The phosphofructokinase of Trypanosoma (Schizotrypanum) cruzi: purification and kinetic mechanism.
The phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, E.C.2.7.1.11) of Trypanosoma (Schizotrypanum) cruzi epimastigotes has been purified 180-fold, to apparent electrophoretic homogeneity, by differential centrifugation, gel filtration chromatography and anionic exchange chromatography. The minimum catalytic unit of the purified enzyme is a polypeptide of 17,000 +/- 1300 molecular weight, as shown by gel filtration chromatography and SDS-gel electrophoresis. Hanes-Woolf plots of initial rates and Mg-ATP or D-fructose-6-phosphate concentrations for varying values of the co-substrate concentration gave intersecting lines which indicate a sequential mechanism. No inhibition is observed at high Mg-ATP concentrations, confirming the result of a previous study using a partially purified enzyme. The pure enzyme displays both negative and positive cooperative kinetics at low (less than 0.8 mM) concentrations of D-fructose-6-phosphate, suggesting aggregation and/or activation phenomena induced by this substrate. Product inhibition studies at saturating and non-saturating concentrations of D-fructose-6-phosphate gave as the only compatible mechanism an iso-ordered bi-bi process with a probable order of entry to and exit from the active site being: D-fructose-6-phosphate followed by Mg-ATP and D-fructose-1,6-bisphosphate followed by Mg-ADP, respectively. The very important differences in both the structural and mechanistic aspects between this enzyme and its vertebrate and bacterial counterparts supports the notion of a highly unusual carbohydrate catabolism in this parasite.
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000302:isolation & purification* / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology*
D010732:Phosphofructokinase-1