The putative effector-binding site of Leishmania mexicana pyruvate kinase studied by site-directed mutagenesis

The activity of pyruvate kinase of Leishmania mexicana is allosterically regulated by fructose 2,6-bisphosphate (F-2,6-P-2), contrary to the pyruvate kinases from other eukaryotes that are usually stimulated by fructose 1,6-bisphosphate (F-1,6-P-2). Based on the comparison of the three-dimensional structure of Saccharomyces cerevisiae pyruvate kinase crystallized with F-1,6-P-2 present at the effector site (R-state) and the L. mexicana enzyme crystallized in the T-state, two residues (Lys453 and His480) were proposed to bind the 2-phospho group of the effector. This hypothesis was tested by site-directed mutagenesis. The allosteric activation by F-2,6-P-2 appeared to be entirely abrogated in the mutated enzymes confirming our predictions. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.