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The in vitro Entamoeba histolytica pyruvate:ferredoxin oxidoreductase (EhPFOR) kinetic properties and the effect of oxidative stress on glycolytic pathway enzymes and fluxes in live trophozoites were evaluated.EhPFOR showed a strong preference for pyruvate as substrate over other oxoacids. | Pyruvate ferredoxin oxidoreductase and bifunctional aldehyde-alcohol dehydrogenase are essential for energy metabolism under oxidative stress in Entamoeba histolytica Erika Pineda1 Rusely Encalada1 Jose S. Rodriguez-Zavala1 Alfonso Olivos-Garcia2 Rafael Moreno-Sanchez1 and Emma Saavedra1 1 Departamento de Bioquimica Institute Nacionalde Cardiologia Ignacio Chavez Mexico D.F. Mexico 2 Departamento de Medicina Experimental Facultad de Medicina Universidad NacionalAutonoma de Mexico Mexico D.F. Mexico Keywords Fe-S cluster glycolysis oxidative stress pyruvate ferredoxin oxidoreductase PFOR reactive oxygen species ROS Correspondence E. Saavedra Departamento de Bioquimica Instituto Nacionalde Cardiologia Ignacio Chavez Juan Badiano No. 1 Col. Seccion XVI CP 14080 Tlalpan Mexico D.F. Mexico Fax 5255 55730994 Tel 5255 5573 2911 ext 1298 E-mail emma_saavedra2002@yahoo.com Received 9 February 2010 revised 4 June 2010 accepted 17 June 2010 doi 10.1111 j.1742-4658.2010.07743.x The in vitro Entamoeba histolytica pyruvate ferredoxin oxidoreductase EhPFOR kinetic properties and the effect of oxidative stress on glycolytic pathway enzymes and fluxes in live trophozoites were evaluated. EhPFOR showed a strong preference for pyruvate as substrate over other oxoacids. The enzyme was irreversibly inactivated by a long period of saturating O2 exposure IC50 0.034 mm whereas short-term exposure 30 min leading to 90 inhibition allowed for partial restoration by addition of Fe2 . CoA and acetyl-CoA prevented whereas pyruvate exacerbated inactivation induced by short-term saturating O2 exposure. Superoxide dismutase was more effective than catalase in preventing the inactivation indicating that reactive oxygen species ROS were involved. Hydrogen peroxide caused inactivation in an Fe2 -reversible fashion that was not prevented by the coenzymes suggesting different mechanisms of enzyme inactivation by ROS. Structural analysis on an EhPFOR 3D model suggested that the protection against ROS .
