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...

Examined here by directed mutation, circular dichroism spectroscopy, and kinetics are the relationships of five residues, Asp 13 , Glu 14 , Lys 16 , His 41 , and Arg 131 , to the catalytic function and structural organization of adenylosuccinate synthetase from Escherichia coli. The D13A mutant has no measurable activity. Mutants E14A and H41N exhibit 1% of the activity of the wild-type enzyme and 2-7-fold increases in the K m of substrates. The mutant K16Q has 34% of the activity of wild-type enzyme and K m values for substrates virtually unchanged from those of the wild-type system. Mutation of Arg 131 to leucine caused only a 4-fold increase in the K m for aspartate relative to the wild-type enzyme. The dramatic effects of the D13A, E14A, and H41N mutations on k cat are consistent with the putative roles assigned to Asp 13 (catalytic base), His 41 (catalytic acid), and Glu 14 (structural organization of the active site). The modest effect of the R131L mutation on the binding of aspartate is also in harmony with recent crystallographic investigations, which suggests that Arg 131 stabilizes the conformation of the loop that binds the ␤-carboxylate of aspartate. The modest effect of the K16Q mutation, however, contrasts with significant changes brought about by the mutation of the corresponding lysines in the P-loop of other GTPand ATP-binding proteins. Crystallographic structures place Lys 16 in a position of direct interaction with the ␥-phosphate of GTP. Furthermore, lysine is present at corresponding positions in all known sequences of adenylosuccinate synthetase. We suggest that along with a modest role in stabilizing the transition state of the phosphotransfer reaction, Lys 16 may stabilize the enzyme structurally. In addition, the modest loss of catalytic activity of the K16Q mutant may confer such a selective disadvantage to E. coli that this seemingly innocuous mutation is not tolerated in nature.Adenylosuccinate synthetase (AMPSase) 1 (see Ref. 1 for review) catalyzes the following reversible reaction in the presence of Mg 2ϩ ions: GTP ϩ IMP ϩ aspartate i GDP ϩ adenylosuccinate ϩ phosphate (P i ). This reaction is the first committed step in the formation of AMP from IMP on the pathway for de novo purine nucleotide biosynthesis and is an integral part of the purine nucleotide cycle in muscle (2). The reaction mechanism of AMPSase centers on 6-phosphoryl-IMP, formed putatively by the nucleophilic attack of the 6-oxyanion of IMP on the ␥-phosphate of GTP. A second nucleophilic substitution reaction by the amino group of aspartate on the C-6 of 6-phosphoryl-IMP yields adenylosuccinate and P i (3). Two Mg 2ϩ ions are involved in the reaction mechanism (4). One Mg 2ϩ is in the active site, associated with the phosphate moiety of the guanine nucleotide and the N-formyl group of hadacidin, an inactive analog of aspartate (5). However, crystallographic investigations have yet to reveal the location of the second Mg 2ϩ

No 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.

Aluminum fluoride has become an important tool for investigating the mechanism of phosphoryl transfer, an essential reaction that controls a host of vital cell functions. Planar AlF 3 or AlF 4 ؊ molecules are proposed to mimic the phosphoryl group in the catalytic transition state. Acetate kinase catalyzes phosphoryl transfer of the ATP ␥-phosphate to acetate. Here we describe the inhibition of acetate kinase from Methanosarcina thermophila by preincubation with MgCl 2 , ADP, AlCl 3 , NaF, and acetate. Preincubation with butyrate in place of acetate did not significantly inhibit the enzyme. Several NTPs can substitute for ATP in the reaction, and the corresponding NDPs, in conjunction with MgCl 2 , AlCl 3 , NaF, and acetate, inhibit acetate kinase activity. Fluorescence quenching experiments indicated an increase in binding affinity of acetate kinase for MgADP in the presence of AlCl 3 , NaF, and acetate. These and other characteristics of the inhibition indicate that the transition state analog, MgADP-aluminum fluoride-acetate, forms an abortive complex in the active site. The protection from inhibition by a non-hydrolyzable ATP analog or acetylphosphate, in conjunction with the strict dependence of inhibition on the presence of both ADP and acetate, supports a direct in-line mechanism for acetate kinase.