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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 TbEK1 (formerly TbC/EK1)(A) EK activity was measured spectrophotometrically as a function of pH as described in the Experimental section. Results are means±S.D. of three measurements. (B, C) Determination of TbEK1 Michaelis–Menten constants for ethanolamine and ATP (insets are Lineweaver–Burk plots). (B) ATP concentration was held constant (5 mM), while ethanolamine concentration was varied. (C) Ethanolamine concentration was held constant (2 mM), while ATP concentration was varied.
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Figure 5: Kinetic analysis of TbEK1 (formerly TbC/EK1)(A) EK activity was measured spectrophotometrically as a function of pH as described in the Experimental section. Results are means±S.D. of three measurements. (B, C) Determination of TbEK1 Michaelis–Menten constants for ethanolamine and ATP (insets are Lineweaver–Burk plots). (B) ATP concentration was held constant (5 mM), while ethanolamine concentration was varied. (C) Ethanolamine concentration was held constant (2 mM), while ATP concentration was varied.

Mentions: Kinetic analysis showed that TbEK1 displayed a pH optimum at pH 8.0 (Figure 5A), with a Km of 18.4±0.9 μM for ethanolamine (Figure 5B) and a Km of 219±29 μM for ATP (Figure 5C), which is in reasonable agreement with a previously reported Km value of 2.75 μM for EK activity from crude trypanosome homogenates [14]. The Vmax of TbEK1 is 7.62±0.07 μmol/min per mg of protein, corresponding to a kcat of 6.56 s−1 (Table 1). This turnover rate/specific activity compares favourably with the previously reported activities of 346 nmol/min per mg of protein for the yeast EK [16] and 10.73 μmol/min per mg of protein for a soya bean EK [25]. Two catalytically inactive mutants of TbEK1 were generated by substitution of alanine for Asp267 and Asp286 (Figures 4B and 4C), thus confirming the crucial importance of these two residues in the catalytic mechanism of this kinase.


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 TbEK1 (formerly TbC/EK1)(A) EK activity was measured spectrophotometrically as a function of pH as described in the Experimental section. Results are means±S.D. of three measurements. (B, C) Determination of TbEK1 Michaelis–Menten constants for ethanolamine and ATP (insets are Lineweaver–Burk plots). (B) ATP concentration was held constant (5 mM), while ethanolamine concentration was varied. (C) Ethanolamine concentration was held constant (2 mM), while ATP concentration was varied.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2552378&req=5

Figure 5: Kinetic analysis of TbEK1 (formerly TbC/EK1)(A) EK activity was measured spectrophotometrically as a function of pH as described in the Experimental section. Results are means±S.D. of three measurements. (B, C) Determination of TbEK1 Michaelis–Menten constants for ethanolamine and ATP (insets are Lineweaver–Burk plots). (B) ATP concentration was held constant (5 mM), while ethanolamine concentration was varied. (C) Ethanolamine concentration was held constant (2 mM), while ATP concentration was varied.
Mentions: Kinetic analysis showed that TbEK1 displayed a pH optimum at pH 8.0 (Figure 5A), with a Km of 18.4±0.9 μM for ethanolamine (Figure 5B) and a Km of 219±29 μM for ATP (Figure 5C), which is in reasonable agreement with a previously reported Km value of 2.75 μM for EK activity from crude trypanosome homogenates [14]. The Vmax of TbEK1 is 7.62±0.07 μmol/min per mg of protein, corresponding to a kcat of 6.56 s−1 (Table 1). This turnover rate/specific activity compares favourably with the previously reported activities of 346 nmol/min per mg of protein for the yeast EK [16] and 10.73 μmol/min per mg of protein for a soya bean EK [25]. Two catalytically inactive mutants of TbEK1 were generated by substitution of alanine for Asp267 and Asp286 (Figures 4B and 4C), thus confirming the crucial importance of these two residues in the catalytic mechanism of this kinase.

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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