Leishmania donovani phosphofructokinase. Gene characterization, biochemical properties and structure-modeling studies.
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 (PPi)-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. PPi, 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/PPi specificity among PFKs.
Instituto de Biología Experimental, Universidad Central de Venezuela, Caracas, Venezuela.
Claudia
C
López
Nathalie
N
Chevalier
Véronique
V
Hannaert
Daniel J
DJ
Rigden
Paul A M
PA
Michels
Jose Luis
JL
Ramirez
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D029968:Escherichia coli Proteins / Q000737:chemistry; D005635:Fructosediphosphates / Q000378:metabolism; D005636:Fructosephosphates / Q000378:metabolism; D017125:Genes, Protozoan; D006019:Glycolysis; D016830:Kinetoplastida / Q000201:enzymology / Q000235:genetics; D007893:Leishmania donovani / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics
D029968:Escherichia coli Proteins; D005635:Fructosediphosphates; D005636:Fructosephosphates; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; C027618:fructose-6-phosphate; D010732:Phosphofructokinase-1; C029063:fructose-1,6-diphosphate