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Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases.

Gibellini F, Hunter WN, Smith TK - Biochem. J. (2008)

Bottom Line: The K(m) values for ethanolamine and ATP were found to be 18.4+/-0.9 and 219+/-29 microM respectively.TbC/EK2 (T. brucei choline/ethanolamine kinase 2), on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a K(m) 80 times lower than that of ethanolamine.Both enzymes recognized analogues with substituents on C-2, but substitutions on C-1 and elongations of the carbon chain were not well tolerated.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.

ABSTRACT
Ethanolamine and choline are major components of the trypanosome membrane phospholipids, in the form of GPEtn (phosphatidylethanolamine) [corrected] and GPCho (phosphatidylcholine) [corrected] . Ethanolamine is also found as an integral component of the GPI (glycosylphosphatidylinositol) anchor that is required for membrane attachment of cell-surface proteins, most notably the variant-surface glycoproteins. The de novo synthesis of GPEtn and GPCho starts with the generation of phosphoethanolamine and phosphocholine by ethanolamine and choline kinases via the Kennedy pathway. Database mining revealed two putative C/EKs (choline/ethanolamine kinases) in the Trypanosoma brucei genome, which were cloned, overexpressed, purified and characterized. TbEK1 (T. brucei ethanolamine kinase 1) was shown to be catalytically active as an ethanolamine-specific kinase, i.e. it had no choline kinase activity. The K(m) values for ethanolamine and ATP were found to be 18.4+/-0.9 and 219+/-29 microM respectively. TbC/EK2 (T. brucei choline/ethanolamine kinase 2), on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a K(m) 80 times lower than that of ethanolamine. The K(m) values for choline, ethanolamine and ATP were 31.4+/-2.6 microM, 2.56+/-0.31 mM and 20.6+/-1.96 microM respectively. Further substrate specificity analysis revealed that both TbEK1 and TbC/EK2 were able to tolerate various modifications at the amino group, with the exception of a quaternary amine for TbEK1 (choline) and a primary amine for TbC/EK2 (ethanolamine). Both enzymes recognized analogues with substituents on C-2, but substitutions on C-1 and elongations of the carbon chain were not well tolerated.

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Kinetic analysis of TbC/EK2(A) CK activity was measured spectrophotometrically as a function of pH as described in the Experimental section. Results are means±S.D. of three measurements. (B–D) Determination of TbC/EK2 Michaelis–Menten constants for choline, ethanolamine and ATP (insets are Lineweaver–Burk plots). ATP concentration was held constant (5 mM), while choline (B) or ethanolamine (EtN) (C) concentrations were varied. (D) Choline concentration was held constant (2 mM), while ATP concentration was varied.
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Figure 7: Kinetic analysis of TbC/EK2(A) CK activity was measured spectrophotometrically as a function of pH as described in the Experimental section. Results are means±S.D. of three measurements. (B–D) Determination of TbC/EK2 Michaelis–Menten constants for choline, ethanolamine and ATP (insets are Lineweaver–Burk plots). ATP concentration was held constant (5 mM), while choline (B) or ethanolamine (EtN) (C) concentrations were varied. (D) Choline concentration was held constant (2 mM), while ATP concentration was varied.

Mentions: TbC/EK2, on the other hand, was found to be able to phosphorylate both ethanolamine (Figure 6B) and choline (Figures 6A and 6C), even though choline was the preferred substrate (Tables 1 and 2), with a Km 80 times lower than that of ethanolamine. TbC/EK2 was also dependent upon ATP and Mg2+ for activity (Figures 6B and 6C) with a pH optimum of 7.8 (Figure 7A). Kinetic analysis showed that TbC/EK2 displayed Km values for choline, ethanolamine and ATP of 31.4±2.6 μM, 2.56±0.31 mM and 20.6±1.96 μM respectively with a Vmax of 10.67±0.18 μmol/min per mg of protein and a kcat of 12.91 s−1 (Figures 7B–7D and Table 1). This value is in the range of activities reported for other eukaryotic C/EKs, and more than ten times higher than the CK from P. falciparum [15].


Biochemical characterization of the initial steps of the Kennedy pathway in Trypanosoma brucei: the ethanolamine and choline kinases.

Gibellini F, Hunter WN, Smith TK - Biochem. J. (2008)

Kinetic analysis of TbC/EK2(A) CK activity was measured spectrophotometrically as a function of pH as described in the Experimental section. Results are means±S.D. of three measurements. (B–D) Determination of TbC/EK2 Michaelis–Menten constants for choline, ethanolamine and ATP (insets are Lineweaver–Burk plots). ATP concentration was held constant (5 mM), while choline (B) or ethanolamine (EtN) (C) concentrations were varied. (D) Choline concentration was held constant (2 mM), while ATP concentration was varied.
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Related In: Results  -  Collection

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Figure 7: Kinetic analysis of TbC/EK2(A) CK activity was measured spectrophotometrically as a function of pH as described in the Experimental section. Results are means±S.D. of three measurements. (B–D) Determination of TbC/EK2 Michaelis–Menten constants for choline, ethanolamine and ATP (insets are Lineweaver–Burk plots). ATP concentration was held constant (5 mM), while choline (B) or ethanolamine (EtN) (C) concentrations were varied. (D) Choline concentration was held constant (2 mM), while ATP concentration was varied.
Mentions: TbC/EK2, on the other hand, was found to be able to phosphorylate both ethanolamine (Figure 6B) and choline (Figures 6A and 6C), even though choline was the preferred substrate (Tables 1 and 2), with a Km 80 times lower than that of ethanolamine. TbC/EK2 was also dependent upon ATP and Mg2+ for activity (Figures 6B and 6C) with a pH optimum of 7.8 (Figure 7A). Kinetic analysis showed that TbC/EK2 displayed Km values for choline, ethanolamine and ATP of 31.4±2.6 μM, 2.56±0.31 mM and 20.6±1.96 μM respectively with a Vmax of 10.67±0.18 μmol/min per mg of protein and a kcat of 12.91 s−1 (Figures 7B–7D and Table 1). This value is in the range of activities reported for other eukaryotic C/EKs, and more than ten times higher than the CK from P. falciparum [15].

Bottom Line: The K(m) values for ethanolamine and ATP were found to be 18.4+/-0.9 and 219+/-29 microM respectively.TbC/EK2 (T. brucei choline/ethanolamine kinase 2), on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a K(m) 80 times lower than that of ethanolamine.Both enzymes recognized analogues with substituents on C-2, but substitutions on C-1 and elongations of the carbon chain were not well tolerated.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK.

ABSTRACT
Ethanolamine and choline are major components of the trypanosome membrane phospholipids, in the form of GPEtn (phosphatidylethanolamine) [corrected] and GPCho (phosphatidylcholine) [corrected] . Ethanolamine is also found as an integral component of the GPI (glycosylphosphatidylinositol) anchor that is required for membrane attachment of cell-surface proteins, most notably the variant-surface glycoproteins. The de novo synthesis of GPEtn and GPCho starts with the generation of phosphoethanolamine and phosphocholine by ethanolamine and choline kinases via the Kennedy pathway. Database mining revealed two putative C/EKs (choline/ethanolamine kinases) in the Trypanosoma brucei genome, which were cloned, overexpressed, purified and characterized. TbEK1 (T. brucei ethanolamine kinase 1) was shown to be catalytically active as an ethanolamine-specific kinase, i.e. it had no choline kinase activity. The K(m) values for ethanolamine and ATP were found to be 18.4+/-0.9 and 219+/-29 microM respectively. TbC/EK2 (T. brucei choline/ethanolamine kinase 2), on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a K(m) 80 times lower than that of ethanolamine. The K(m) values for choline, ethanolamine and ATP were 31.4+/-2.6 microM, 2.56+/-0.31 mM and 20.6+/-1.96 microM respectively. Further substrate specificity analysis revealed that both TbEK1 and TbC/EK2 were able to tolerate various modifications at the amino group, with the exception of a quaternary amine for TbEK1 (choline) and a primary amine for TbC/EK2 (ethanolamine). Both enzymes recognized analogues with substituents on C-2, but substitutions on C-1 and elongations of the carbon chain were not well tolerated.

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