Novel pyrophosphate-forming acetate kinase from the protist Entamoeba histolytica

Abstract

Acetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protist Entamoeba histolytica. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PP(i))/inorganic phosphate (P(i)) as the sole phosphoryl donor/acceptor. Detection of ACK activity in E. histolytica cell extracts in the direction of acetate/PP(i) formation but not in the direction of acetyl phosphate/P(i) formation suggests that the physiological direction of the reaction is toward acetate/PP(i) production. Kinetic parameters determined for each direction of the reaction are consistent with this observation. The E. histolytica PP(i)-forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterized Methanosarcina thermophila ATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for the M. thermophila ACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway in Entamoeba.

Fig 1

Utilization of different phosphoryl donors and acceptors by EhACK. (A) The enzymatic activity in the acetyl phosphate-forming direction of the reaction in the presence of 1 M acetate with various concentrations of each phosphoryl donor was measured in a hydroxamate assay. ●, PP_i; ○, ADP; △, ATP. (B) The enzymatic activity in the direction of acetate formation in the presence of 2 mM acetyl phosphate with various concentrations of each phosphoryl acceptor was determined in a modified reverse hydroxamate assay. ●, P_i; ○, PP_i; △, ADP; □, AMP. The data for PP_i (A) and P_i (B) were fit to the Michaelis-Menten equation by using nonlinear regression.

Fig 2

Double-reciprocal plot of acetate-forming activity of EhACK. (A) The reciprocal of the sodium phosphate concentration (40 mM, 50 mM, 60 mM, and 70 mM) versus the reciprocal specific activity at acetyl phosphate concentrations of 0.5 mM (●), 0.7 mM (○), 1.0 mM (■), and 1.5 mM (□). (B) The reciprocal of the acetyl phosphate concentration of 0.5 mM, 0.7 mM, 1.0 mM, and 1.5 mM versus the reciprocal specific activity at sodium phosphate concentrations of 40 mM (●), 50 mM (○), 60mM (■), and 70 mM (□).

Fig 3

Acetate kinase partial sequence alignment. ACK sequences were aligned using Clustal X. Only those regions surrounding the residues altered in EhACK are shown. Numbers above the alignment indicate the positions of residues in MtACK, and those below indicate the position of residues in EhACK. EcACK, Escherichia coli ACK; PrACK, Phytophthora ramorum ACK; CnACK, Cryptococcus neoformans ACK.

Fig 4

Putative acetate fermentation pathways in Entamoeba. Potential partners for the amoebal acetate kinase that have not been identified in genome searches are shown. ADP-ACS, ADP-forming acetyl-CoA synthetase; PFOR, pyruvate:ferredoxin oxidoreductase; FD, ferredoxin; HYD, hydrogenase.