Department of Genetics and Biochemistry and the Eukaryotic Pathogens Innovation Center, Clemson University, Clemson, SC, 29634, USA.
Sci Rep. 2017 Jul 19;7(1):5912. doi: 10.1038/s41598-017-06156-5.
Acetate kinase (ACK; E.C. 2.7.2.1), which catalyzes the interconversion of acetate and acetyl phosphate, is nearly ubiquitous in bacteria but is present only in one genus of archaea and certain eukaryotic microbes. All ACKs utilize ATP/ADP as the phosphoryl donor/acceptor in the respective directions of the reaction (acetate + ATP [Formula: see text] acetyl phosphate + ADP), with the exception of the Entamoeba histolytica ACK (EhACK) which uses pyrophosphate (PP)/inorganic phosphate (P) (acetyl phosphate + P [Formula: see text] acetate + PP). Structural analysis and modeling of EhACK indicated steric hindrance by active site residues constricts entry to the adenosine pocket as compared to ATP-utilizing Methanosarcina thermophila ACK (MtACK). Reciprocal alterations were made to enlarge the adenosine pocket of EhACK and reduce that of MtACK. The EhACK variants showed a step-wise increase in ADP and ATP binding but were still unable to use these as substrates, and enzymatic activity with P/PP was negatively impacted. Consistent with this, ATP utilization by MtACK variants was negatively affected but the alterations were not sufficient to convert this enzyme to P/PP utilization. Our results suggest that controlling access to the adenosine pocket can contribute to substrate specificity but is not the sole determinant.
乙酰激酶 (ACK; EC 2.7.2.1) 催化醋酸盐和乙酰磷酸之间的相互转化，几乎存在于所有细菌中，但仅存在于古菌的一个属和某些真核微生物中。所有 ACK 都利用 ATP/ADP 作为反应的磷酸供体/受体（醋酸盐 + ATP [Formula: see text] 乙酰磷酸 + ADP），除了溶组织内阿米巴 ACK（EhACK），它使用焦磷酸 (PP)/无机磷酸 (P)（乙酰磷酸 + P [Formula: see text] 醋酸盐 + PP）。EhACK 的结构分析和建模表明，与利用 ATP 的 Methanosarcina thermophila ACK（MtACK）相比，活性位点残基的空间位阻限制了腺苷口袋的进入。对 EhACK 扩大腺苷口袋和减小 MtACK 腺苷口袋进行了相互改变。EhACK 变体的 ADP 和 ATP 结合呈逐步增加，但仍不能将其用作底物，并且对 P/PP 的酶活性产生负面影响。与此一致，MtACK 变体的 ATP 利用受到负面影响，但这些改变不足以使该酶转化为 P/PP 利用。我们的结果表明，控制进入腺苷口袋的通道可以有助于底物特异性，但不是唯一的决定因素。
