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Literature summary for 6.3.1.9 extracted from

  • Leroux, A.E.; Haanstra, J.R.; Bakker, B.M.; Krauth-Siegel, R.L.
    Dissecting the catalytic mechanism of Trypanosoma brucei trypanothione synthetase by kinetic analysis and computational modeling (2013), J. Biol. Chem., 288, 23751-23764.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 6.3.1.9

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His-tagged enzyme in Escherichia coli strain Tuner(DE3) Trypanosoma brucei

Inhibitors

Inhibitors Comment Organism Structure
glutathione substrate inhibition Trypanosoma brucei
trypanothione competitive feedback inhibition Trypanosoma brucei

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0066
-
 ATP recombinant enzyme, pH 8.0, 25°C, HEPES buffer, with spermidine Trypanosoma brucei
0.01
-
 ATP recombinant enzyme, pH 8.0, 37°C, HEPES buffer, with spermidine Trypanosoma brucei
0.012
-
 ATP recombinant enzyme, pH 7.0, 37°C, phosphate buffer, with glutathionylspermidine Trypanosoma brucei
0.012
-
 N1-glutathionylspermidine recombinant enzyme, pH 8.0, 25°C, HEPES buffer Trypanosoma brucei
0.012
-
 N8-glutathionylspermidine recombinant enzyme, pH 8.0, 25°C, HEPES buffer Trypanosoma brucei
0.016
-
 N1-glutathionylspermidine recombinant enzyme, pH 8.0, 37°C, HEPES buffer Trypanosoma brucei
0.016
-
 N8-glutathionylspermidine recombinant enzyme, pH 8.0, 37°C, HEPES buffer Trypanosoma brucei
0.018
-
 ATP recombinant enzyme, pH 7.0, 37°C, phosphate buffer, with spermidine Trypanosoma brucei
0.032
-
 N1-glutathionylspermidine recombinant enzyme, pH 7.0, 37°C, phosphate buffer Trypanosoma brucei
0.032
-
 N8-glutathionylspermidine recombinant enzyme, pH 7.0, 37°C, phosphate buffer Trypanosoma brucei
0.032
-
 glutathione recombinant enzyme, pH 8.0, 25°C, HEPES buffer, with spermidine Trypanosoma brucei
0.034
-
 glutathione recombinant enzyme, pH 7.0, 37°C, phosphate buffer, with glutathionylspermidine Trypanosoma brucei
0.045
-
 glutathione recombinant enzyme, pH 8.0, 37°C, HEPES buffer, with spermidine Trypanosoma brucei
0.069
-
 glutathione recombinant enzyme, pH 7.0, 37°C, phosphate buffer, with spermidine Trypanosoma brucei
0.092
-
 spermidine recombinant enzyme, pH 8.0, 25°C, HEPES buffer Trypanosoma brucei
0.139
-
 spermidine recombinant enzyme, pH 8.0, 37°C, HEPES buffer Trypanosoma brucei
0.687
-
 spermidine recombinant enzyme, pH 7.0, 37°C, phosphate buffer Trypanosoma brucei

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Trypanosoma brucei

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2 glutathione + spermidine + 2 ATP Trypanosoma brucei overall reaction, the enzyme catalyzes the formation of N1,N8-bis(glutathionyl)spermidine in two reaction steps, exhibiting activity of EC 6.3.1.8, glutathionylspermidine synthase, and 6.3.1.9, trypanothione synthase N1,N8-bis(glutathionyl)spermidine + 2 ADP + 2 phosphate
-
 ?
2 glutathione + spermidine + 2 ATP Trypanosoma brucei 449 overall reaction, the enzyme catalyzes the formation of N1,N8-bis(glutathionyl)spermidine in two reaction steps, exhibiting activity of EC 6.3.1.8, glutathionylspermidine synthase, and 6.3.1.9, trypanothione synthase N1,N8-bis(glutathionyl)spermidine + 2 ADP + 2 phosphate
-
 ?
glutathione + spermidine + ATP Trypanosoma brucei reaction of EC 6.3.1.8, step one N1-glutathionylspermidine + N8-glutathionylspermidine + ADP + phosphate
-
 ?
glutathione + spermidine + ATP Trypanosoma brucei 449 reaction of EC 6.3.1.8, step one N1-glutathionylspermidine + N8-glutathionylspermidine + ADP + phosphate
-
 ?
additional information Trypanosoma brucei in pathogenic trypanosomes, trypanothione synthetase catalyzes the synthesis of both glutathionylspermidine and trypanothione (bis(glutathionyl)spermidine). Trypanothione is synthesized from two molecules of GSH that are covalently bridged by a spermidine in two ATP-dependent reactions. The enzyme also shows trypanothione amidase activity catalyzed at a second distinct active site ?
-
 ?
additional information Trypanosoma brucei 449 in pathogenic trypanosomes, trypanothione synthetase catalyzes the synthesis of both glutathionylspermidine and trypanothione (bis(glutathionyl)spermidine). Trypanothione is synthesized from two molecules of GSH that are covalently bridged by a spermidine in two ATP-dependent reactions. The enzyme also shows trypanothione amidase activity catalyzed at a second distinct active site ?
-
 ?
N1-glutathionylspermidine + glutathione + ATP Trypanosoma brucei step two N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
-
 ?
N1-glutathionylspermidine + glutathione + ATP Trypanosoma brucei 449 step two N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
-
 ?
N8-glutathionylspermidine + glutathione + ATP Trypanosoma brucei step two N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
-
 ?
N8-glutathionylspermidine + glutathione + ATP Trypanosoma brucei 449 step two N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
-
 ?

Organism

Organism UniProt Comment Textmining
Trypanosoma brucei
-
-
-
Trypanosoma brucei 449
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged enzyme from Escherichia coli strain Tuner(DE3) by nickel affinity chromatography and tag cleavage by tobacco etch virus protease, followed by a second nickel affinity chromatography Trypanosoma brucei

Reaction

Reaction Comment Organism Reaction ID
glutathione + spermidine + ATP = glutathionylspermidine + ADP + phosphate ter-reactant mechanism of TryS catalysis, modeling, overview Trypanosoma brucei
a

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 glutathione + spermidine + 2 ATP overall reaction, the enzyme catalyzes the formation of N1,N8-bis(glutathionyl)spermidine in two reaction steps, exhibiting activity of EC 6.3.1.8, glutathionylspermidine synthase, and 6.3.1.9, trypanothione synthase Trypanosoma brucei N1,N8-bis(glutathionyl)spermidine + 2 ADP + 2 phosphate
-
 ?
2 glutathione + spermidine + 2 ATP overall reaction, the enzyme catalyzes the formation of N1,N8-bis(glutathionyl)spermidine in two reaction steps, exhibiting activity of EC 6.3.1.8, glutathionylspermidine synthase, and 6.3.1.9, trypanothione synthase Trypanosoma brucei 449 N1,N8-bis(glutathionyl)spermidine + 2 ADP + 2 phosphate
-
 ?
glutathione + spermidine + ATP reaction of EC 6.3.1.8, step one Trypanosoma brucei N1-glutathionylspermidine + N8-glutathionylspermidine + ADP + phosphate
-
 ?
glutathione + spermidine + ATP reaction of EC 6.3.1.8, step one Trypanosoma brucei 449 N1-glutathionylspermidine + N8-glutathionylspermidine + ADP + phosphate
-
 ?
additional information in pathogenic trypanosomes, trypanothione synthetase catalyzes the synthesis of both glutathionylspermidine and trypanothione (bis(glutathionyl)spermidine). Trypanothione is synthesized from two molecules of GSH that are covalently bridged by a spermidine in two ATP-dependent reactions. The enzyme also shows trypanothione amidase activity catalyzed at a second distinct active site Trypanosoma brucei ?
-
 ?
additional information in pathogenic trypanosomes, trypanothione synthetase catalyzes the synthesis of both glutathionylspermidine and trypanothione (bis(glutathionyl)spermidine). Trypanothione is synthesized from two molecules of GSH that are covalently bridged by a spermidine in two ATP-dependent reactions. The enzyme also shows trypanothione amidase activity catalyzed at a second distinct active site Trypanosoma brucei 449 ?
-
 ?
N1-glutathionylspermidine + glutathione + ATP step two Trypanosoma brucei N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
-
 ?
N1-glutathionylspermidine + glutathione + ATP step two Trypanosoma brucei 449 N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
-
 ?
N8-glutathionylspermidine + glutathione + ATP step two Trypanosoma brucei N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
-
 ?
N8-glutathionylspermidine + glutathione + ATP step two Trypanosoma brucei 449 N1,N8-bis(glutathionyl)spermidine + ADP + phosphate
-
 ?

Synonyms

Synonyms Comment Organism
Trypanothione synthetase
-
 Trypanosoma brucei
TryS
-
 Trypanosoma brucei

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25 37 assay at Trypanosoma brucei

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.8
-
 glutathione recombinant enzyme, pH 8.0, 25°C, HEPES buffer, with spermidine Trypanosoma brucei
2.1
-
 glutathione recombinant enzyme, pH 7.0, 37°C, phosphate buffer, with glutathionylspermidine Trypanosoma brucei
2.8
-
 glutathione recombinant enzyme, pH 7.0, 37°C, phosphate buffer, with spermidine Trypanosoma brucei
5.2
-
 glutathione recombinant enzyme, pH 8.0, 37°C, HEPES buffer, with spermidine Trypanosoma brucei

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7 8 reaction step 1 Trypanosoma brucei
7
-
 reaction step 2, no activity at pH 8.0 Trypanosoma brucei

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Trypanosoma brucei

Ki Value [mM]

Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
0.132
-
 glutathione recombinant enzyme, pH 8.0, 25°C, HEPES buffer, with spermidine Trypanosoma brucei
0.223
-
 trypanothione recombinant enzyme, pH 7.0, 37°C, phosphate buffer, with glutathione as substrate, versus spermidine Trypanosoma brucei
0.228
-
 trypanothione recombinant enzyme, pH 7.0, 37°C, phosphate buffer, with spermidine as substrate, versus spermidine Trypanosoma brucei
0.332
-
 glutathione recombinant enzyme, pH 8.0, 37°C, HEPES buffer, with spermidine Trypanosoma brucei
0.346
-
 trypanothione recombinant enzyme, pH 7.0, 37°C, phosphate buffer, with glutathione as substrate, versus glutathione Trypanosoma brucei
0.377
-
 trypanothione recombinant enzyme, pH 7.0, 37°C, phosphate buffer, with spermidine as substrate, versus glutathione Trypanosoma brucei
0.849
-
 glutathione recombinant enzyme, pH 7.0, 37°C, phosphate buffer, with spermidine Trypanosoma brucei
1.085
-
 glutathione recombinant enzyme, pH 7.0, 37°C, phosphate buffer, versus glutathionylspermidine Trypanosoma brucei

General Information

General Information Comment Organism
additional information the steady-state kinetic analysis discard a ping-pong mechanism Trypanosoma brucei