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Literature summary for 3.1.3.48 extracted from

  • Yun, H.Y.; Lee, J.; Kim, H.; Ryu, H.; Shin, H.C.; Oh, B.H.; Ku, B.; Kim, S.J.
    Structural study reveals the temperature-dependent conformational flexibility of Tk-PTP, a protein tyrosine phosphatase from Thermococcus kodakaraensis KOD1 (2018), PLoS ONE, 13, e0197635 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) RIL Thermococcus kodakarensis

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant enzyme Tk-PTP(form I) Tk-PTP(form II) Tk-PTP(G95A), sitting-drop vapor diffusion method, for enzyme form I: mixing 0.001 ml of 10 mg/ml protein solution with 0.001 ml of precipitant solution containing 0.1 M sodium citrate, pH 5.4, 8% w/v PEG 10000, and 14% v/v dioxane, for enzyme form II: mixing of 0.001 ml of 10 mg/ml protein solution with 0.001 ml of precipitant solution containing 0.03 M citric acid, 0.07 M Bis-Tris propane, pH 7.6, 8% w/v PEG 3350, and 0.08 M spermine tetrahydrochloride, and for mutant Tk-PTP(G95A): mixing of 0.001 ml of 10 mg/ml protein solution with 0.001 ml of precipitant solution containing 0.1 M Bis-Tris, pH 6.25, and 0.8 M magnesium formate dehydrate, all at 18°C, X-ray diffraction structure determination and analysis at 1.7-2.3 A resolution, molecular replacement method, model building Thermococcus kodakarensis

Protein Variants

Protein Variants Comment Organism
C93S site-directed mutagenesis, inactive mutant Thermococcus kodakarensis
D63A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Thermococcus kodakarensis
D63N site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Thermococcus kodakarensis
D63N/E132A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Thermococcus kodakarensis
E132A site-directed mutagenesis, the mutant shows about 1.5fold increased activity compared to wild-type Thermococcus kodakarensis
E132L site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Thermococcus kodakarensis
E132Q site-directed mutagenesis, the mutant shows about 2.5fold increased activity compared to wild-type Thermococcus kodakarensis
G95A site-directed mutagenesis, the mutant shows about 10fold increased activity compared to wild-type Thermococcus kodakarensis
Q136A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Thermococcus kodakarensis
R124A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Thermococcus kodakarensis
R124E site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Thermococcus kodakarensis

Inhibitors

Inhibitors Comment Organism Structure
Sodium vanadate a PTP inhibitor targeting the catalytic site pocket. Vanadate is anchored in the active site of Tk-PTP(form II), stabilized by electrostatic interaction with the guanidinium group of Arg109 and by its oxygen atom-mediated hydrogen bonds with the main chain amides of Met94, Gly95, Leu97, Gly98, and Arg99. The Tk-PTP(form II) P-loop is structurally similar to those of catalytically active DUSP proteins Thermococcus kodakarensis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.023
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant E132A Thermococcus kodakarensis
0.0274
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant D63A Thermococcus kodakarensis
0.0658
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant E132A Thermococcus kodakarensis
0.0666
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant D63A Thermococcus kodakarensis
0.0682
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant G95A Thermococcus kodakarensis
0.106
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant G95A Thermococcus kodakarensis
0.135
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant Q136A Thermococcus kodakarensis
0.15
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant E132Q Thermococcus kodakarensis
0.223
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant D63N/E132A Thermococcus kodakarensis
0.251
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant E132L Thermococcus kodakarensis
0.252
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant D63N Thermococcus kodakarensis
0.258
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant R124E Thermococcus kodakarensis
0.276
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, wild-type enzyme Thermococcus kodakarensis
0.301
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant R124A Thermococcus kodakarensis
0.307
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant E132Q Thermococcus kodakarensis
0.363
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant Q136A Thermococcus kodakarensis
0.363
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant R124E Thermococcus kodakarensis
0.466
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant D63N/E132A Thermococcus kodakarensis
0.56
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant D63N Thermococcus kodakarensis
0.611
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant R124A Thermococcus kodakarensis
0.719
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, wild-type enzyme Thermococcus kodakarensis
0.862
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant E132L Thermococcus kodakarensis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
[a protein]-tyrosine phosphate + H2O Thermococcus kodakarensis
-
[a protein]-tyrosine + phosphate
-
?
[a protein]-tyrosine phosphate + H2O Thermococcus kodakarensis JCM 12380
-
[a protein]-tyrosine + phosphate
-
?
[a protein]-tyrosine phosphate + H2O Thermococcus kodakarensis ATCC BAA-918
-
[a protein]-tyrosine + phosphate
-
?

Organism

Organism UniProt Comment Textmining
Thermococcus kodakarensis Q8X270 i.e. Pyrococcus kodakaraensis
-
Thermococcus kodakarensis ATCC BAA-918 Q8X270 i.e. Pyrococcus kodakaraensis
-
Thermococcus kodakarensis JCM 12380 Q8X270 i.e. Pyrococcus kodakaraensis
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) RIL by nickel affinity chromatography, tag cleavage by thrombin, and gel filtration Thermococcus kodakarensis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6,8-difluoro-4-methylumbelliferyl phosphate + H2O
-
Thermococcus kodakarensis 6,8-difluoro-4-methylumbelliferone + phosphate
-
?
6,8-difluoro-4-methylumbelliferyl phosphate + H2O
-
Thermococcus kodakarensis JCM 12380 6,8-difluoro-4-methylumbelliferone + phosphate
-
?
6,8-difluoro-4-methylumbelliferyl phosphate + H2O
-
Thermococcus kodakarensis ATCC BAA-918 6,8-difluoro-4-methylumbelliferone + phosphate
-
?
additional information the enzyme shows dual specificity, protein tyrosine/serine/threonine phosphatase activity Thermococcus kodakarensis ?
-
?
additional information the enzyme shows dual specificity, protein tyrosine/serine/threonine phosphatase activity Thermococcus kodakarensis JCM 12380 ?
-
?
additional information the enzyme shows dual specificity, protein tyrosine/serine/threonine phosphatase activity Thermococcus kodakarensis ATCC BAA-918 ?
-
?
[a protein]-tyrosine phosphate + H2O
-
Thermococcus kodakarensis [a protein]-tyrosine + phosphate
-
?
[a protein]-tyrosine phosphate + H2O
-
Thermococcus kodakarensis JCM 12380 [a protein]-tyrosine + phosphate
-
?
[a protein]-tyrosine phosphate + H2O
-
Thermococcus kodakarensis ATCC BAA-918 [a protein]-tyrosine + phosphate
-
?

Synonyms

Synonyms Comment Organism
dual-specificity phosphatase
-
Thermococcus kodakarensis
DUSP
-
Thermococcus kodakarensis
More cf. EC 3.1.3.16 Thermococcus kodakarensis
protein tyrosine phosphatase
-
Thermococcus kodakarensis
PTP
-
Thermococcus kodakarensis
Tk-PTP
-
Thermococcus kodakarensis
TK0241
-
Thermococcus kodakarensis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
80
-
-
Thermococcus kodakarensis

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
the hyperthermostability of Tk-PTP depends on its intramolecular hydrophobic interactions, structure comparisons, overview Thermococcus kodakarensis
60
-
the enzymatic activity of Tk-PTP is not affected by pre-heating the protein at 60°C for 3 h Thermococcus kodakarensis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0143
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant D63N Thermococcus kodakarensis
0.0371
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant R124E Thermococcus kodakarensis
0.0415
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant D63A Thermococcus kodakarensis
0.0652
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant D63N/E132A Thermococcus kodakarensis
0.102
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant Q136A Thermococcus kodakarensis
0.188
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant R124E Thermococcus kodakarensis
0.197
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant E132L Thermococcus kodakarensis
0.248
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant R124A Thermococcus kodakarensis
0.46
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant Q136A Thermococcus kodakarensis
0.591
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant E132A Thermococcus kodakarensis
0.688
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant D63N/E132A Thermococcus kodakarensis
1.29
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant D63A Thermococcus kodakarensis
1.59
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant R124A Thermococcus kodakarensis
2.92
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant D63N Thermococcus kodakarensis
2.97
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant E132L Thermococcus kodakarensis
3.83
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant E132A Thermococcus kodakarensis
4.17
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant G95A Thermococcus kodakarensis
4.74
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, wild-type enzyme Thermococcus kodakarensis
5.64
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant E132Q Thermococcus kodakarensis
8.82
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant E132Q Thermococcus kodakarensis
30
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, wild-type enzyme Thermococcus kodakarensis
35.8
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant G95A Thermococcus kodakarensis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
4.5 5
-
Thermococcus kodakarensis

General Information

General Information Comment Organism
additional information Tk-PTP adopts a common dual-specificity phosphatase (DUSP) fold, but it undergoes an atypical temperature-dependent conformational change in its P-loop and alpha4-alpha5 loop regions, switching between the inactive and active forms. Tk-PTP contains a PTP signature motif HCxxGxxR, HC93MGGLGR99 in Tk-PTP constituting the phosphate binding loop (or simply called P-loop), contains the catalytic cysteine residue (Cys93) that functions as a nucleophile for dephosphorylation and the conserved arginine residue (Arg99) that anchors the phosphate group of the substrate during the enzyme reaction. Tk-PTP contains dual general acid/base residues, Asp63 or Glu132. Structural analysis of the conformation of the P-loop of Tk-PTP(form I), structure comparisons of PTPs, structure-function analysis of the two enzyme forms, overview. Molecular dynamics simulations Thermococcus kodakarensis

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.102
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant R124E Thermococcus kodakarensis
0.14
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant D63N/E132A Thermococcus kodakarensis
0.229
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant E132L Thermococcus kodakarensis
0.281
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant Q136A Thermococcus kodakarensis
0.406
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant R124A Thermococcus kodakarensis
0.623
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant D63A Thermococcus kodakarensis
0.729
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant R124E Thermococcus kodakarensis
3.09
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant D63N/E132A Thermococcus kodakarensis
3.41
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant Q136A Thermococcus kodakarensis
5.28
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant R124A Thermococcus kodakarensis
6.59
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, wild-type enzyme Thermococcus kodakarensis
8.98
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant E132A Thermococcus kodakarensis
11.6
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant D63N Thermococcus kodakarensis
11.8
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant E132L Thermococcus kodakarensis
18.4
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant E132Q Thermococcus kodakarensis
47.1
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant D63A Thermococcus kodakarensis
58.8
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant E132Q Thermococcus kodakarensis
61.1
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant G95A Thermococcus kodakarensis
109
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, wild-type enzyme Thermococcus kodakarensis
167
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant E132A Thermococcus kodakarensis
255
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 20°C, mutant D63N Thermococcus kodakarensis
338
-
6,8-difluoro-4-methylumbelliferyl phosphate pH 5.0, 60°C, mutant G95A Thermococcus kodakarensis