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.
Hannaert
Véronique
V
Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry, Université Catholique de Louvain, ICP-TROP 74.39 Avenue Hippocrate, B-1200 Brussels, Belgium. hannaert@trop.ucl.ac.be
Yernaux
Cédric
C
Rigden
Daniel J
DJ
Fothergill-Gilmore
Linda A
LA
Opperdoes
Fred R
FR
Michels
Paul A M
PA
D000494:Allosteric Regulation / Q000187:drug effects / Q000502:physiology; D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics; D005635:Fructosediphosphates / Q000494:pharmacology; D007700:Kinetics; D007894:Leishmania mexicana / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship
D005635:Fructosediphosphates; D015800:Protozoan Proteins; C027652:fructose 2,6-diphosphate; D011770:Pyruvate Kinase