Investigating the role of ADP-forming acetyl-CoA synthetase from the protozoan parasite Entamoeba histolytica.

摘要

ADP-forming acetyl-CoA synthetase (ACD; EC 6.2.1.13) catalyzes the reversible conversion of acetyl-CoA to acetate coupled to the production of ATP. This enzyme is present only in certain acetate-producing archaea and a limited number of bacteria and eukaryotes. ACD belongs to the same NDP-forming acyl-CoA synthetase enzyme superfamily as succinyl-CoA synthetase (SCS; EC 6.2.1.4) from the citric acid cycle, and a similar three-step mechanism involving a phosphoenzyme intermediate was originally proposed for this enzyme.;ACD has been postulated to be a major acetate-producing enzyme in the protozoan parasite Entamoeba histolytica and may contribute to ATP production. Biochemical and kinetic characterization of recombinant E. histolytica ACD (EhACD) revealed that this enzyme may function in the direction of acetate production for generation of ATP and CoA during growth in the high glucose environment of the small intestine, and in acetate assimilation to acetyl-CoA in the high acetate environment of the lower intestine during colonization. EhACD utilizes multiple substrates including propionate and propionyl-CoA supporting an additional proposed role in amino acid degradation. EhACD activity is regulated by both ATP and PPi, important energy molecules in E. histolytica.;The ACD mechanism has been controversial, as a required second phosphorylation step was proposed for the Pyrococcus furiosus enzyme. Investigation of the catalytic role of the two proposed phosphorylation sites in EhACD revealed that His252, the site of phosphorylation in the original three-step mechanism, is essential for activity and His533, the proposed second phosphorylation site, is important but not essential. Likewise, Glu213, proposed to play a role in phosphorylation/ dephosphorylation of His252, is also required but Asp674 thought to stabilize the phosphohistidine is not. These results suggest that EhACD follows a three-step mechanism with a single phosphoenzyme intermediate.;Additional conserved active site residues were examined for their role in catalysis. Asp314 was shown to be essential for activity, possibly in both a catalytic role and a structural role. Alteration at this position resulted in complete loss of activity, and computational modeling based on the Candidatus Korarchaeum cryptofilum ACD-I structure suggests that this residue may be critical for dimerization. Future directions for understanding the complex mechanism of ACD and its physiological role are presented.