Acetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the ␥-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.A cetate kinase (ACK; EC 2.7.2.12; CH 3 COO Ϫ ϩ ATP % CH 3 COPO 4 2Ϫ ϩ ADP) is a key enzyme in prokaryotic metabolism for the activation of acetate as a carbon and energy source or for the generation of ATP during fermentative growth. ACK is a member of the ASKHA phosphotransferase superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, as well as the Hsc70 heat shock cognate and actin (5,6,14,15). In 2001, Buss et al. (9) published the first structure for an ACK, that from the archaeon Methanosarcina thermophila, and they suggested that ACK is the urkinase for the ASKHA superfamily.Several crystal structures have now been solved for the well-characterized M. thermophila ACK (9, 13), and the roles of a number of active site residues in substrate binding and catalysis have been examined experimentally (16,17,21,22,28,29). Kinetic and structural studies support a direct in-line transfer of the phosphoryl group of ATP to acetate. An MgADP-AlF 3 -acetate transition state analog resulted in an abortive complex (22) and was found to be in a linear array in the active site (13). Based on analysis of site-altered enzyme variants and structural studies, Gorrell et al. (13) postulated a mechanism detailing the roles of active site residues in catalysis. The active site residues implicated in this mechanism are well conserved among the ACKs, consistent with their key roles in catalysis.Here we report the biochemical...