show Functional expression of a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster and its C-terminal deletion mutants
Munch-Petersen, B.; Knecht, W.; Lenz, C.; Sondergaard, L.; Piskur, J.; J. Biol. Chem. 275, 6673-6679 (2000)
Data extracted from this reference:
Cloned(Commentary)
EC Number
Commentary
Organism
2.7.1.145
expression in Escherichia coli; wild-type and truncated enzyme forms with removal of 10, 20 or 30 C-terminal amino acids, expression as a fusion protein with glutathione S-transferase
Drosophila melanogaster
Engineering
EC Number
Amino acid exchange
Commentary
Organism
2.7.1.145
additional information
the deletion mutants rDm-dNKDELTAC10 and rDm-dNKDELTAC20 show the same substrate activity pattern as the recombinant wild-type enzyme. Relative phosphorylation of 2'-deoxycytidine and 2-chloro-2'-deoxyadenosine increases with increasing C-terminal truncation. The relative activities of rDm-dNKDELTAC10 and rDm-dNKDELTAC20 with deoxyribonucleosides remains largely unchanged, whereas there is a substantial decrease in the phosphorylation of the purine ribonucleosides adenosine and guanosine, as well as of all dideoxyribonucleosides and 3'-azido-2',3'-dideoxythymidine. The relative activities with the pyrimidine ribonucleosides and 1-beta-D-arabinofuranosylcytosine and 1-beta-D-arabinofuranosylthymine are not affected by the C-terminal deletions
Drosophila melanogaster
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.7.1.145
0.0012
-
thymidine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
2.7.1.145
0.0023
-
2'-deoxycytidine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
2.7.1.145
0.225
-
2'-deoxyadenosine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
2.7.1.145
0.665
-
2'-deoxyguanosine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
2.7.1.145
29000
-
1 * 29000, SDS-PAGE
Drosophila melanogaster
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
2.7.1.145
Drosophila melanogaster
Q9XZT6
-
-
Purification (Commentary)
EC Number
Commentary
Organism
2.7.1.145
recombinant enzyme
Drosophila melanogaster
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
2.7.1.145
0.35
-
C-terminal truncated enzyme rDm-dNKDELTAC30
Drosophila melanogaster
2.7.1.145
23.8
-
C-terminal truncated enzyme rDm-dNKDELTAC10
Drosophila melanogaster
2.7.1.145
36.5
-
recombinant enzyme
Drosophila melanogaster
2.7.1.145
51.6
-
C-terminal truncated enzyme rDm-dNKDELTAC20
Drosophila melanogaster
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.7.1.145
(E)-5-(2-bromovinyl)-2'-deoxyuridine + ATP
54% of the activity with thymidine
636842
Drosophila melanogaster
(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
1-beta-D-arabinofuranosylcytosine + ATP
58.2% of the activity with thymidine
636842
Drosophila melanogaster
1-beta-D-arabinofuranosylcytosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
1-beta-D-arabinofuranosylthymine + ATP
53.9% of the activity with thymidine
636842
Drosophila melanogaster
1-beta-D-arabinofuranosylthymine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2',3'-dideoxyadenosine + ATP
0.9% of the activity with thymidine
636842
Drosophila melanogaster
2',3'-dideoxyadenosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2',3'-dideoxycytidine + ATP
11.1% of the activity with thymidine
636842
Drosophila melanogaster
2',3'-dideoxycytidine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2',3'-dideoxythymidine + ATP
4.44% of the activity with thymidine
636842
Drosophila melanogaster
2',3'-dideoxythymidine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2'-deoxyadenosine + ATP
96% of the activity with thymidine
636842
Drosophila melanogaster
2'-deoxyadenosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2'-deoxycytidine + ATP
134% of the activity with thymidine
636842
Drosophila melanogaster
2'-deoxycytidine 5'-phosphate + ADP
-
636842
Drosophila melanogaster
?
2.7.1.145
2'-deoxyguanosine + ATP
53% of the activity with thymidine
636842
Drosophila melanogaster
2'-deoxyguanosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2'-deoxyuridine + ATP
113% of the activity with thymidine
636842
Drosophila melanogaster
2'-deoxyuridine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2-chloro-2'-deoxyadenosine + ATP
126% of the activity with thymidine
636842
Drosophila melanogaster
2-chloro-2'-deoxyadenosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
3'-azido-2',3'-dideoxythymidine + ATP
2% of the activity with thymidine
636842
Drosophila melanogaster
3'-azido-2',3'-dideoxythymidine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
5-fluoro-2'-deoxyuridine + ATP
92% of the activity with thymidine
636842
Drosophila melanogaster
5-fluoro-2'-deoxyuridine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
adenosine + ATP
0.82% of the activity with thymidine
636842
Drosophila melanogaster
adenosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
cytidine + ATP
16% of the activity with thymidine
636842
Drosophila melanogaster
cytidine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
guanosine + ATP
1.18% of the activity with thymidine
636842
Drosophila melanogaster
guanosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
thymidine + ATP
-
636842
Drosophila melanogaster
thymidine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
uridine + ATP
6.45% of the activity with thymidine
636842
Drosophila melanogaster
uridine 5'-phosphate + ADP
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
2.7.1.145
monomer
1 * 29000, SDS-PAGE
Drosophila melanogaster
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.7.1.145
14.2
-
thymidine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
2.7.1.145
15.1
-
2'-deoxyguanosine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
2.7.1.145
16.5
-
2'-deoxycytidine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
2.7.1.145
20.6
-
2'-deoxyadenosine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
2.7.1.145
expression in Escherichia coli; wild-type and truncated enzyme forms with removal of 10, 20 or 30 C-terminal amino acids, expression as a fusion protein with glutathione S-transferase
Drosophila melanogaster
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
2.7.1.145
additional information
the deletion mutants rDm-dNKDELTAC10 and rDm-dNKDELTAC20 show the same substrate activity pattern as the recombinant wild-type enzyme. Relative phosphorylation of 2'-deoxycytidine and 2-chloro-2'-deoxyadenosine increases with increasing C-terminal truncation. The relative activities of rDm-dNKDELTAC10 and rDm-dNKDELTAC20 with deoxyribonucleosides remains largely unchanged, whereas there is a substantial decrease in the phosphorylation of the purine ribonucleosides adenosine and guanosine, as well as of all dideoxyribonucleosides and 3'-azido-2',3'-dideoxythymidine. The relative activities with the pyrimidine ribonucleosides and 1-beta-D-arabinofuranosylcytosine and 1-beta-D-arabinofuranosylthymine are not affected by the C-terminal deletions
Drosophila melanogaster
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.7.1.145
0.0012
-
thymidine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
2.7.1.145
0.0023
-
2'-deoxycytidine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
2.7.1.145
0.225
-
2'-deoxyadenosine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
2.7.1.145
0.665
-
2'-deoxyguanosine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
2.7.1.145
29000
-
1 * 29000, SDS-PAGE
Drosophila melanogaster
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
2.7.1.145
recombinant enzyme
Drosophila melanogaster
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
2.7.1.145
0.35
-
C-terminal truncated enzyme rDm-dNKDELTAC30
Drosophila melanogaster
2.7.1.145
23.8
-
C-terminal truncated enzyme rDm-dNKDELTAC10
Drosophila melanogaster
2.7.1.145
36.5
-
recombinant enzyme
Drosophila melanogaster
2.7.1.145
51.6
-
C-terminal truncated enzyme rDm-dNKDELTAC20
Drosophila melanogaster
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.7.1.145
(E)-5-(2-bromovinyl)-2'-deoxyuridine + ATP
54% of the activity with thymidine
636842
Drosophila melanogaster
(E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
1-beta-D-arabinofuranosylcytosine + ATP
58.2% of the activity with thymidine
636842
Drosophila melanogaster
1-beta-D-arabinofuranosylcytosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
1-beta-D-arabinofuranosylthymine + ATP
53.9% of the activity with thymidine
636842
Drosophila melanogaster
1-beta-D-arabinofuranosylthymine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2',3'-dideoxyadenosine + ATP
0.9% of the activity with thymidine
636842
Drosophila melanogaster
2',3'-dideoxyadenosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2',3'-dideoxycytidine + ATP
11.1% of the activity with thymidine
636842
Drosophila melanogaster
2',3'-dideoxycytidine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2',3'-dideoxythymidine + ATP
4.44% of the activity with thymidine
636842
Drosophila melanogaster
2',3'-dideoxythymidine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2'-deoxyadenosine + ATP
96% of the activity with thymidine
636842
Drosophila melanogaster
2'-deoxyadenosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2'-deoxycytidine + ATP
134% of the activity with thymidine
636842
Drosophila melanogaster
2'-deoxycytidine 5'-phosphate + ADP
-
636842
Drosophila melanogaster
?
2.7.1.145
2'-deoxyguanosine + ATP
53% of the activity with thymidine
636842
Drosophila melanogaster
2'-deoxyguanosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2'-deoxyuridine + ATP
113% of the activity with thymidine
636842
Drosophila melanogaster
2'-deoxyuridine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
2-chloro-2'-deoxyadenosine + ATP
126% of the activity with thymidine
636842
Drosophila melanogaster
2-chloro-2'-deoxyadenosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
3'-azido-2',3'-dideoxythymidine + ATP
2% of the activity with thymidine
636842
Drosophila melanogaster
3'-azido-2',3'-dideoxythymidine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
5-fluoro-2'-deoxyuridine + ATP
92% of the activity with thymidine
636842
Drosophila melanogaster
5-fluoro-2'-deoxyuridine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
adenosine + ATP
0.82% of the activity with thymidine
636842
Drosophila melanogaster
adenosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
cytidine + ATP
16% of the activity with thymidine
636842
Drosophila melanogaster
cytidine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
guanosine + ATP
1.18% of the activity with thymidine
636842
Drosophila melanogaster
guanosine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
thymidine + ATP
-
636842
Drosophila melanogaster
thymidine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
uridine + ATP
6.45% of the activity with thymidine
636842
Drosophila melanogaster
uridine 5'-phosphate + ADP
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
2.7.1.145
monomer
1 * 29000, SDS-PAGE
Drosophila melanogaster
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.7.1.145
14.2
-
thymidine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
2.7.1.145
15.1
-
2'-deoxyguanosine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
2.7.1.145
16.5
-
2'-deoxycytidine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster
2.7.1.145
20.6
-
2'-deoxyadenosine
pH 7.8, 22°C, recombinant enzyme
Drosophila melanogaster