RESUMO
ADP-forming acetyl-CoA synthetase (ACD) catalyzes the interconversion of acetyl-CoA and acetate. The related succinyl-CoA synthetase follows a three-step mechanism involving a single phosphoenzyme, but a novel four-step mechanism with two phosphoenzyme intermediates was proposed for Pyrococcus ACD. Characterization of enzyme variants of Entamoeba ACD in which the two proposed phosphorylated His residues were individually altered revealed that only His252 is essential for enzymatic activity. Analysis of variants altered at two residues proposed to interact with the phosphohistidine loop that swings between distinct parts of the active site are consistent with a mechanism involving a single phosphoenzyme intermediate. Our results suggest ACDs with different subunit structures may employ slightly different mechanisms to bridge the span between active sites I and II.
Assuntos
Difosfato de Adenosina/metabolismo , Coenzima A Ligases/metabolismo , Entamoeba histolytica/enzimologia , Proteínas de Protozoários/metabolismo , Acetatos/metabolismo , Acetilcoenzima A/metabolismo , Sequência de Aminoácidos , Biocatálise , Domínio Catalítico , Coenzima A Ligases/química , Coenzima A Ligases/genética , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Histidina/análogos & derivados , Histidina/química , Histidina/genética , Histidina/metabolismo , Cinética , Modelos Moleculares , Mutação de Sentido Incorreto , Fosforilação , Domínios Proteicos , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Especificidade por SubstratoRESUMO
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.