title: Roles of acetyl-CoA synthetase (ADP-forming) and acetate kinase (PPi-forming) in ATP and PPi supply in Entamoeba histolytica.
abstract: BACKGROUND
  Acetate is an end-product of the PPi-dependent fermentative glycolysis in Entamoeba histolytica; it is synthesized from acetyl-CoA by ADP-forming acetyl-CoA synthetase (ACS) with net ATP synthesis or from acetyl-phosphate by a unique PPi-forming acetate kinase (AcK). The relevance of these enzymes to the parasite ATP and PPi supply, respectively, are analyzed here.
  
  
  METHODS
  The recombinant enzymes were kinetically characterized and their physiological roles were analyzed by transcriptional gene silencing and further metabolic analyses in amoebae.
  
  
  RESULTS
  Recombinant ACS showed higher catalytic efficiencies (Vmax/Km) for acetate formation than for acetyl-CoA formation and high acetyl-CoA levels were found in trophozoites. Gradual ACS gene silencing (49-93%) significantly decreased the acetate flux without affecting the levels of glycolytic metabolites and ATP in trophozoites. However, amoebae lacking ACS activity were unable to reestablish the acetyl-CoA/CoA ratio after an oxidative stress challenge. Recombinant AcK showed activity only in the acetate formation direction; however, its substrate acetyl-phosphate was undetected in axenic parasites. AcK gene silencing did not affect acetate production in the parasites but promoted a slight decrease (10-20%) in the hexose phosphates and PPi levels.
  
  
  CONCLUSIONS
  These results indicated that the main role of ACS in the parasite energy metabolism is not ATP production but to recycle CoA for glycolysis to proceed under aerobic conditions. AcK does not contribute to acetate production but might be marginally involved in PPi and hexosephosphate homeostasis.
  
  
  CONCLUSIONS
  The previous, long-standing hypothesis that these enzymes importantly contribute to ATP and PPi supply in amoebae can now be ruled out.
authors.affiliation: Departamento de Bioquímica, Instituto Nacional de Cardiología Ignacio Chávez. Mexico D.F. 14080, Mexico.
authors.forename: Erika
authors.initials: E
authors.lastname: Pineda
authors.affiliation: Departamento de Bioquímica, Instituto Nacional de Cardiología Ignacio Chávez. Mexico D.F. 14080, Mexico.
authors.forename: Citlali
authors.initials: C
authors.lastname: Vázquez
authors.affiliation: Departamento de Bioquímica, Instituto Nacional de Cardiología Ignacio Chávez. Mexico D.F. 14080, Mexico.
authors.forename: Rusely
authors.initials: R
authors.lastname: Encalada
authors.affiliation: Department of Parasitology, National Institute of Infectious Diseases. Tokyo 162-8640, Japan.
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Nozaki
authors.affiliation: Department of Parasitology, National Institute of Infectious Diseases. Tokyo 162-8640, Japan.
authors.forename: Emi
authors.initials: E
authors.lastname: Sato
authors.affiliation: Department of Parasitology, National Institute of Infectious Diseases. Tokyo 162-8640, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Hanadate
authors.affiliation: Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México. Mexico D.F. 04510, Mexico.
authors.forename: Mario
authors.initials: M
authors.lastname: Néquiz
authors.affiliation: Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de México. Mexico D.F. 04510, Mexico.
authors.forename: Alfonso
authors.initials: A
authors.lastname: Olivos-García
authors.affiliation: Departamento de Bioquímica, Instituto Nacional de Cardiología Ignacio Chávez. Mexico D.F. 14080, Mexico.
authors.forename: Rafael
authors.initials: R
authors.lastname: Moreno-Sánchez
authors.affiliation: Departamento de Bioquímica, Instituto Nacional de Cardiología Ignacio Chávez. Mexico D.F. 14080, Mexico. Electronic address: emma_saavedra2002@yahoo.com.
authors.forename: Emma
authors.initials: E
authors.lastname: Saavedra
mesh_terms: D000084:Acetate Kinase / Q000235:genetics / Q000502:physiology*; D000106:Acetate-CoA Ligase / Q000235:genetics / Q000502:physiology*; D000085:Acetates / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism*; D004734:Energy Metabolism; D004748:Entamoeba histolytica / Q000378:metabolism*; D000431:Ethanol / Q000378:metabolism; D006019:Glycolysis
chemical_list: D000085:Acetates; D011756:Diphosphates; D000431:Ethanol; C107241:diphosphoric acid; D000255:Adenosine Triphosphate; D000084:Acetate Kinase; D000106:Acetate-CoA Ligase
keywords: Acetate kinase (PPi forming); Acetate metabolism; Acetyl-CoA synthetase (ADP-forming); Oxidative stress; PPi-dependent glycolysis; Pyrophosphate