Biochemical characterisation of the initial steps of the kennedy pathway in Trypanosoma brucei - the ethanolamine and choline kinases
Résumé
Ethanolamine (EtN) and choline (Cho) are major components of the trypanosome membrane phospholipids, in the form of phosphatidylethanolamine (GPEtn) and phosphatidylcholine (GPCho). Ethanolamine is also found as an integral component of the glycosylphosphatidylinositol (GPI) anchor that is required for membrane attachment of cell surface proteins, most notably the variant surface glycoproteins. The de novo synthesis of GPEth and GPCho starts with the generation of phosphoethanolamine and phosphocholine by ethanolamine and choline kinases via the Kennedy pathway. Database mining revealed two putative choline/ethanolamine kinases (C/EKs) in the T. brucei genome, which were cloned, over-expressed, purified and characterised. TbEK1 was shown to be catalytically active as an ethanolamine-specific kinase, i.e. no choline kinase activity. The Km values for ethanolamine and ATP were found to be 18.4 ± 0.9 μM and 219 ± 29 μM, respectively. TbC/EK2, on the other hand, was found to be able to phosphorylate both ethanolamine and choline, even though choline was the preferred substrate, with a Km 80 times lower than that of ethanolamine. The Km values for choline, ethanolamine and ATP were; 31.4 ± 2.6 μM, 2.56 ± 0.31 mM and 20.6 ± 1.96 μM respectively. Further substrate specificity analysis revealed 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 C2 but substitutions on C1 and elongations of the carbon chain were not well tolerated.
Origine : Fichiers produits par l'(les) auteur(s)
Loading...