Literature summary for 2.7.1.32 extracted from

Kall, S.L.; Delikatny, E.J.; Lavie, A.
Identification of a unique inhibitor-binding site on choline kinase alpha (2018), Biochemistry, 57, 1316-1325 .

Application

Application	Comment	Organism
drug development	choline kinase is a promising target for development of inhibitors and inhibition of tumor growth	Homo sapiens

Cloned(Commentary)

Cloned (Comment)	Organism
gene CHKA, recombinant expression of His-SUMO-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta (DE3)pLysS	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant isozyme ChoKalpha truncated mutant DELTA79 in apoform and in complex with inhibitor TCD-717, sitting drop vapor diffusion method, mixing of 0.002 ml of 10 mg/ml protein solution with 0.002 ml of reservoir solution containing 0.1 M MES, pH 6.5, and 20% PEG 3350, 2-3 days, 20°C, crystals are soaked with TCD-717 by mixing a 50 mM solution of 100% DMSO-resuspended compound in a 1:4 ratio with the reservoir buffer, X-ray diffraction structure determination and analysis at 2.35 A resolution	Homo sapiens

Crystallization (Comment)

Organism

purified recombinant isozyme ChoKalpha truncated mutant DELTA79 in apoform and in complex with inhibitor TCD-717, sitting drop vapor diffusion method, mixing of 0.002 ml of 10 mg/ml protein solution with 0.002 ml of reservoir solution containing 0.1 M MES, pH 6.5, and 20% PEG 3350, 2-3 days, 20°C, crystals are soaked with TCD-717 by mixing a 50 mM solution of 100% DMSO-resuspended compound in a 1:4 ratio with the reservoir buffer, X-ray diffraction structure determination and analysis at 2.35 A resolution

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
additional information	construction of enzyme N-terminally truncated mutant ChoKalpha1-DELTA79, inhibitor TCD-717 binding structure, overview	Homo sapiens

Inhibitors

Inhibitors	Comment	Organism
(1-azabicyclo[2.2.2]octan-3-yl)bis(5-chlorothiophen-2-yl)methanol	-	Homo sapiens
(1S)-1-[(3R)-1-azabicyclo[2.2.2]octan-3-yl]-3-[2-[(oxan-4-yl)oxy]phenyl]-1-phenylpropan-1-ol	-	Homo sapiens
1,1'-[butane-1,4-diylbis[(4,1-phenylene)methylene]]bis[4-(dimethylamino)pyridin-1-ium]	-	Homo sapiens
1-[(4-[4-[4-([4-[(4-chlorophenyl)(methyl)amino]pyridin-1-ium-1-yl]methyl)phenyl]butyl]phenyl)methyl]-4-(dimethylamino)pyridin-1-ium	-	Homo sapiens
1-[[4-[4-[[4-(4-chloro-N-methylanilino)quinolin-1-ium-1-yl]methyl]phenyl]phenyl]methyl]-N-(4-chlorophenyl)-N-methylquinolin-1-ium-4-amine	i.e. TCD-717 or RSM-932A, strong inhibition of ChoKalpha, TCD-717 does not bind directly in the choline pocket but rather in a proximal location near the surface of the enzyme. Most notably, residues Tyr148, Ala176, Met177, Glu180, Phe200, Pro201, Trp248, Thr252, Tyr256, Glu332, Tyr333, Phe341, and Leu419 serve to orient and hold TCD-717 in place. Interaction of TCD-717 with ChoKalpha is driven by hydrophobic interactions across the N- and C-terminal domains. Binding of TCD-717 induces conformational changes compared to the unbound ChoKalpha structure. TCD-717 has completed phase I clinical trials	Homo sapiens
6-amino-3-[(4'-[[4-(dimethylamino)pyridin-1-ium-1-yl]methyl][1,1'-biphenyl]-4-yl)methyl]-9H-purin-3-ium	-	Homo sapiens
hemicholinium-3	HC-3	Homo sapiens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	steady-state kinetics	Homo sapiens

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + choline	Homo sapiens	-	ADP + phosphocholine	-	?

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	P35790	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-SUMO-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta (DE3)pLysS by nickel affinity chromatography, and dialysis, if necessary the tag is cleaved, followd by another nickel affinity chromatography step, and dialysis, the last step for all is gel filtration	Homo sapiens

Source Tissue

Source Tissue	Comment	Organism	Textmining
carcinoma cell	-	Homo sapiens	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + choline	-	Homo sapiens	ADP + phosphocholine	-	?

Synonyms

Synonyms	Comment	Organism
choline kinase alpha	-	Homo sapiens

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Homo sapiens

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
4.5	-	ATP	wild-type enzyme ChoKalpha, pH 7.5, 37°C	Homo sapiens
26.5	-	ATP	recombinant mutant ChoKalphaDELTA79, pH 7.5, 37°C	Homo sapiens
71	-	choline	wild-type enzyme ChoKalpha, pH 7.5, 37°C	Homo sapiens
91.1	-	choline	recombinant mutant ChoKalphaDELTA79, pH 7.5, 37°C	Homo sapiens

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Homo sapiens

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor	Structure
0.00052	-	wild-type enzyme ChoKalpha, pH 7.5, 37°C	Homo sapiens	1-[[4-[4-[[4-(4-chloro-N-methylanilino)quinolin-1-ium-1-yl]methyl]phenyl]phenyl]methyl]-N-(4-chlorophenyl)-N-methylquinolin-1-ium-4-amine

General Information

General Information	Comment	Organism
physiological function	choline kinase alpha (ChoKalpha) is an enzyme that is upregulated in many types of cancer and has been shown to be tumorigenic	Homo sapiens