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Literature summary for 2.7.1.32 extracted from

  • Gibellini, F.; Hunter, W.N.; Smith, T.K.
    Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases (2008), Biochem. J., 415, 135-144.
    View publication on PubMedView publication on EuropePMC

Activating Compound

Activating Compound Comment Organism Structure
isopropyl-beta-D-thiogalactoside recombinant protein expression is induced with 1 mM Trypanosoma brucei

Cloned(Commentary)

Cloned (Comment) Organism
molecular cloning and recombinant expression in Escherichia coli strains DH5alpha, XL-1 blue and TOP10. BL21-Gold(DE3) is used for protein overexpression, RNA from bloodstream form of Trypanosoma brucei is used for cDNA synthesis. Trypanosoma brucei

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.0206
-
ATP
-
Trypanosoma brucei
0.0314
-
choline
-
Trypanosoma brucei
2.56
-
ethanolamine
-
Trypanosoma brucei

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
72610
-
determined by MALDI-TOF-MS Trypanosoma brucei

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
choline + ATP Trypanosoma brucei first step in the biosynthesis of the glycerophospholipid glycerophosphocholine O-phosphocholine + ADP
-
?
ethanolamine + ATP Trypanosoma brucei first step in the biosynthesis of the glycerophospholipid glycerophosphoethanolamine O-phosphoethanolamine + ADP
-
?

Organism

Organism UniProt Comment Textmining
Trypanosoma brucei
-
protozoan parasite which causes African sleeping sickness
-

Purification (Commentary)

Purification (Comment) Organism
cleared lysate is applied to a HisTrap column column pre-loaded with Ni2+. Unbound proteins are removed by washing the column with 50 mM Tris/HCl, pH 8.0, 300 mM NaCl and 10 mM imidazole, whereas TbC/EK1 and TbC/EK2 are eluted with an imidazole gradient in the same buffer. Fractions containing TbC/EK1 or TbC/EK2 are pooled, dialysed against 50 mM Tris/HCl and 300 mM NaCl and purified further through a Superdex 200 16/60 gel-filtration column. Trypanosoma brucei

Source Tissue

Source Tissue Comment Organism Textmining
cell culture strainn 427 is used for isolation of RNA Trypanosoma brucei
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
10.67
-
2 microgram of purified protein is incubated with a reaction mixture of 100 mM MOPS (pH 7.8), 6 mM MgCl2 and 5 mM ATP, with either 2 mM ethanolamine and 200 nanoCi of [3H]ethanolamine or 2 mM choline and 200 nanoCi of (3H)choline at 37°C for 20 min and quenched by boiling at 100°C for 5 min. Substrates and products are separated by high-performance TLC using silica 60 plates with methanol/0.6% NaCl/ammonium hydroxide as solvent. Radiolabelled species are detected by fluorography at -70°C. Trypanosoma brucei

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
choline + ATP first step in the biosynthesis of the glycerophospholipid glycerophosphocholine Trypanosoma brucei O-phosphocholine + ADP
-
?
diethanolamine + ATP
-
Trypanosoma brucei ADP + O-phospho-diethanolamine
-
?
ethanolamine + ATP first step in the biosynthesis of the glycerophospholipid glycerophosphoethanolamine Trypanosoma brucei O-phosphoethanolamine + ADP
-
?
additional information Further substrate specificity analysis reveals that TbC/EK2 are able to tolerate various modifications at the amino group, with the exception of a quaternary amine for TbEK1 (choline). The enzyme recognizes analogues with substituents on C-2, but substitutions on C-1 and elongations of the carbon chain are not well tolerate. Secondary and tertiary amino modifications are better than choline. A reduction of activity is seen with the elongation of the carbon chain, but it is not abolished even when increased by an ethylene unit. Modifications at the hydroxy group result in no detectable reactivity. Trypanosoma brucei ?
-
?
N,N-diethylethanolamine + ATP
-
Trypanosoma brucei ADP + O-phospho-N-diethylethanolamine
-
?
N,N-dimethylethanolamine + ATP
-
Trypanosoma brucei ADP + O-phospho-N,N-dimethylethanolamine
-
?
N-ethylethanolamine + ATP
-
Trypanosoma brucei ADP + O-phospho-N-ethylethanolamine
-
?
N-methylethanolamine + ATP
-
Trypanosoma brucei ADP + O-phospho-N-methylethanolamine
-
?

Subunits

Subunits Comment Organism
dimer Elution from the gel-filtration column shows that TbC/EK2 exhibited a dimeric state in solution. Trypanosoma brucei

Synonyms

Synonyms Comment Organism
choline kinase
-
Trypanosoma brucei
choline/ethanolamine kinase 2
-
Trypanosoma brucei
TbC/EK2
-
Trypanosoma brucei

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
12.91
-
choline pH 7.8, 37°C, assay with recombinantly expressed enzyme Trypanosoma brucei

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.8
-
The pH optimum is assessed by measuring choline kinase activities at various pH values from pH 5.0 to pH 9.0 Trypanosoma brucei

Cofactor

Cofactor Comment Organism Structure
ATP
-
Trypanosoma brucei