IBM and NeSC workshop on Protein Science
National e-Science Centre, Edinburgh, March
15-16 2002
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The effector site of leishmania pyruvate kinase as a
chemotherapeutic target Linda A Fothergill-Gilmore1, Véronique Hannaert2 & Paul A M Michels2, (1) ICMB, University of Edinburgh, Edinburgh EH9 3JR, Scotland, and (2) Christian de Duve Institute of Cellular Pathology, Université Catholique de Louvain, B-1200 Brussels, Belgium. Glycolytic enzymes have been identified as promising targets for new drugs against trypanosomatids because these enzymes play an essential role in ATP supply. The effector site of parasite pyruvate kinase (PYK) displays important differences from human PYKs, and thus offers opportunities for the development of parasite-specific inhibitors. Among the differences is the presence of two basic residues (Lys453 and His480) that are unique to trypanosomatid PYKs. The crystal structure of leishmania PYK has been solved in the inactive T-state in which the effector site has no bound ligand. However, it can be inferred by comparative modelling that Lys453 and His480 define the effector specificity of the parasite enzyme. The allosteric activator is fructose bisphosphate that has a phospho group in the 2-position instead of the 1-position as for PYKs from all other species, including humans. We have verified the putative binding role of Lys453 and His480 by making a series of site-directed mutants. All mutants show complete loss of the capability to respond to activation by fructose 2,6-bisphosphate, thus confirming our prediction. In addition, we have prepared two mutants for use as tools to screen the large number of compounds we anticipate from ligand docking, database mining and combinatorial chemistry. Wild-type trypanosomatid PYK has no tryptophan residues, and we have introduced this residue into two different positions near the effector site (F442W and E451W). Both mutants show fluorescence quenching in response to substrates and effectors, and will thus play an important role in screening combinatorial libraries. This work was supported by the European Commission through its INCO-DC programme. |
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