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 ␥-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...

Atypical Glycolysis in Clostridium thermocellum

“…(ii) PP i yielding reactions in the conversion of pyruvate to fermentation products such as PP i -dependent acetate kinase (acetylphosphate ϩ P i N acetate ϩ PP i ) (EC 2.7.2.12) as occurs in E. histolytica (47) or PP i -linked PEP carboxykinase (PEP ϩ P i ϩ CO 2 N OAA ϩ PP i ) (EC 4.1.1.38) as occurs in P. shermanii (48) and E. histolytica (49). In C. thermocellum, however, acetate kinase has been shown to be ATP dependent (50) and P i could not replace GDP in the PEP carboxykinase reaction (Table 1).…”

Crystal structures of acetate kinases from the eukaryotic pathogens Entamoeba histolytica and Cryptococcus neoformans

“…Genes encoding putative ACKs have since been identified within the genomes of other eukaryotic pathogens, including the basidomycete Cryptococcus neoformans (Ingram-Smith et al, 2006). At least in E. histolytica , the organism does not appear to have homologs for other proteins required for the known bioenergetic pathways that use ACK (Fowler et al, 2012). This suggests that the biological function of the E. histolytica ACK may be different from that demonstrated in prokaryotes, but at present, that function remains unknown.…”

“…Interestingly, the E. histolytica ACK has been shown to use P i /PP i , rather than ADP/ATP, as the phosphoryl acceptor/donor pair for phosphoryl transfer (Fowler et al, 2012; Reeves and Guthrie, 1975). Further, this enzyme has a strong kinetic advantage for catalyzing the dephosphorylation of acetyl phosphate (Fowler et al, 2012; Reeves and Guthrie, 1975). Although a kinetic characterization has not yet been reported in the literature, C. neoformans ACK has also been shown to kinetically favor acetate formation (Ingram-Smith, C., personal communication).…”

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

“…9 . MtACK and its variants were produced in E. coli Rosetta2 (DE3) pLysS and purified as described in Fowler et al .…”