shows very high 5,5'-dithiobis(2-nitrobenzoic acid) reductase activity, as do the alanine insertion mutants Drosophila melanogaster
additional information where insertion of alanine between the redox-active Cys residues of the C-terminal redox center has very little effect on DTNB reductase activity Caenorhabditis elegans
Sec489C pH-optimum shifts from pH 7.0 to 8.0 Mus musculus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information KM-values of semisynthetic truncated mTR3 enzymes Mus musculus
additional information
-
 additional information KM-values of truncated enzymes forms Drosophila melanogaster
0.173
-
 thioredoxin disulfide pH 7, 25°C Drosophila melanogaster
0.75
-
 5,5'-dithiobis(2-nitrobenzoic acid) pH 7, 25°C Drosophila melanogaster

Localization

Localization Comment Organism GeneOntology No. Textmining
mitochondrion
-
 Caenorhabditis elegans 5739
-
mitochondrion
-
 Mus musculus 5739
-

Organism

Organism UniProt Comment Textmining
Caenorhabditis elegans
-
-
-
Drosophila melanogaster
-
-
-
Mus musculus Q8BTW3
-
-

Purification (Commentary)

Purification (Comment) Organism
wild-type and mutant enzymes Caenorhabditis elegans

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5,5'-dithiobis(2-nitrobenzoic acid) + NADPH + H+
-
 Drosophila melanogaster 2-nitro-5-thiobenzoate + NADP+
-
 ?
5,5'-dithiobis(2-nitrobenzoic acid) + NADPH + H+
-
 Caenorhabditis elegans 2-nitro-5-thiobenzoate + NADP+
-
 ?
5,5'-dithiobis(2-nitrobenzoic acid) + NADPH + H+ C-terminal tetrapeptide sequences is Gly-Cys-Sec-Gly. Changing the C-terminal carboxylate of mTR3 to a carboxamide increases the activity of the enzyme. If the selenium content is normalized for both samples, the carboxamide mutant has nearly twice the activity as the semisynthetic wild-type carboxylate enzyme Mus musculus 2-nitro-5-thiobenzoate + NADP+
-
 ?
thioredoxin disulfide + NADPH + H+
-
 Drosophila melanogaster thioredoxin + NADP+
-
 ?
thioredoxin disulfide + NADPH + H+
-
 Caenorhabditis elegans thioredoxin + NADP+
-
 ?
thioredoxin disulfide + NADPH + H+ C-terminal tetrapeptide sequences is Gly-Cys-Sec-Gly. Changing the C-terminal carboxylate of mTR3 to a carboxamide increases the activity of the enzyme. If the selenium content is normalized for both samples, the carboxamide mutant has nearly twice the activity as the semisynthetic wild-type carboxylate enzyme Mus musculus thioredoxin + NADP+
-
 ?

Synonyms

Synonyms Comment Organism
CeTR2
-
 Caenorhabditis elegans
DmTR
-
 Drosophila melanogaster
mTR3
-
 Mus musculus
thioredoxin reductase
-
 Drosophila melanogaster
thioredoxin reductase
-
 Caenorhabditis elegans
thioredoxin reductase
-
 Mus musculus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
additional information
-
 additional information turnover number of semisynthetic truncated mTR3 enzymes Mus musculus
additional information
-
 additional information turnover number of truncated enzyme forms Drosophila melanogaster
2.62
-
 5,5'-dithiobis(2-nitrobenzoic acid) pH 7, 25°C Drosophila melanogaster
4.99
-
 thioredoxin disulfide pH 7, 25°C Drosophila melanogaster

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 wild-type enzyme Mus musculus
7.5
-
-
 Drosophila melanogaster
8
-
 mutant enzyme Sec489C Mus musculus

pH Range

pH Minimum pH Maximum Comment Organism
6.5 9 pH 6.5: about 40% of maximal activity, pH 9.0: about 50% of maximal activity, wild-type enzyme Mus musculus
6.5 9.5 pH 6.5: 45% of maximal activity, pH 9.5: about 50% of maximal activity, mutant enzyme Sec489C Mus musculus

Cofactor

Cofactor Comment Organism Structure
NADPH
-
 Drosophila melanogaster
NADPH
-
 Caenorhabditis elegans
NADPH
-
 Mus musculus