Entamoeba histolytica: kinetic and molecular evidence of a previously unidentified pyruvate kinase.
We report the kinetic characterization of a previously unidentified pyruvate kinase (PK) activity in extracts from Entamoeba histolytica trophozoites. This activity was about 74% of the activity of pyruvate phosphate dikinase. EhPK differed from most PKs in that its pH optimum was 5.5-6.5 and was inhibited by high PEP concentrations (1-5mM); these are concentrations at which PK is usually assayed. The optimal temperature was above 40 degrees C with negligible activity below 20 degrees C. EhPK exhibited hyperbolic kinetics with respect to both PEP (K(m) = 0.018 mM) and ADP (K(m) = 1.05 mM). However, it exhibited a sigmoidal behavior with respect to PEP at sub-saturating ADP concentrations. EhPK did not require monovalent cations for activity. Fructose-1,6 bisphosphate was a potent non-essential activator; it increased the affinity for ADP without modification of the V(max) or the affinity for PEP. Phosphate, citrate, malate, and alpha-ketoglutarate significantly inhibited EhPK activity. A putative EhPK gene fragment found in EhDNA was analyzed. The data indicate that E. histolytica trophozoites contain an active PK, which might contribute to the generation of glycolytic ATP for parasite survival.
Saavedra
Emma
E
Departamento de Bioquímica, Instituto Nacional de Cardiología, México D.F. 14080, Mexico. emma_saavedra2002@yahoo.com
Olivos
Alfonso
A
Encalada
Rusely
R
Moreno-Sánchez
Rafael
R
D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008101:Liver Abscess, Amebic / Q000469:parasitology; D008969:Molecular Sequence Data; D010728:Phosphoenolpyruvate / Q000378:metabolism; D011770:Pyruvate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011772:Pyruvate, Orthophosphate Dikinase / Q000378:metabolism; D011817:Rabbits; D016415:Sequence Alignment; D013696:Temperature
D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; D011772:Pyruvate, Orthophosphate Dikinase