Characterization of the Acetate Binding Pocket in theMethanosarcina thermophilaAcetate Kinase

Abstract

Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP ␥-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val 93 , Leu 122 , Phe 179 , and Pro 232 in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with K m values for acetate that were 7-to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe 179 is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.Acetate kinase catalyzes the reversible magnesium-dependent phosphorylation of acetate with ATP (equation 1) and is very important for the energy-yielding metabolism of anaerobic microbes.In most fermentative anaerobes this enzyme is responsible for production of a major portion of the ATP (reverse of equation 1). Acetate kinase functions in the energy-yielding pathway for conversion of the methyl group of acetate to methane (equation 2) in Methanosarcina species.

Novel Pyrophosphate-Forming Acetate Kinase from the Protist Entamoeba histolytica

“…The extraordinarily broad acyl substrate range for EhACK and the strong preference for the acetate/PP i -forming direction suggest differences between EhACK and MtACK in acetate/acetyl phosphate binding. Acyl substrate binding in MtACK appears to be mediated primarily through hydrophobic interaction between the methyl group of acetate and residues within the acetate binding pocket, with the side chains of the binding pocket residues also serving to properly position the carboxyl group of acetate in proximity to the ␥-phosphate of ATP (13,17). Our results and those of IngramSmith et al (17) Pro variant showed a similar acyl substrate range and kinetic parameters to the unaltered enzyme in the direction of acyl phosphate formation.…”

“…MtACK site-altered enzyme variants were produced and purified as previously described (16,17). Kinetic parameters in the acetate/ATP-forming direction of the reaction were determined using a coupled enzyme assay in which ATP formation was coupled to the reduction of NADP to NADPH (1).…”

“…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.…”

“…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.…”

Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase

“…Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya 6 . The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila [7][8][9][10][11][12][13][14] . An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus 15,16 .…”