Literature summary for 2.7.1.30 extracted from

Balogun, E.O.; Inaoka, D.K.; Shiba, T.; Tokuoka, S.M.; Tokumasu, F.; Sakamoto, K.; Kido, Y.; Michels, P.A.M.; Watanabe, Y.I.; Harada, S.; Kita, K.
Glycerol kinase of African trypanosomes possesses an intrinsic phosphatase activity (2017), Biochim. Biophys. Acta, 1861, 2830-2842 .

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain JM109	Trypanosoma brucei gambiense
recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain JM109	Trypanosoma brucei rhodesiense

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant His6-tagged enzyme in complex with pNPP, sitting drop method, mixing of 0.001 ml of 5 mg/mL TbgGK in 10 mM MgSO4, 20 mM NaCl, 0.001% glycerol and 10 mM MOPS, pH 6.8, with 0.001 ml of 11% 1,6-hexanediol, 25% PEG 400, and 0.1 M HEPES, pH 7.5, at 20°C, crystals of the TbgGK-pNPP complex are obtained by soaking TbgGK crystals in a cryoprotectant solution containing 5.5% 1,6-hexanediol, 40% PEG 400, and 0.1 M HEPES, pH 7.5, that is supplemented with 20 mM pNPP, at 20°C, for 6-14 h, X-ray diffraction structure determination and analysis, molecular replacement method using the protein structure of the TbgGK-ADP complex (PDB ID 3WXL) as a search model	Trypanosoma brucei gambiense
purified recombinant His6-tagged enzyme in complex with pNPP, sitting drop method, mixing of 0.001 ml of 5 mg/mL TbgGK in 10 mM MgSO4, 20 mM NaCl, 0.001% glycerol and 10 mM MOPS, pH 6.8, with 0.001 ml of 11% 1,6-hexanediol, 25% PEG 400, and 0.1 M HEPES, pH 7.5, at 20°C, crystals of the TbgGK-pNPP complex are obtained by soaking TbgGK crystals in a cryoprotectant solution containing 5.5% 1,6-hexanediol, 40% PEG 400, and 0.1 M HEPES, pH 7.5, that is supplemented with 20 mM pNPP, at 20°C, for 6-14 h, X-ray diffraction structure determination and analysis, molecular replacement method using the protein structure of the TbgGK-ADP complex (PDB ID 3WXL) as a search model	Trypanosoma brucei rhodesiense

Crystallization (Comment)

Organism

purified recombinant His6-tagged enzyme in complex with pNPP, sitting drop method, mixing of 0.001 ml of 5 mg/mL TbgGK in 10 mM MgSO4, 20 mM NaCl, 0.001% glycerol and 10 mM MOPS, pH 6.8, with 0.001 ml of 11% 1,6-hexanediol, 25% PEG 400, and 0.1 M HEPES, pH 7.5, at 20°C, crystals of the TbgGK-pNPP complex are obtained by soaking TbgGK crystals in a cryoprotectant solution containing 5.5% 1,6-hexanediol, 40% PEG 400, and 0.1 M HEPES, pH 7.5, that is supplemented with 20 mM pNPP, at 20°C, for 6-14 h, X-ray diffraction structure determination and analysis, molecular replacement method using the protein structure of the TbgGK-ADP complex (PDB ID 3WXL) as a search model

Trypanosoma brucei gambiense

Trypanosoma brucei rhodesiense

Inhibitors

Inhibitors	Comment	Organism
ADP	competitive inhibition of the phosphatase activity	Trypanosoma brucei gambiense
ADP	competitive inhibition of the phosphatase activity	Trypanosoma brucei rhodesiense
sn-glycerol 3-phosphate	shows both competitive and non-competitive mixed-type inhibition	Trypanosoma brucei gambiense
sn-glycerol 3-phosphate	shows both competitive and non-competitive mixed-type inhibition	Trypanosoma brucei rhodesiense

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	classical Michaelis-Menten kinetic pattern for the phosphatase reaction	Trypanosoma brucei gambiense
additional information	-	additional information	classical Michaelis-Menten kinetic pattern for the phosphatase reaction	Trypanosoma brucei rhodesiense

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Trypanosoma brucei gambiense
Mg2+	required	Trypanosoma brucei rhodesiense

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + glycerol	Trypanosoma brucei gambiense	-	ADP + sn-glycerol 3-phosphate	-	?
ATP + glycerol	Trypanosoma brucei rhodesiense	-	ADP + sn-glycerol 3-phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Trypanosoma brucei gambiense	D3KVM3	-	-
Trypanosoma brucei rhodesiense	D3KVM4	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain JM109 by nickel affinity chromatography and gel filtration	Trypanosoma brucei gambiense
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain JM109 by nickel affinity chromatography and gel filtration	Trypanosoma brucei rhodesiense

Reaction

Reaction	Comment	Organism	Reaction ID
ATP + glycerol = ADP + sn-glycerol 3-phosphate	the structure of the TGK-pNPP complex, and structure-guided mutagenesis implicate that enzyme residue T276 is important for the catalysis. The enzyme is a bifunctional kinase/phosphatase. Proposed catalytic mechanism for the phosphatase activity of TbgGK	Trypanosoma brucei gambiense	a
ATP + glycerol = ADP + sn-glycerol 3-phosphate	the structure of the TGK-pNPP complex, and structure-guided mutagenesis implicate that enzyme residue T276 is important for the catalysis. The enzyme is a bifunctional kinase/phosphatase. Proposed catalytic mechanism for the phosphatase activity of TbgGK	Trypanosoma brucei rhodesiense	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + glycerol	-	Trypanosoma brucei gambiense	ADP + sn-glycerol 3-phosphate	-	?
ATP + glycerol	-	Trypanosoma brucei rhodesiense	ADP + sn-glycerol 3-phosphate	-	?
additional information	in addition to its widely known and expected phosphotransferase (class II) activity, TbgGK can efficiently facilitate the hydrolytic cleavage of phosphoric anhydride bonds (a class III property). 4-Nitrophenylphosphate (pNPP) dephosphorylation is TbgGK-mediated, LC-MS analysis of the TbgGK-catalyzed dephosphorylation of its physiological substrate	Trypanosoma brucei gambiense	?	-	-
additional information	in addition to its widely known and expected phosphotransferase (class II) activity, TbgGK can efficiently facilitate the hydrolytic cleavage of phosphoric anhydride bonds (a class III property). 4-Nitrophenylphosphate (pNPP) dephosphorylation is TbgGK-mediated, LC-MS analysis of the TbgGK-catalyzed dephosphorylation of its physiological substrate	Trypanosoma brucei rhodesiense	?	-	-

Synonyms

Synonyms	Comment	Organism
TbgGK	-	Trypanosoma brucei gambiense

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	recombinant enzyme, phosphatase activity	Trypanosoma brucei gambiense
37	-	recombinant enzyme, phosphatase activity	Trypanosoma brucei rhodesiense

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
5	-	recombinant enzyme, the pH optima of the phosphatase and kinase activities coincide, but the kinase activity is 7fold higher and also evident over a wider range of pH values (pH 5 to 8)	Trypanosoma brucei gambiense
5	-	recombinant enzyme, the pH optima of the phosphatase and kinase activities coincide, but the kinase activity is 7fold higher and also evident over a wider range of pH values (pH 5 to 8)	Trypanosoma brucei rhodesiense

pH Range

pH Minimum	pH Maximum	Comment	Organism
5	8	kinase activity	Trypanosoma brucei gambiense
5	8	kinase activity	Trypanosoma brucei rhodesiense

General Information

General Information	Comment	Organism
evolution	although there are two African human pathogenic Trypanosoma subspecies: Trypanosoma brucei gambiense (Tbg) and Trypanosoma brucei rhodesiense (Tbr), it is reported in that the amino acid sequences of their GKs are exactly identical. Hence, TbgGK represents the glycerol kinase of both subspecies	Trypanosoma brucei gambiense
evolution	although there are two African human pathogenic Trypanosoma subspecies: Trypanosoma brucei gambiense (Tbg) and Trypanosoma brucei rhodesiense (Tbr), it is reported in that the amino acid sequences of their GKs are exactly identical. Hence, TbgGK represents the glycerol kinase of both subspecies	Trypanosoma brucei rhodesiense
additional information	the enzyme uses a common catalytic site for both activities, phosphatase and kinase	Trypanosoma brucei gambiense
additional information	the enzyme uses a common catalytic site for both activities, phosphatase and kinase	Trypanosoma brucei rhodesiense