title: Biochemical and kinetic characterization of the recombinant ADP-forming acetyl coenzyme A synthetase from the amitochondriate protozoan Entamoeba histolytica.
abstract: Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PPi-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PPi were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PPi displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed.
authors.affiliation: Eukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, USA.
authors.forename: Cheryl P
authors.initials: CP
authors.lastname: Jones
authors.affiliation: Eukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, USA cheryli@clemson.edu.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Ingram-Smith
mesh_terms: D000106:Acetate-CoA Ligase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000105:Acetyl Coenzyme A / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D011756:Diphosphates / Q000737:chemistry; D004748:Entamoeba histolytica / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D011756:Diphosphates; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C107241:diphosphoric acid; D000244:Adenosine Diphosphate; D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate; D000106:Acetate-CoA Ligase; D008274:Magnesium