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Structural and kinetic characterization of human deoxycytidine kinase variants able to phosphorylate 5-substituted deoxycytidine and thymidine analogues

Hazra, S.; Ort, S.; Konrad, M.; Lavie, A.; Biochemistry 49, 6784-6790 (2010)

Data extracted from this reference:

Application
EC Number
Application
Commentary
Organism
2.7.1.74
medicine
ability of the designer enzymes to activate 5-substitued pyrimidines makes it possible to utilize such nucleoside analogs in suicide gene therapy or protein therapy applications that target cancer cells
Homo sapiens
Cloned(Commentary)
EC Number
Commentary
Organism
2.7.1.74
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain C41(DE3)
Homo sapiens
Crystallization (Commentary)
EC Number
Crystallization
Organism
2.7.1.74
purified recombinanat Wild-type enzyme in complex with 5-methyldeoxycytidine and ADP, hanging drop vapour diffusion method, mixing of 0.001 ml of 8 mg/ml protein mixed with 0.001 ml reservoir solution containing 0.9-1.5 M trisodium citrate dihydrate and 100 mM Tris, pH 7.5, room temperature, X-ray diffraction structure determination and analysis at 1.96 A resolution
Homo sapiens
Engineering
EC Number
Amino acid exchange
Commentary
Organism
2.7.1.74
R104I/D133A
site-directed mutagenesis, the mutations render the enzyme active with 5-substituted deoxycytidine and thymidine, altered substrate specificity compared to the wild-type enzyme
Homo sapiens
2.7.1.74
R104L/D133A
site-directed mutagenesis, the mutations render the enzyme active with 5-substituted deoxycytidine and thymidine, altered substrate specificity compared to the wild-type enzyme
Homo sapiens
2.7.1.74
R104M/D133A
site-directed mutagenesis, the mutations render the enzyme active with 5-substituted deoxycytidine and thymidine, altered substrate specificity compared to the wild-type enzyme
Homo sapiens
2.7.1.74
R104Q/D133A
site-directed mutagenesis, the mutations render the enzyme active with 5-substituted deoxycytidine and thymidine, altered substrate specificity compared to the wild-type enzyme
Homo sapiens
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
2.7.1.74
Mg2+
required
Homo sapiens
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
2.7.1.74
ATP + 5-methyldeoxycytidine
Homo sapiens
-
ADP + 5-methyl-dCMP
-
-
?
2.7.1.74
ATP + deoxycytidine
Homo sapiens
-
ADP + dCMP
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
2.7.1.74
Homo sapiens
P27707
-
-
Purification (Commentary)
EC Number
Commentary
Organism
2.7.1.74
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain C41(DE3) by nickel affinity chromatography and gel filtration
Homo sapiens
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.7.1.74
ATP + (E)-5-(2-bromovinyl)-2'-deoxyuridine
substrate of enzyme mutant R104M/D133A, poor substrate of the wild-type enzyme
721622
Homo sapiens
ADP + ?
-
-
-
?
2.7.1.74
ATP + 5-bromodeoxycytidine
-
721622
Homo sapiens
ADP + 5-bromo-dCMP
-
-
-
?
2.7.1.74
ATP + 5-iododeoxycytidine
-
721622
Homo sapiens
ADP + 5-iodo-dCMP
-
-
-
?
2.7.1.74
ATP + 5-methyldeoxycytidine
-
721622
Homo sapiens
ADP + 5-methyl-dCMP
-
-
-
?
2.7.1.74
ATP + 5-propynyldeoxycytidine
-
721622
Homo sapiens
ADP + 5-propynyl-dCMP
-
-
-
?
2.7.1.74
ATP + D-deoxycytidine
-
721622
Homo sapiens
ADP + D-dCMP
-
-
-
?
2.7.1.74
ATP + D-deoxythymidine
-
721622
Homo sapiens
ADP + D-dTMP
-
-
-
?
2.7.1.74
ATP + D-deoxyuridine
-
721622
Homo sapiens
ADP + D-dUMP
-
-
-
?
2.7.1.74
ATP + deoxycytidine
-
721622
Homo sapiens
ADP + dCMP
-
-
-
?
2.7.1.74
ATP + gemcitabine
-
721622
Homo sapiens
ADP + phospho-gemcitabine
-
-
-
?
2.7.1.74
ATP + L-deoxyuridine
-
721622
Homo sapiens
ADP + L-dUMP
-
-
-
?
2.7.1.74
ATP + L-thymidine
-
721622
Homo sapiens
ADP + L-dTMP
-
-
-
?
2.7.1.74
additional information
substrate specificity or wild-type an dmutant enzymes, overview
721622
Homo sapiens
?
-
-
-
-
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
2.7.1.74
37
-
assay at
Homo sapiens
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
2.7.1.74
7.5
-
assay at
Homo sapiens
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
2.7.1.74
ATP
-
Homo sapiens
Application (protein specific)
EC Number
Application
Commentary
Organism
2.7.1.74
medicine
ability of the designer enzymes to activate 5-substitued pyrimidines makes it possible to utilize such nucleoside analogs in suicide gene therapy or protein therapy applications that target cancer cells
Homo sapiens
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
2.7.1.74
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain C41(DE3)
Homo sapiens
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
2.7.1.74
ATP
-
Homo sapiens
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
2.7.1.74
purified recombinanat Wild-type enzyme in complex with 5-methyldeoxycytidine and ADP, hanging drop vapour diffusion method, mixing of 0.001 ml of 8 mg/ml protein mixed with 0.001 ml reservoir solution containing 0.9-1.5 M trisodium citrate dihydrate and 100 mM Tris, pH 7.5, room temperature, X-ray diffraction structure determination and analysis at 1.96 A resolution
Homo sapiens
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
2.7.1.74
R104I/D133A
site-directed mutagenesis, the mutations render the enzyme active with 5-substituted deoxycytidine and thymidine, altered substrate specificity compared to the wild-type enzyme
Homo sapiens
2.7.1.74
R104L/D133A
site-directed mutagenesis, the mutations render the enzyme active with 5-substituted deoxycytidine and thymidine, altered substrate specificity compared to the wild-type enzyme
Homo sapiens
2.7.1.74
R104M/D133A
site-directed mutagenesis, the mutations render the enzyme active with 5-substituted deoxycytidine and thymidine, altered substrate specificity compared to the wild-type enzyme
Homo sapiens
2.7.1.74
R104Q/D133A
site-directed mutagenesis, the mutations render the enzyme active with 5-substituted deoxycytidine and thymidine, altered substrate specificity compared to the wild-type enzyme
Homo sapiens
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
2.7.1.74
Mg2+
required
Homo sapiens
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
2.7.1.74
ATP + 5-methyldeoxycytidine
Homo sapiens
-
ADP + 5-methyl-dCMP
-
-
?
2.7.1.74
ATP + deoxycytidine
Homo sapiens
-
ADP + dCMP
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
2.7.1.74
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain C41(DE3) by nickel affinity chromatography and gel filtration
Homo sapiens
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.7.1.74
ATP + (E)-5-(2-bromovinyl)-2'-deoxyuridine
substrate of enzyme mutant R104M/D133A, poor substrate of the wild-type enzyme
721622
Homo sapiens
ADP + ?
-
-
-
?
2.7.1.74
ATP + 5-bromodeoxycytidine
-
721622
Homo sapiens
ADP + 5-bromo-dCMP
-
-
-
?
2.7.1.74
ATP + 5-iododeoxycytidine
-
721622
Homo sapiens
ADP + 5-iodo-dCMP
-
-
-
?
2.7.1.74
ATP + 5-methyldeoxycytidine
-
721622
Homo sapiens
ADP + 5-methyl-dCMP
-
-
-
?
2.7.1.74
ATP + 5-propynyldeoxycytidine
-
721622
Homo sapiens
ADP + 5-propynyl-dCMP
-
-
-
?
2.7.1.74
ATP + D-deoxycytidine
-
721622
Homo sapiens
ADP + D-dCMP
-
-
-
?
2.7.1.74
ATP + D-deoxythymidine
-
721622
Homo sapiens
ADP + D-dTMP
-
-
-
?
2.7.1.74
ATP + D-deoxyuridine
-
721622
Homo sapiens
ADP + D-dUMP
-
-
-
?
2.7.1.74
ATP + deoxycytidine
-
721622
Homo sapiens
ADP + dCMP
-
-
-
?
2.7.1.74
ATP + gemcitabine
-
721622
Homo sapiens
ADP + phospho-gemcitabine
-
-
-
?
2.7.1.74
ATP + L-deoxyuridine
-
721622
Homo sapiens
ADP + L-dUMP
-
-
-
?
2.7.1.74
ATP + L-thymidine
-
721622
Homo sapiens
ADP + L-dTMP
-
-
-
?
2.7.1.74
additional information
substrate specificity or wild-type an dmutant enzymes, overview
721622
Homo sapiens
?
-
-
-
-
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
2.7.1.74
37
-
assay at
Homo sapiens
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
2.7.1.74
7.5
-
assay at
Homo sapiens
General Information
EC Number
General Information
Commentary
Organism
2.7.1.74
additional information
structure-function analysis and substrate specificity, overview
Homo sapiens
General Information (protein specific)
EC Number
General Information
Commentary
Organism
2.7.1.74
additional information
structure-function analysis and substrate specificity, overview
Homo sapiens