Glycolysis in Entamoeba histolytica. Biochemical characterization of recombinant glycolytic enzymes and flux control analysis.
The synthesis of ATP in the human parasite Entamoeba histolytica is carried out solely by the glycolytic pathway. Little kinetic and structural information is available for most of the pathway enzymes. We report here the gene cloning, overexpression and purification of hexokinase, hexose-6-phosphate isomerase, inorganic pyrophosphate-dependent phosphofructokinase, fructose-1,6 bisphosphate aldolase (ALDO), triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, phosphoglycerate mutase (PGAM), enolase, and pyruvate phosphate dikinase (PPDK) enzymes from E. histolytica. Kinetic characterization of these 10 recombinant enzymes was made, establishing the kinetic constants at optimal and physiological pH values, analyzing the effect of activators and inhibitors, and investigating the storage stability and oligomeric state. Determination of the catalytic efficiencies at the pH optimum and at pH values that resemble those of the amoebal trophozoites was performed for each enzyme to identify possible controlling steps. This analysis suggested that PGAM, ALDO, GAPDH, and PPDK might be flux control steps, as they showed the lowest catalytic efficiencies. An in vitro reconstruction of the final stages of glycolysis was made to determine their flux control coefficients. Our results indicate that PGAM and PPDK exhibit high control coefficient values at physiological pH.
Saavedra
Emma
E
Departamento de Bioquímica, Instituto Nacional de Cardiología, Tlalpan, México DF, México. emma_saavedra2002@yahoo.com
Encalada
Rusely
R
Pineda
Erika
E
Jasso-Chávez
Ricardo
R
Moreno-Sánchez
Rafael
R
D000818:Animals; D003001:Cloning, Molecular; D004748:Entamoeba histolytica / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D004798:Enzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006019:Glycolysis / Q000235:genetics / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D020539:Sequence Analysis, Protein
D004798:Enzymes; D011994:Recombinant Proteins