Investigation of pyrophosphate versus ATP substrate selection in the Entamoeba histolytica acetate kinase

Abstract

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 acetyl phosphate + ADP), with the exception of the Entamoeba histolytica ACK (EhACK) which uses pyrophosphate (PPi)/inorganic phosphate (Pi) (acetyl phosphate + Pi acetate + PPi). 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 Pi/PPi 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 Pi/PPi utilization. Our results suggest that controlling access to the adenosine pocket can contribute to substrate specificity but is not the sole determinant.

Identification of a pyrophosphate-dependent kinase and its donor selectivity determinants

“…In PPi-PFK, the conserved aspartate residue (e.g., D175 in the enzyme from E. histolytica ) is suggested to prevent ATP binding, because its replacement with glycine, which is conserved in ATP-PFK, led to an 18-fold better K m value toward ATP 6 . In PPi-ACK from E. histolytica , the five residues (T201, D322, Q323, M324, and E327) occlude the ATP-binding cleft of ATP-ACK 17 , 18 . These residues were introduced to ATP-dependent kinases in order to transform them into PPi-dependent ones, but these attempts were unsuccessful 6 , 17 , 18 .…”

“…In PPi-ACK from E. histolytica , the five residues (T201, D322, Q323, M324, and E327) occlude the ATP-binding cleft of ATP-ACK 17 , 18 . These residues were introduced to ATP-dependent kinases in order to transform them into PPi-dependent ones, but these attempts were unsuccessful 6 , 17 , 18 . Our identification of the three basic residues of TM0415 may enable such transformation on ATP-dependent ribokinase family members, which phosphorylate various acceptors.…”

“…For example, the aspartate residue at the phosphate-donor-binding site in PPi-PFK prevents the ATP binding 6 , 15 , 16 . In PPi-ACK from E. histolytica , the five residues in the donor-binding site occlude the ATP-binding cleft 17 , 18 . Thus, only mechanisms for interfering with ATP binding have been suggested, while the residues that specifically recognize PPi remain unclear.…”