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Literature summary for 3.5.1.1 extracted from

  • Bansal, S.; Srivastava, A.; Mukherjee, G.; Pandey, R.; Verma, A.K.; Mishra, P.; Kundu, B.
    Hyperthermophilic asparaginase mutants with enhanced substrate affinity and antineoplastic activity: structural insights on their mechanism of action (2012), FASEB J., 26, 1161-1171.
    View publication on PubMed
Search for more literature for 3.5.1.1

Cloned(Commentary)

Cloned (Comment) Organism
expression of His-tagged mutant enzymes in Escherichia coli strain Rosetta (DE3) Pyrococcus furiosus

Protein Variants

Protein Variants Comment Organism
K274E site-directed mutagenesis, the active site mutant shows improved enzymatic properties at physiological conditions compared to the wild-type. The mutant is thermodynamically stable and resistant to proteolytic digestion, displays no glutaminase activity, and shows increased and more significant killing of human cell lines HL60, MCF7, and K562 as compared to the Escherichia coli L-asparaginase Pyrococcus furiosus
T53Q site-directed mutagenesis, the active site mutant shows improved enzymatic properties at physiological conditions compared to the wild-type. The mutant is thermodynamically stable and resistant to proteolytic digestion, displays no glutaminase activity, and shows increased and more significant killing of human cell lines HL60, MCF7, and K562 as compared to the Escherichia coli L-asparaginase Pyrococcus furiosus
T53Q/K274E site-directed mutagenesis, the active site mutant shows improved enzymatic properties at physiological conditions compared to the wild-type. The mutant is thermodynamically stable and resistant to proteolytic digestion, displays no glutaminase activity, and shows increased and more significant killing of human cell lines HL60, MCF7, and K562 as compared to the Escherichia coli L-asparaginase Pyrococcus furiosus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information steady-state enzyme kinetics at different conditions of wild-type and mutant enzymes, overview Pyrococcus furiosus
2.1
-
 L-asparagine pH 8.2, 80°C, mutant K274E Pyrococcus furiosus
4.3
-
 L-asparagine pH 7.4, 37°C, mutant K274E Pyrococcus furiosus
5.41
-
 L-asparagine pH 7.4, 37°C, mutant T53Q/K274E Pyrococcus furiosus
7.52
-
 L-asparagine pH 7.4, 37°C, mutant T53Q Pyrococcus furiosus
8.12
-
 L-asparagine pH 7.4, 37°C, wild-type enzyme Pyrococcus furiosus
8.2
-
 L-asparagine pH 8.2, 80°C, mutant T53Q/K274E Pyrococcus furiosus
8.3
-
 L-asparagine pH 8.2, 80°C, mutant T53Q Pyrococcus furiosus
12.1
-
 L-asparagine pH 8.2, 80°C, wild-type enzyme Pyrococcus furiosus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-asparagine + H2O Pyrococcus furiosus
-
 L-aspartate + NH3
-
 ?

Organism

Organism UniProt Comment Textmining
Pyrococcus furiosus Q8TZE8
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged mutant enzymes from Escherichia coli strain Rosetta (DE3) by nickel affinity chromatography Pyrococcus furiosus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-asparagine + H2O
-
 Pyrococcus furiosus L-aspartate + NH3
-
 ?
additional information increased substrate accessibility through the active site loop plays a major role in determining activity, dynamic flipping of a critical Tyr residue is responsible for the activity of thermophilic L-asparaginases, molecular dynamic simulation and reaction mechanism, overview Pyrococcus furiosus ?
-
 ?

Synonyms

Synonyms Comment Organism
L-asparaginase
-
 Pyrococcus furiosus
PfA
-
 Pyrococcus furiosus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at in phosphate buffer Pyrococcus furiosus
80
-
 assay at in Tris-HCl buffer Pyrococcus furiosus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
17.98
-
 L-asparagine pH 7.4, 37°C, wild-type enzyme Pyrococcus furiosus
23.8
-
 L-asparagine pH 7.4, 37°C, mutant T53Q/K274E Pyrococcus furiosus
24.9
-
 L-asparagine pH 7.4, 37°C, mutant K274E Pyrococcus furiosus
35.2
-
 L-asparagine pH 7.4, 37°C, mutant T53Q Pyrococcus furiosus
199.2
-
 L-asparagine pH 8.2, 80°C, mutant K274E Pyrococcus furiosus
246.7
-
 L-asparagine pH 8.2, 80°C, mutant T53Q Pyrococcus furiosus
277.9
-
 L-asparagine pH 8.2, 80°C, mutant T53Q/K274E Pyrococcus furiosus
888.6
-
 L-asparagine pH 8.2, 80°C, wild-type enzyme Pyrococcus furiosus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.4
-
 assay at in phosphate buffer Pyrococcus furiosus
8.2
-
 assay at in Tris-HCl buffer Pyrococcus furiosus

General Information

General Information Comment Organism
additional information increased substrate accessibility through the active site loop plays a major role in determining activity, dynamic flipping of a critical Tyr residue is responsible for the activity of thermophilic L-asparaginases, molecular dynamic simulation and reaction mechanism, overview. Structure-function analysis of active site residues T53 and K274 Pyrococcus furiosus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
22.1
-
 L-asparagine pH 7.4, 37°C, wild-type enzyme Pyrococcus furiosus
30
-
 L-asparagine pH 8.2, 80°C, mutant T53Q Pyrococcus furiosus
34
-
 L-asparagine pH 8.2, 80°C, mutant T53Q/K274E Pyrococcus furiosus
44
-
 L-asparagine pH 7.4, 37°C, mutant T53Q/K274E Pyrococcus furiosus
47.2
-
 L-asparagine pH 7.4, 37°C, mutant T53Q Pyrococcus furiosus
57.8
-
 L-asparagine pH 7.4, 37°C, mutant K274E Pyrococcus furiosus
73
-
 L-asparagine pH 8.2, 80°C, wild-type enzyme Pyrococcus furiosus
96
-
 L-asparagine pH 8.2, 80°C, mutant K274E Pyrococcus furiosus