Open AccessLeishmania donovani phosphofructokinase
Author(s) -
López Claudia,
Chevalier Nathalie,
Hannaert Véronique,
Rigden Daniel J.,
Michels Paul A. M.,
Ramirez Jose Luis
Publication year - 2002
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2002.03086.x
Subject(s) - phosphofructokinase , biology , biochemistry , microbiology and biotechnology , entamoeba histolytica , leishmania donovani , phosphofructokinase 2 , gene , enzyme , glycolysis , genetics , leishmaniasis , visceral leishmaniasis
The characterization of the gene encoding Leishmania donovani phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported. L. donovani has a single PFK gene copy per haploid genome that encodes a polypeptide with a deduced molecular mass of 53 988 and a pI of 9.26. The predicted amino acid sequence contains a C‐terminal tripeptide that conforms to an established signal for glycosome targeting. L. donovani PFK showed most sequence similarity to inorganic pyrophosphate (PP i )‐dependent PFKs, despite being ATP‐dependent. It thereby resembles PFKs from other Kinetoplastida such as Trypanosoma brucei , Trypanoplasma borreli (characterized in this study), and a PFK found in Entamoeba histolytica . It exhibited hyperbolic kinetics with respect to ATP whereas the binding of the other substrate, fructose 6‐phosphate, showed slight positive cooperativity. PP i , even at high concentrations, did not have any effect. AMP acted as an activator of PFK, shifting its kinetics for fructose 6‐phosphate from slightly sigmoid to hyperbolic, and increasing considerably the affinity for this substrate, whereas GDP did not have any effect. Modelling studies and site‐directed mutagenesis were employed to shed light on the structural basis for the AMP effector specificity and on ATP/PP i specificity among PFKs.