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Functional studies of active-site mutants from Drosophila melanogaster deoxyribonucleoside kinase. Investigations of the putative catalytic glutamate-arginine pair and of residues responsible for substrate specificity

Egeblad-Welin, L.; Sonntag, Y.; Eklund, H.; Munch-Petersen, B.; FEBS J. 274, 1542-1551 (2007)

Data extracted from this reference:

Crystallization (Commentary)
EC Number
Crystallization
Organism
2.7.1.145
crystals of a C-terminally truncated (D20) recombinant Dm-dNK mutant E52D are grown using the vapour diffusion method by hanging drop geometry. The E52D mutant is crystallized with its feedback inhibitor dTTP. The backbone conformation remains unchanged, and coordination between D52 and the dTTP–Mg complex is observed
Drosophila melanogaster
Engineering
EC Number
Amino acid exchange
Commentary
Organism
2.7.1.145
E52D
markedly decreased kcat
Drosophila melanogaster
2.7.1.145
E52H
markedly decreased kcat
Drosophila melanogaster
2.7.1.145
E52Q
inactive mutant protein
Drosophila melanogaster
2.7.1.145
Q81N
mutation shows a 200fold and 100fold increase in Km. kcat is decreased 5fold and 2fold for dThd and dCyd
Drosophila melanogaster
2.7.1.145
R105H
markedly decreased kcat
Drosophila melanogaster
2.7.1.145
R105K
inactive mutant protein
Drosophila melanogaster
2.7.1.145
Y70W
mutant enzyme loses activity towards purines. Negative cooperativity towards dThd and dCyd is observed
Drosophila melanogaster
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.7.1.145
0.0012
-
2'-deoxythymidine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0022
-
5-(E)-(2-bromvinyl)-2'-deoxyuridine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0023
-
2'-deoxycytidine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0037
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme E52D
Drosophila melanogaster
2.7.1.145
0.0037
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme E52H
Drosophila melanogaster
2.7.1.145
0.0038
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme E52D
Drosophila melanogaster
2.7.1.145
0.0049
-
5-(E)-(2-bromvinyl)-2'-deoxyuridine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
0.0058
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme E52H
Drosophila melanogaster
2.7.1.145
0.0089
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme R105H
Drosophila melanogaster
2.7.1.145
0.0243
-
1-(beta-D-arabinofuranosyl)-cytosine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.062
-
1-(beta-D-arabinofuranosyl)-thymine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.113
-
2'-deoxycytidine
37°C, pH 7.5, mutant ezyme R105H
Drosophila melanogaster
2.7.1.145
0.205
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme Q81N
Drosophila melanogaster
2.7.1.145
0.231
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme Q81N
Drosophila melanogaster
2.7.1.145
0.246
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
0.251
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
0.357
-
1-(beta-D-arabinofuranosyl)-thymine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
1.441
-
1-(beta-D-arabinofuranosyl)-cytosine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
2.7.1.145
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
2'-deoxycytidine + ATP
-
673584
Drosophila melanogaster
2'-deoxycytidine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
ATP + 1-(beta-D-arabinofuranosyl)-cytosine
-
673584
Drosophila melanogaster
ADP + 1-(beta-D-arabinofuranosyl)-cytosine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 1-(beta-D-arabinofuranosyl)-thymine
-
673584
Drosophila melanogaster
ADP + 1-(beta-D-arabinofuranosyl)-thymine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 2'-deoxythymidine
-
673584
Drosophila melanogaster
ADP + 2'-deoxythymidine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 5-(E)-(2-bromvinyl)-2'-deoxyuridine
-
673584
Drosophila melanogaster
ADP + 5-(E)-(2-bromvinyl)-2'-deoxyuridine 5'-phosphate
-
-
-
?
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.7.1.145
0.0003
-
1-(beta-D-arabinofuranosyl)-thymine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.00071
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme E52D
Drosophila melanogaster
2.7.1.145
0.00072
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme E52D
Drosophila melanogaster
2.7.1.145
0.0012
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme E52H
Drosophila melanogaster
2.7.1.145
0.0029
-
1-(beta-D-arabinofuranosyl)-cytosine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0059
-
5-(E)-(2-bromvinyl)-2'-deoxyuridine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0067
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme R105H
Drosophila melanogaster
2.7.1.145
0.012
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme E52H
Drosophila melanogaster
2.7.1.145
0.0142
-
2'-deoxythymidine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0162
-
2'-deoxycytidine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.055
-
2'-deoxycytidine
37°C, pH 7.5, mutant ezyme R105H
Drosophila melanogaster
2.7.1.145
0.4
-
5-(E)-(2-bromvinyl)-2'-deoxyuridine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
1
-
1-(beta-D-arabinofuranosyl)-thymine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
2.1
-
1-(beta-D-arabinofuranosyl)-cytosine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
2.3
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
5.4
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme Q81N
Drosophila melanogaster
2.7.1.145
6.6
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme Q81N
Drosophila melanogaster
2.7.1.145
6.8
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
2.7.1.145
crystals of a C-terminally truncated (D20) recombinant Dm-dNK mutant E52D are grown using the vapour diffusion method by hanging drop geometry. The E52D mutant is crystallized with its feedback inhibitor dTTP. The backbone conformation remains unchanged, and coordination between D52 and the dTTP–Mg complex is observed
Drosophila melanogaster
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
2.7.1.145
E52D
markedly decreased kcat
Drosophila melanogaster
2.7.1.145
E52H
markedly decreased kcat
Drosophila melanogaster
2.7.1.145
E52Q
inactive mutant protein
Drosophila melanogaster
2.7.1.145
Q81N
mutation shows a 200fold and 100fold increase in Km. kcat is decreased 5fold and 2fold for dThd and dCyd
Drosophila melanogaster
2.7.1.145
R105H
markedly decreased kcat
Drosophila melanogaster
2.7.1.145
R105K
inactive mutant protein
Drosophila melanogaster
2.7.1.145
Y70W
mutant enzyme loses activity towards purines. Negative cooperativity towards dThd and dCyd is observed
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
-
2'-deoxythymidine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0022
-
5-(E)-(2-bromvinyl)-2'-deoxyuridine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0023
-
2'-deoxycytidine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0037
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme E52D
Drosophila melanogaster
2.7.1.145
0.0037
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme E52H
Drosophila melanogaster
2.7.1.145
0.0038
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme E52D
Drosophila melanogaster
2.7.1.145
0.0049
-
5-(E)-(2-bromvinyl)-2'-deoxyuridine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
0.0058
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme E52H
Drosophila melanogaster
2.7.1.145
0.0089
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme R105H
Drosophila melanogaster
2.7.1.145
0.0243
-
1-(beta-D-arabinofuranosyl)-cytosine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.062
-
1-(beta-D-arabinofuranosyl)-thymine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.113
-
2'-deoxycytidine
37°C, pH 7.5, mutant ezyme R105H
Drosophila melanogaster
2.7.1.145
0.205
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme Q81N
Drosophila melanogaster
2.7.1.145
0.231
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme Q81N
Drosophila melanogaster
2.7.1.145
0.246
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
0.251
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
0.357
-
1-(beta-D-arabinofuranosyl)-thymine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
1.441
-
1-(beta-D-arabinofuranosyl)-cytosine
37°C, pH 7.5, mutant enzyme Y70W
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
2'-deoxycytidine + ATP
-
673584
Drosophila melanogaster
2'-deoxycytidine 5'-phosphate + ADP
-
-
-
?
2.7.1.145
ATP + 1-(beta-D-arabinofuranosyl)-cytosine
-
673584
Drosophila melanogaster
ADP + 1-(beta-D-arabinofuranosyl)-cytosine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 1-(beta-D-arabinofuranosyl)-thymine
-
673584
Drosophila melanogaster
ADP + 1-(beta-D-arabinofuranosyl)-thymine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 2'-deoxythymidine
-
673584
Drosophila melanogaster
ADP + 2'-deoxythymidine 5'-phosphate
-
-
-
?
2.7.1.145
ATP + 5-(E)-(2-bromvinyl)-2'-deoxyuridine
-
673584
Drosophila melanogaster
ADP + 5-(E)-(2-bromvinyl)-2'-deoxyuridine 5'-phosphate
-
-
-
?
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
0.0003
-
1-(beta-D-arabinofuranosyl)-thymine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.00071
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme E52D
Drosophila melanogaster
2.7.1.145
0.00072
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme E52D
Drosophila melanogaster
2.7.1.145
0.0012
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme E52H
Drosophila melanogaster
2.7.1.145
0.0029
-
1-(beta-D-arabinofuranosyl)-cytosine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0059
-
5-(E)-(2-bromvinyl)-2'-deoxyuridine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0067
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme R105H
Drosophila melanogaster
2.7.1.145
0.012
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme E52H
Drosophila melanogaster
2.7.1.145
0.0142
-
2'-deoxythymidine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.0162
-
2'-deoxycytidine
37°C, pH 7.5, wild-type enzyme
Drosophila melanogaster
2.7.1.145
0.055
-
2'-deoxycytidine
37°C, pH 7.5, mutant ezyme R105H
Drosophila melanogaster
2.7.1.145
0.4
-
5-(E)-(2-bromvinyl)-2'-deoxyuridine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
1
-
1-(beta-D-arabinofuranosyl)-thymine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
2.1
-
1-(beta-D-arabinofuranosyl)-cytosine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
2.3
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster
2.7.1.145
5.4
-
2'-deoxythymidine
37°C, pH 7.5, mutant enzyme Q81N
Drosophila melanogaster
2.7.1.145
6.6
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme Q81N
Drosophila melanogaster
2.7.1.145
6.8
-
2'-deoxycytidine
37°C, pH 7.5, mutant enzyme Y70W
Drosophila melanogaster