BRENDA - Enzyme Database

Deoxycytidylate hydroxymethylase: purification, properties, and the role of a thiol group in catalysis

Lee, M.H.; Gautam-Basak, M.; Wooley, C.; Sander, E.G.; Biochemistry 27, 1367-1373 (1988)

Data extracted from this reference:

Activating Compound
EC Number
Activating Compound
Commentary
Organism
Structure
2.1.2.8
dithiothreitol
low specific activity samples can be activated by a 30 min room temperature incubation in 50 mM DTT
Escherichia virus T4
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
2.1.2.8
5,5'-dithiobis(2-nitrobenzoic acid)
complete loss of activity, second-order kinetics, inactivation is reversed by dithiothreitol, inactivation involves the modification of one thiol group per mol of dimeric enzyme
Escherichia virus T4
2.1.2.8
aminopterin
competitive inhibitor with respect to tetrahydrofolate
Escherichia virus T4
2.1.2.8
dihydrofolate
competitive inhibitor with respect to tetrahydrofolate
Escherichia virus T4
2.1.2.8
methotrexate
competitive inhibitor with respect to tetrahydrofolate
Escherichia virus T4
2.1.2.8
N-ethylmaleimide
complete loss of activity, second-order kinetics, pH-dependent inactivation, dCMP protects against inactivation, dCMP plus either methotrexate or aminopterin greatly enhances protection, inactivation involves the modification of one thiol group per mol of dimeric enzyme
Escherichia virus T4
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.1.2.8
0.04
-
tetrahydrofolate
-
Escherichia virus T4
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
2.1.2.8
28000
-
2 * 28000, two equally sized subunits are arranged asymmetrically, SDS-PAGE
Escherichia virus T4
2.1.2.8
51400
-
gel filtration
Escherichia virus T4
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
2.1.2.8
Escherichia virus T4
-
Escherichia coli strains Cr 63 and B infected with bacteriophage T4
-
Purification (Commentary)
EC Number
Commentary
Organism
2.1.2.8
1026fold purification; Escherichia coli B infected with
Escherichia virus T4
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
2.1.2.8
additional information
-
-
Escherichia virus T4
Storage Stability
EC Number
Storage Stability
Organism
2.1.2.8
frozen, 20 mM KH2PO4, pH 6.8, 6 months
Escherichia virus T4
2.1.2.8
room temperature, 24 h, stable
Escherichia virus T4
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.1.2.8
5,10-methylenetetrahydrofolate + H2O + deoxycytidylate
stoichiometry of dCMP binding to the dimeric enzyme is 1:1 and depends upon a reduced thiol group, one thiol-containing active site per mol of dimeric enzyme, sequential order of substrate binding
485785
Escherichia virus T4
tetrahydrofolate + 5-hydroxymethyldeoxycytidylate
-
485785
Escherichia virus T4
r
Subunits
EC Number
Subunits
Commentary
Organism
2.1.2.8
dimer
2 * 28000, two equally sized subunits are arranged asymmetrically, SDS-PAGE
Escherichia virus T4
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
2.1.2.8
30
-
assay at
Escherichia virus T4
Temperature Stability [°C]
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
2.1.2.8
20
-
room temperature, 24 h, stable
Escherichia virus T4
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
2.1.2.8
7.6
-
assay at
Escherichia virus T4
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
2.1.2.8
5,10-methylenetetrahydrofolate
active cofactor is likely 5,10-methylenetetrahydrofolate
Escherichia virus T4
2.1.2.8
tetrahydrofolate
-
Escherichia virus T4
Ki Value [mM]
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
2.1.2.8
0.001
-
aminopterin
-
Escherichia virus T4
2.1.2.8
0.016
-
methotrexate
-
Escherichia virus T4
2.1.2.8
0.021
-
dihydrofolate
-
Escherichia virus T4
Activating Compound (protein specific)
EC Number
Activating Compound
Commentary
Organism
Structure
2.1.2.8
dithiothreitol
low specific activity samples can be activated by a 30 min room temperature incubation in 50 mM DTT
Escherichia virus T4
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
2.1.2.8
5,10-methylenetetrahydrofolate
active cofactor is likely 5,10-methylenetetrahydrofolate
Escherichia virus T4
2.1.2.8
tetrahydrofolate
-
Escherichia virus T4
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
2.1.2.8
5,5'-dithiobis(2-nitrobenzoic acid)
complete loss of activity, second-order kinetics, inactivation is reversed by dithiothreitol, inactivation involves the modification of one thiol group per mol of dimeric enzyme
Escherichia virus T4
2.1.2.8
aminopterin
competitive inhibitor with respect to tetrahydrofolate
Escherichia virus T4
2.1.2.8
dihydrofolate
competitive inhibitor with respect to tetrahydrofolate
Escherichia virus T4
2.1.2.8
methotrexate
competitive inhibitor with respect to tetrahydrofolate
Escherichia virus T4
2.1.2.8
N-ethylmaleimide
complete loss of activity, second-order kinetics, pH-dependent inactivation, dCMP protects against inactivation, dCMP plus either methotrexate or aminopterin greatly enhances protection, inactivation involves the modification of one thiol group per mol of dimeric enzyme
Escherichia virus T4
Ki Value [mM] (protein specific)
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
2.1.2.8
0.001
-
aminopterin
-
Escherichia virus T4
2.1.2.8
0.016
-
methotrexate
-
Escherichia virus T4
2.1.2.8
0.021
-
dihydrofolate
-
Escherichia virus T4
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.1.2.8
0.04
-
tetrahydrofolate
-
Escherichia virus T4
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
2.1.2.8
28000
-
2 * 28000, two equally sized subunits are arranged asymmetrically, SDS-PAGE
Escherichia virus T4
2.1.2.8
51400
-
gel filtration
Escherichia virus T4
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
2.1.2.8
1026fold purification; Escherichia coli B infected with
Escherichia virus T4
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
2.1.2.8
additional information
-
-
Escherichia virus T4
Storage Stability (protein specific)
EC Number
Storage Stability
Organism
2.1.2.8
frozen, 20 mM KH2PO4, pH 6.8, 6 months
Escherichia virus T4
2.1.2.8
room temperature, 24 h, stable
Escherichia virus T4
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.1.2.8
5,10-methylenetetrahydrofolate + H2O + deoxycytidylate
stoichiometry of dCMP binding to the dimeric enzyme is 1:1 and depends upon a reduced thiol group, one thiol-containing active site per mol of dimeric enzyme, sequential order of substrate binding
485785
Escherichia virus T4
tetrahydrofolate + 5-hydroxymethyldeoxycytidylate
-
485785
Escherichia virus T4
r
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
2.1.2.8
dimer
2 * 28000, two equally sized subunits are arranged asymmetrically, SDS-PAGE
Escherichia virus T4
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
2.1.2.8
30
-
assay at
Escherichia virus T4
Temperature Stability [°C] (protein specific)
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
2.1.2.8
20
-
room temperature, 24 h, stable
Escherichia virus T4
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
2.1.2.8
7.6
-
assay at
Escherichia virus T4