BRENDA - Enzyme Database
show all sequences of 2.7.4.13

The study of the bacteriophage T5 deoxynucleoside monophosphate kinase active site by site-directed mutagenesis

Mikoulinskaia, G.; Taran, S.; Skoblov, Y.; Feofanov, S.; Russ. J. Bioorg. Chem. 39, 607-618 (2013)
No PubMed abstract available

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
gene dnk, recombinant expression of wild-type and mutant enzymes in Escherichia coli strains BL21(DE3) and XL10Gold
Escherichia virus T5
Engineering
Amino acid exchange
Commentary
Organism
D16N
site-directed mutagenesis of the P loop-related residue, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
D170N
site-directed mutagenesis of the core residue, almost inactive mutant
Escherichia virus T5
E176Q
site-directed mutagenesis of the core residue, almost inactive mutant
Escherichia virus T5
G137A
site-directed mutagenesis of the dNMP-binding residue, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
K131E
site-directed mutagenesis, of the dNMP-binding residue, the mutant shows slightly increased activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
additional information
all enzyme mutants but T17S show an increased pH optimum compared to the wild-type enzyme
Escherichia virus T5
Q134A
site-directed mutagenesis of the dNMP-binding residue, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
R130K
site-directed mutagenesis of the dNMP-binding residue, almost inactive mutant
Escherichia virus T5
R172I
site-directed mutagenesis of the core residue, almost inactive mutant
Escherichia virus T5
S13A
site-directed mutagenesis of the P loop-related residue, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
T138A
site-directed mutagenesis of the dNMP-binding residue, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
T17N
site-directed mutagenesis of the P loop-related residue, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
T17S
site-directed mutagenesis, of the P loop-related residue, the mutant shows reduced activity compared to the wild-type enzyme
Escherichia virus T5
W150A
site-directed mutagenesis of the core residue
Escherichia virus T5
W150F
site-directed mutagenesis of the conserved residue, the mutant shows unaltered activity compared to the wild-type enzyme
Escherichia virus T5
Metals/Ions
Metals/Ions
Commentary
Organism
Structure
Mg2+
required
Escherichia virus T5
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ATP + dAMP
Escherichia virus T5
-
ADP + dADP
-
-
?
ATP + dCMP
Escherichia virus T5
-
ADP + dCDP
-
-
?
ATP + dGMP
Escherichia virus T5
-
ADP + dGDP
-
-
?
ATP + dTMP
Escherichia virus T5
-
ADP + dTDP
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Escherichia virus T5
Q6QGP4
gene dnk
-
Purification (Commentary)
Commentary
Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange and affinity chromatography
Escherichia virus T5
Specific Activity [micromol/min/mg]
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
additional information
-
specific activties of wild-type enzyme and enzyme mutants with different dNMP acceptor substrates, overview
Escherichia virus T5
25.78
-
pH 7.5, 37°C, recombinant wild-type enzyme, substrate dCMP
Escherichia virus T5
36.54
-
pH 7.5, 37°C, recombinant wild-type enzyme, substrate dGMP
Escherichia virus T5
41.24
-
pH 7.5, 37°C, recombinant wild-type enzyme, substrate dTMP
Escherichia virus T5
73.17
-
pH 7.5, 37°C, recombinant wild-type enzyme, substrate dAMP
Escherichia virus T5
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ATP + dAMP
-
739645
Escherichia virus T5
ADP + dADP
-
-
-
?
ATP + dAMP
best substrate dAMP, wild-type enzyme
739645
Escherichia virus T5
ADP + dADP
-
-
-
?
ATP + dCMP
-
739645
Escherichia virus T5
ADP + dCDP
-
-
-
?
ATP + dCMP
35.2% of the activity with dAMP, wild-type enzyme
739645
Escherichia virus T5
ADP + dCDP
-
-
-
?
ATP + dGMP
-
739645
Escherichia virus T5
ADP + dGDP
-
-
-
?
ATP + dGMP
49.9% of the activity with dAMP, wild-type enzyme
739645
Escherichia virus T5
ADP + dGDP
-
-
-
?
ATP + dTMP
-
739645
Escherichia virus T5
ADP + dTDP
-
-
-
?
ATP + dTMP
56.4% of the activity with dAMP, wild-type enzyme
739645
Escherichia virus T5
ADP + dTDP
-
-
-
?
additional information
substrate specificities of wild-type and mutant enzymes, structure-function analysis, overview
739645
Escherichia virus T5
?
-
-
-
-
Subunits
Subunits
Commentary
Organism
monomer
-
Escherichia virus T5
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Escherichia virus T5
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
wild-type enzyme and mutant T17S
Escherichia virus T5
7.5
8.5
all enzyme mutants but T17S
Escherichia virus T5
Cofactor
Cofactor
Commentary
Organism
Structure
ATP
-
Escherichia virus T5
Cloned(Commentary) (protein specific)
Commentary
Organism
gene dnk, recombinant expression of wild-type and mutant enzymes in Escherichia coli strains BL21(DE3) and XL10Gold
Escherichia virus T5
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
ATP
-
Escherichia virus T5
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
D16N
site-directed mutagenesis of the P loop-related residue, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
D170N
site-directed mutagenesis of the core residue, almost inactive mutant
Escherichia virus T5
E176Q
site-directed mutagenesis of the core residue, almost inactive mutant
Escherichia virus T5
G137A
site-directed mutagenesis of the dNMP-binding residue, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
K131E
site-directed mutagenesis, of the dNMP-binding residue, the mutant shows slightly increased activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
additional information
all enzyme mutants but T17S show an increased pH optimum compared to the wild-type enzyme
Escherichia virus T5
Q134A
site-directed mutagenesis of the dNMP-binding residue, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
R130K
site-directed mutagenesis of the dNMP-binding residue, almost inactive mutant
Escherichia virus T5
R172I
site-directed mutagenesis of the core residue, almost inactive mutant
Escherichia virus T5
S13A
site-directed mutagenesis of the P loop-related residue, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
T138A
site-directed mutagenesis of the dNMP-binding residue, the mutant shows reduced activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
T17N
site-directed mutagenesis of the P loop-related residue, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type enzyme
Escherichia virus T5
T17S
site-directed mutagenesis, of the P loop-related residue, the mutant shows reduced activity compared to the wild-type enzyme
Escherichia virus T5
W150A
site-directed mutagenesis of the core residue
Escherichia virus T5
W150F
site-directed mutagenesis of the conserved residue, the mutant shows unaltered activity compared to the wild-type enzyme
Escherichia virus T5
Metals/Ions (protein specific)
Metals/Ions
Commentary
Organism
Structure
Mg2+
required
Escherichia virus T5
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
ATP + dAMP
Escherichia virus T5
-
ADP + dADP
-
-
?
ATP + dCMP
Escherichia virus T5
-
ADP + dCDP
-
-
?
ATP + dGMP
Escherichia virus T5
-
ADP + dGDP
-
-
?
ATP + dTMP
Escherichia virus T5
-
ADP + dTDP
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by anion exchange and affinity chromatography
Escherichia virus T5
Specific Activity [micromol/min/mg] (protein specific)
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
additional information
-
specific activties of wild-type enzyme and enzyme mutants with different dNMP acceptor substrates, overview
Escherichia virus T5
25.78
-
pH 7.5, 37°C, recombinant wild-type enzyme, substrate dCMP
Escherichia virus T5
36.54
-
pH 7.5, 37°C, recombinant wild-type enzyme, substrate dGMP
Escherichia virus T5
41.24
-
pH 7.5, 37°C, recombinant wild-type enzyme, substrate dTMP
Escherichia virus T5
73.17
-
pH 7.5, 37°C, recombinant wild-type enzyme, substrate dAMP
Escherichia virus T5
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ATP + dAMP
-
739645
Escherichia virus T5
ADP + dADP
-
-
-
?
ATP + dAMP
best substrate dAMP, wild-type enzyme
739645
Escherichia virus T5
ADP + dADP
-
-
-
?
ATP + dCMP
-
739645
Escherichia virus T5
ADP + dCDP
-
-
-
?
ATP + dCMP
35.2% of the activity with dAMP, wild-type enzyme
739645
Escherichia virus T5
ADP + dCDP
-
-
-
?
ATP + dGMP
-
739645
Escherichia virus T5
ADP + dGDP
-
-
-
?
ATP + dGMP
49.9% of the activity with dAMP, wild-type enzyme
739645
Escherichia virus T5
ADP + dGDP
-
-
-
?
ATP + dTMP
-
739645
Escherichia virus T5
ADP + dTDP
-
-
-
?
ATP + dTMP
56.4% of the activity with dAMP, wild-type enzyme
739645
Escherichia virus T5
ADP + dTDP
-
-
-
?
additional information
substrate specificities of wild-type and mutant enzymes, structure-function analysis, overview
739645
Escherichia virus T5
?
-
-
-
-
Subunits (protein specific)
Subunits
Commentary
Organism
monomer
-
Escherichia virus T5
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Escherichia virus T5
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7
-
wild-type enzyme and mutant T17S
Escherichia virus T5
7.5
8.5
all enzyme mutants but T17S
Escherichia virus T5
General Information
General Information
Commentary
Organism
additional information
essential catalytic site residue, active site modelling, overview. The charged amino acid residues of the NMP binding domain and the presence of an OH-group at position 17 are important for the catalytic activity. Arginine residues at positions 130 and 172 are involved in the binding to the donor gamma-phosphoryl and acceptor alpha-phosphoryl groups, as well as the aspartic acid residue at position 16 of the ATP-binding site (P-loop), in the binding to some acceptors, first of all dTMP. The NMPK-P loop, or a glycine enriched loop, forms an anion hole interacting with donor phosphoryl groups. It contains a GX1X2X3X4GKX5T(S) consensus called the Walker A motif
Escherichia virus T5
General Information (protein specific)
General Information
Commentary
Organism
additional information
essential catalytic site residue, active site modelling, overview. The charged amino acid residues of the NMP binding domain and the presence of an OH-group at position 17 are important for the catalytic activity. Arginine residues at positions 130 and 172 are involved in the binding to the donor gamma-phosphoryl and acceptor alpha-phosphoryl groups, as well as the aspartic acid residue at position 16 of the ATP-binding site (P-loop), in the binding to some acceptors, first of all dTMP. The NMPK-P loop, or a glycine enriched loop, forms an anion hole interacting with donor phosphoryl groups. It contains a GX1X2X3X4GKX5T(S) consensus called the Walker A motif
Escherichia virus T5
Other publictions for EC 2.7.4.13
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
739645
Mikoulinskaia
-
The study of the bacteriophage ...
Escherichia virus T5
Russ. J. Bioorg. Chem.
39
607-618
2013
-
-
1
-
15
-
-
-
-
1
-
4
-
1
-
-
1
-
-
-
5
-
9
1
1
-
-
-
2
-
-
1
-
-
-
-
-
1
1
-
15
-
-
-
-
-
-
1
-
4
-
-
-
1
-
-
5
-
9
1
1
-
-
-
2
-
-
-
-
1
1
-
-
-
661371
Bao
Cloning of deoxynucleoside mon ...
Saccharomyces cerevisiae
Biotechnol. Bioeng.
93
572-580
2006
-
-
1
-
-
-
-
8
-
1
4
1
-
4
-
-
-
-
-
-
4
-
5
-
-
-
-
-
2
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
8
-
1
4
1
-
-
-
-
-
-
4
-
5
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
663294
Mikoulinskaia
Identification, cloning, and e ...
Escherichia virus T5
Protein Expr. Purif.
33
166-175
2004
-
-
1
-
-
-
-
-
-
-
2
1
-
3
-
-
1
-
-
-
8
-
5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
2
1
-
-
-
1
-
-
8
-
5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
642544
Mikoulinskaia
Purification and characterizat ...
Escherichia virus T5, no activity in Escherichia coli
Protein Expr. Purif.
27
195-201
2003
-
-
-
-
-
-
-
-
-
-
2
1
-
4
-
-
1
-
-
-
2
1
5
1
-
-
4
-
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
1
-
-
-
1
-
-
2
1
5
1
-
-
4
-
1
1
-
1
-
-
-
-
-
-
209854
Allen
T4 phage deoxyribonucleotide-s ...
Escherichia virus T4, no activity in Escherichia coli
J. Biol. Chem.
258
5746-5753
1983
-
-
-
-
-
-
-
-
-
-
1
1
-
2
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
642542
Bessman
The enzymology of virus-infect ...
Escherichia virus T5, no activity in Escherichia coli
J. Biol. Chem.
240
439-445
1965
-
-
-
-
-
-
3
6
-
5
-
1
-
2
-
-
1
-
-
-
1
2
12
-
1
-
-
-
2
3
-
-
3
-
-
-
-
-
-
-
-
-
-
3
3
6
-
5
-
1
-
-
-
1
-
-
1
2
12
-
1
-
-
-
2
3
-
-
-
-
-
-
-
-