Structural and Kinetic Analyses of Arginine Residues in the Active Site of the Acetate Kinase from Methanosarcina thermophila

Abstract

Phosphoryl transfer is a key reaction in numerous biological processes, playing roles in signaling mechanisms, energy transfer, and energy storage in both eukaryotic and prokaryotic cells (1). One of the earliest phosphoryl transfers identified was the phosphorylation of acetate by ATP to form acetyl phosphate (AcP) 1 and ADP, described in 1944 by Lippman (2). This reversible reaction is catalyzed by acetate kinase, which is widely distributed among anaerobic prokaryotes playing a central role in energy-yielding metabolism by synthesizing ATP from acetyl phosphate generated in fermentation pathways. The enzyme also plays an essential role in the fermentation of acetate to methane, which accounts for most of the one billion metric tons of methane produced annually from the decomposition of organic matter by anaerobic microbial consortia (3). In Methanosarcina thermophila, acetate kinase catalyzes the first step in the pathway by activating acetate to acetyl phosphate prior to transfer of the acetyl moiety to CoA catalyzed by phosphotransacetylase (4, 5). In later steps of the pathway, the acetyl moiety is further metabolized to methane and carbon dioxide (6). Although acetate kinase was one of the first enzymes to be investigated mechanistically, details remain elusive; indeed, the first crystal structure was obtained only recently for the M. thermophila enzyme, identifying acetate kinase as a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) structural superfamily and the best candidate for the common ancestor of this family (7). The earliest kinetic studies of the enzyme from Escherichia coli suggested a ping-pong mechanism (8), and evidence for a covalent phosphoryl intermediate supported this mechanism (9, 10); however, it was later shown that the phosphoryl-enzyme complex is not kinetically competent (11). Additionally, the discovery that the E. coli acetate kinase is able to phosphorylate enzyme I of the phosphotransferase system (12) and CheY (13) in vitro indicates the phosphoenzyme functions in sugar transport. Later investigations reported inversion of the stereochemistry about the phosphorous (14) and isotope exchange kinetics inconsistent with the covalent kinase mechanism (15) and supporting a direct in-line phosphoryl transfer. More recently, the acetate kinase from M. thermophila was shown to be inhibited by components of a putative transition state analogue ADP-AlF x -acetate (16) in which the AlF x is proposed to mimic the meta-phosphate in a direct phosphoryl transfer mechanism. No structural evidence for either the covalent or in-line mechanism has been reported previously.Access to the crystal structure (7) and production of the M. thermophila acetate kinase in E. coli (17) have allowed experimental approaches not previously employed to investigate the catalytic mechanism of this enzyme. The structure of the homodimeric acetate kinase co-crystallized with ATP (the ATP-AK structure) reveals ADP in a cleft with contacts that are conserved in the nucleotide binding sites of other ASKHA f...

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

“…As expected, this experiment demonstrates that the enzyme follows standard Michaelis-Menten-like kinetics for each substrate and produced a hyperbolic saturation curve when plotting activity versus acetyl phosphate concentration. The apparent Km value for acetyl phosphate was 0.27 ± 0.01 mmol/L, which compares favorably to the published Km value of 0.47 ± 0.01 mmol/L using the coupled assay 13 .…”

“…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 .…”

Characterization of the Acetate Binding Pocket in theMethanosarcina thermophilaAcetate Kinase

“…A recent crystal structure of the M. thermophila acetate kinase containing acetate, ADP, and the transition state analog AlF 3 further supported the hypothesis that there is a direct in-line mechanism and showed the methyl group of acetate located in the previously proposed binding pocket (14). Kinetic analyses of active site replacement variants also supported the hypothesis that there is a direct in-line mechanism for the M. thermophila enzyme and suggested roles for conserved arginine, histidine, and glutamate residues (16,26,27,32,33).…”

“…Located in the M. thermophila acetate kinase active site cleft are the residues Val 93 , Phe 179 , and Pro 232 , which form a hydrophobic pocket ( Fig. 1) predicted to bind the methyl group of acetate (8,14). Sequence alignments (data not shown) revealed that Val 93 , Phe 179 , and Pro 232 of the M. thermophila enzyme are strictly conserved in all other acetate kinases obtained from the databases, implying that these residues have important roles, possibly in substrate binding.…”

“…The hydroxamate assay, an adaptation of the methods of Lipmann and Rose et al (1,24,31), detects acetyl phosphate formation from acetate and ATP and was used to measure the initial acetate kinase activities and kinetic parameters in the forward (ADP/acetyl phosphate-producing) direction. By utilizing the pyruvate kinase (PK)-lactate dehydrogenase (LDH) coupled assay system (1), hydroxylamine was found to be an inhibitor of acetate kinase activity (14); therefore, kinetic parameters of wild-type and variant acetate kinases were determined by utilizing the PK-LDH coupled assay system. Briefly, each assay solution contained 60 mM HEPES (pH 7.0), 5 mM MgCl 2 , 16.7 U of PK, 36 U of LDH, 3 mM phosphoenolpyruvate, and 0.2 mM NADH along with a fixed concentration of substrate (200 mM acetate or 1 to 2 mM equimolar ATP-MgCl 2 ) as appropriate.…”