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Literature summary for 2.7.1.21 extracted from

  • Wu, C.C.; Hsu, T.Y.; Chen, J.Y.
    Characterization of three essential residues in the conserved ATP-binding region of Epstein-Barr virus thymidine kinase (2005), Biochemistry, 44, 4785-4793.
    View publication on PubMed

Application

Application Comment Organism
medicine enzyme is a target for antiviral therapy and treatment of EBV-associated malignancies Human gammaherpesvirus 4

Cloned(Commentary)

Cloned (Comment) Organism
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Human gammaherpesvirus 4

Protein Variants

Protein Variants Comment Organism
G294A site-directed mutagenesis, about 80% reduced activity compared to the wild-type enzyme, no activity with TTP as cofactor Human gammaherpesvirus 4
G294V site-directed mutagenesis, nearly inactive mutant, no ATP binding Human gammaherpesvirus 4
K297E site-directed mutagenesis, nearly inactive mutant, no ATP binding Human gammaherpesvirus 4
K297Q site-directed mutagenesis, nearly inactive mutant, no ATP binding Human gammaherpesvirus 4
K297R site-directed mutagenesis, about 90% reduced activity compared to the wild-type enzyme, prefers GTP as cofactor, no activity with TTP as cofactor Human gammaherpesvirus 4
T298A site-directed mutagenesis, about 90% reduced activity compared to the wild-type enzyme Human gammaherpesvirus 4
T298S site-directed mutagenesis, about 20% reduced activity compared to the wild-type enzyme Human gammaherpesvirus 4

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information kinetics, ATP-binding kinetics of wild-type and mutant enzymes at room temperature Human gammaherpesvirus 4
0.03
-
ATP recombinant wild-type enzyme, pH 7.5, 37°C Human gammaherpesvirus 4
0.041
-
GTP recombinant wild-type enzyme, pH 7.5, 37°C Human gammaherpesvirus 4
0.041
-
ATP recombinant mutant T298S, pH 7.5, 37°C Human gammaherpesvirus 4
0.043
-
GTP about, recombinant mutant K297R, pH 7.5, 37°C Human gammaherpesvirus 4
0.045
-
GTP recombinant mutant T298S, pH 7.5, 37°C Human gammaherpesvirus 4
0.049
-
CTP recombinant wild-type enzyme, pH 7.5, 37°C Human gammaherpesvirus 4
0.049
-
ATP recombinant mutant G294A, pH 7.5, 37°C Human gammaherpesvirus 4
0.057
-
CTP about, recombinant mutant T298S, pH 7.5, 37°C Human gammaherpesvirus 4
0.069
-
GTP recombinant mutant G294A, pH 7.5, 37°C Human gammaherpesvirus 4
0.072
-
TTP recombinant wild-type enzyme, pH 7.5, 37°C Human gammaherpesvirus 4
0.088
-
CTP recombinant mutant G294A, pH 7.5, 37°C Human gammaherpesvirus 4
0.088
-
ATP recombinant mutant K297R, pH 7.5, 37°C Human gammaherpesvirus 4
0.108
-
CTP recombinant mutant K297R, pH 7.5, 37°C Human gammaherpesvirus 4
0.127
-
TTP recombinant mutant T298S, pH 7.5, 37°C Human gammaherpesvirus 4

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required, most efficient metal ion, can be substituted by Mn2+ or Zn2+ Human gammaherpesvirus 4
Mn2+ can partially substitute for Mg2+ Human gammaherpesvirus 4
Zn2+ can partially substitute for Mg2+ Human gammaherpesvirus 4

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + thymidine Human gammaherpesvirus 4
-
ADP + thymidine 5'-phosphate
-
?

Organism

Organism UniProt Comment Textmining
Human gammaherpesvirus 4
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) are immobilized on nickel beads and purified Human gammaherpesvirus 4

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + 3'-azido-2',3'-deoxythymidine
-
Human gammaherpesvirus 4 ADP + 3'-azido-2',3'-deoxythymidine 5'-phosphate
-
?
ATP + 3'-deoxy-2',3'-didehydro-thymidine
-
Human gammaherpesvirus 4 ADP + 3'-deoxy-2',3'-didehydro-thymidine 5'-phosphate
-
?
ATP + aciclovir
-
Human gammaherpesvirus 4 ADP + aciclovir 5'-phosphate
-
?
ATP + dideoxyinosine
-
Human gammaherpesvirus 4 ADP + dideoxyinosine 5'-phosphate
-
?
ATP + ganciclovir
-
Human gammaherpesvirus 4 ADP + ganciclovir phosphate
-
?
ATP + iododeoxyuridine
-
Human gammaherpesvirus 4 ADP + iododeoxyuridine 5'-phosphate
-
?
ATP + thymidine
-
Human gammaherpesvirus 4 ADP + thymidine 5'-phosphate
-
?
CTP + thymidine
-
Human gammaherpesvirus 4 CDP + thymidine 5'-phosphate
-
?
GTP + thymidine
-
Human gammaherpesvirus 4 GDP + thymidine 5'-phosphate
-
?
additional information substrate specificity of wild-type and mutant enzymes, overview Human gammaherpesvirus 4 ?
-
?
TTP + thymidine
-
Human gammaherpesvirus 4 TDP + thymidine 5'-phosphate
-
?

Synonyms

Synonyms Comment Organism
TK
-
Human gammaherpesvirus 4

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
assay at Human gammaherpesvirus 4

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2 8 ATP recombinant wild-type enzyme, pH 7.5, 37°C Human gammaherpesvirus 4
7
-
ATP recombinant mutant K297R, pH 7.5, 37°C Human gammaherpesvirus 4
7
-
TTP recombinant mutant T298S, pH 7.5, 37°C Human gammaherpesvirus 4
8
-
GTP recombinant mutant G294A, pH 7.5, 37°C Human gammaherpesvirus 4
9
-
CTP recombinant mutant G294A, pH 7.5, 37°C Human gammaherpesvirus 4
11
-
ATP recombinant mutant G294A, pH 7.5, 37°C Human gammaherpesvirus 4
11
-
TTP recombinant wild-type enzyme, pH 7.5, 37°C Human gammaherpesvirus 4
13.5
-
CTP about, recombinant mutant T298S, pH 7.5, 37°C Human gammaherpesvirus 4
14
-
CTP recombinant mutant K297R, pH 7.5, 37°C Human gammaherpesvirus 4
17
-
GTP recombinant mutant T298S, pH 7.5, 37°C Human gammaherpesvirus 4
18.5
-
GTP about, recombinant mutant K297R, pH 7.5, 37°C Human gammaherpesvirus 4
19
-
CTP recombinant wild-type enzyme, pH 7.5, 37°C Human gammaherpesvirus 4
21
-
GTP recombinant wild-type enzyme, pH 7.5, 37°C Human gammaherpesvirus 4
24
-
ATP recombinant mutant T298S, pH 7.5, 37°C Human gammaherpesvirus 4

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
assay at Human gammaherpesvirus 4

Cofactor

Cofactor Comment Organism Structure
ATP preferred triphosphate cosubstrate, 3 essential residues T298, K297, and G294 in the conserved ATP-binding region, enzyme binding kinetics Human gammaherpesvirus 4
CTP less active than ATP Human gammaherpesvirus 4
GTP less active tahn ATP Human gammaherpesvirus 4
additional information activity with NTP cofactors in descending order ATP, GTP, CTP, TTP Human gammaherpesvirus 4
TTP low activity with wild-type and mutant enzymes Human gammaherpesvirus 4