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At least 19 recordsLinked to original sources
PubMed2005

Characterization of the acetate binding pocket in the Methanosarcina thermophila acetate kinase.

Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP gamma-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↗
PubMed1979

Stereochemical course of phosphokinases. The use of adenosine [gamma-(S)-16O,17O,18O]triphosphate and the mechanistic consequences for the reactions catalyzed by glycerol kinase, hexokinase, pyruvate kinase, and acetate kinase.

We report the synthesis of adenosine [gamma-(S)-16O,17O,18O]triphosphate, an isotopically labeled species of ATP that is chiral at the gamma-phosphoryl group, the configuration of which has been confirmed by independent stereochemical analysis. This molecule has been used as a substrate in the reactions catalyzed by glycerol kinase and by acetate kinase. The resulting samples of isotopically labeled sn-glycerol 3-phosphate and of acetyl phosphate have been used as substrates in the alkaline phosphatase mediated transfer of the chiral phosphoryl groups to (S)-propane-1,2-diol, whence the configuration at phosphorus has been determined [Abbott, S. J., Jones, S. R., Weinman, S. A., & Knowles, J. R. (1978) J. Am. Chem. Soc. 100, 2558]. It is shown that glycerol kinase and acetate kinase (and, by virtue of an earlier correlation, pyruvate kinase and hexokinase) proceed by pathways that result in inversion of the configuration at phosphorus. The sterochemical approach provides an access to the otherwise cryptic events that are involved in phosphoryl-group transfer within the ternary complexes of these kinases and their substrates.

Acetate Kinase↗
PubMed1988

Purification and characterization of acetate kinase from acetate-grown Methanosarcina thermophila. Evidence for regulation of synthesis.

Acetate kinase was purified 102-fold to a specific activity of 656 mumol of ADP formed/min/mg of protein from acetate-grown Methanosarcina thermophila. The enzyme was not intrinsically membrane bound. The native enzyme (Mr 94,000) was an alpha 2 homodimer with a subunit Mr of 53,000. The activity was optimum between pH 7.0 and 7.4. A pI of 4.7 was determined. The enzyme was stable to O2 and stable to heating at 70 degrees C for 15 min but was rapidly inactivated at higher temperatures. The apparent Km for acetate was 22 mM and for ATP was 2.8 mM. The enzyme phosphorylated propionate at 60% of the rate with acetate but was unable to use formate. TTP, ITP, UTP, GTP, and CTP replaced ATP as the phosphoryl donor to acetate. The enzyme required one of several divalent cations for activity; the maximum rate was obtained with Mn2+. Western blots of cell extract proteins showed that acetate grown cells synthesized higher quantities of the acetate kinase than did methanol grown cells.

Acetate Kinase↗
PubMed1981

Evidence for an essential arginine residue at the active site of Escherichia coli acetate kinase.

Escherichia coli acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1.) was inactivated in the presence of either 2,3-butanedione in borate buffer or phenylglyoxal in triethanolamine buffer. When incubated with 9.4 mM phenylglyoxal or 5.1 mM butanedione, the enzyme lost its activity with an apparent rate constant of inactivation of 0.079 min-1, respectively. The loss of enzymatic activity was concomitant with the loss of an arginine residue per active site. Phosphorylated substrates of acetate kinase, ATP, ADP and acetylphosphate as well as AMP markedly decreased the rate of inactivation by both phenylglyoxal and butanedione. Acetate neither provided any protection nor affected the protection rendered by the adenine nucleotides. However, it interfered with the protection afforded by acetylphosphate. These data suggest that an arginine residue is located at the active site of acetate kinase and is essential for its catalytic activity, probably as a binding site for the negatively charged phosphate group of the substrates.

Acetate Kinase↗
PubMed1988

Inactivation of Acinetobacter calcoaceticus acetate kinase by diethylpyrocarbonate.

Acetate kinase purified from Acinetobacter calcoaceticus was inhibited by diethylpyrocarbonate with a second-order rate constant of 620 M-1.min-1 at pH 7.4 at 30 degrees C and showed a concomitant increase in absorbance at 240 nm due to the formation of N-carbethoxyhistidyl derivative. Activity could be restored by hydroxylamine and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.64. Complete inactivation of acetate kinase required the modification of seven residues per molecule of enzyme. Statistical analysis showed that among the seven modifiable residues, only one is essential for activity. 5,5'-dithiobis(2-nitrobenzoic acid), p-chloromercuryphenylsulfonate, N-ethylmaleimide and phenylglyoxal did not affect the enzyme activity. These results suggest that the inactivation is due to the modification of one histidine residue. The substrates, acetate and ATP, protected the enzyme against inactivation, indicating that the modified histidine residue is located at or near the active site.

4-Chloromercuribenzenesulfonate↗
PubMed1998

Identification of essential glutamates in the acetate kinase from Methanosarcina thermophila.

Acetate kinase catalyzes the reversible phosphorylation of acetate (CH3COO- + ATP<-->CH3CO2PO3(2-) + ADP). A mechanism which involves a covalent phosphoryl-enzyme intermediate has been proposed, and chemical modification studies of the enzyme from Escherichia coli indicate an unspecified glutamate residue is phosphorylated (J. A. Todhunter and D. L. Purich, Biochem. Biophys. Res. Commun. 60:273-280, 1974). Alignment of the amino acid sequences for the acetate kinases from E. coli (Bacteria domain), Methanosarcina thermophila (Archaea domain), and four other phylogenetically divergent microbes revealed high identity which included five glutamates. These glutamates were replaced in the M. thermophila enzyme to determine if any are essential for catalysis. The histidine-tagged altered enzymes were produced in E. coli and purified to electrophoretic homogeneity by metal affinity chromatography. Replacements of E384 resulted in either undetectable or extremely low kinase activity, suggesting E384 is essential for catalysis which supports the proposed mechanism. Replacement of E385 influenced the Km values for acetate and ATP with only moderate decreases in k(cat), which suggests that this residue is involved in substrate binding but not catalysis. The unaltered acetate kinase was not inactivated by N-ethylmaleimide; however, replacement of E385 with cysteine conferred sensitivity to N-ethylmaleimide which was prevented by preincubation with acetate, acetyl phosphate, ATP, or ADP, suggesting that E385 is located near the active site. Replacement of E97 decreased the Km value for acetate but not ATP, suggesting this residue is involved in binding acetate. Replacement of either E32 or E334 had no significant effects on the kinetic constants, which indicates that neither residue is essential for catalysis or significantly influences the binding of acetate or ATP.

Acetate Kinase↗
PubMed2001

Site-directed mutational analysis of active site residues in the acetate kinase from Methanosarcina thermophila.

Acetate kinase catalyzes the magnesium-dependent transfer of the gamma-phosphate of ATP to acetate. The recently determined crystal structure of the Methanosarcina thermophila enzyme identifies it as a member of the sugar kinase/Hsc70/actin superfamily based on the fold and the presence of five putative nucleotide and metal binding motifs that characterize the superfamily. Residues from four of these motifs in M. thermophila acetate kinase were selected for site-directed replacement and analysis of the variants. Replacement of Asp(148) and Asn(7) resulted in variants with catalytic efficiencies less than 1% of that of the wild-type enzyme, indicating that these residues are essential for activity. Glu(384) was also found to be essential for catalysis. A 30-fold increase in the magnesium concentration required for half-maximal activity of the E384A variant relative to that of the wild type implicated Glu(384) in magnesium binding. The kinetic analysis of variants and structural data is consistent with nonessential roles for active site residues Ser(10), Ser(12), and Lys(14) in catalysis. The results are discussed with respect to the acetate kinase catalytic mechanism and the relationship to other sugar kinase/Hsc70/actin superfamily members.

Acetate Kinase↗
PubMed1990

Preparation of a mixture of nucleoside triphosphates suitable for use in synthesis of nucleotide phosphate sugars from ribonucleic acid using nuclease P1, a mixture of nucleoside monophosphokinases and acetate kinase.

This paper (1) describes the enzymatic synthesis of a mixture of adenosine, guanosine, cytidine, and uridine triphosphates (ATP, GTP, CTP, and UTP) from ribonucleic acid (RNA). RNA was hydrolyzed by nuclease P1 to a mixture of 5'-nucleoside monophosphates. This mixture was converted to the nucleoside triphosphates using a mixture of nucleoside monophosphate kinases and acetate kinase, with acetyl phosphate as the ultimate phosphoryl donor. The nucleoside monophosphokinases were extracted from brewer's yeast in a four-step procedure. The specific activity of the yeast enzyme preparation after gel permeation chromatography was sufficiently high that the yeast kinases could be immobilized in volumes that were practical for laboratory scale syntheses. Conversions from NMP to NTP in a mixture containing 0.34 mol of total nucleoside phosphates were: ATP, 90%; GTP, 90%; CTP, 60%; and UTP, 40%.

Acetate Kinase↗
PubMed2005

Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila.

Acetate kinase catalyzes transfer of the gamma-phosphate of ATP to acetate. The only crystal structure reported for acetate kinase is the homodimeric enzyme from Methanosarcina thermophila containing ADP and sulfate in the active site (Buss, K. A., Cooper, D. C., Ingram-Smith, C., Ferry, J. G., Sanders, D. A., and Hasson, M. S. (2001) J. Bacteriol. 193, 680-686). Here we report two new crystal structure of the M. thermophila enzyme in the presence of substrate and transition state analogs. The enzyme co-crystallized with the ATP analog adenosine 5'-[gamma-thio]triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B. The enzyme co-crystallized with ADP, acetate, Al(3+), and F(-) contained a linear array of ADP-AlF(3)-acetate in the active site cleft of monomer B. Together, the structures clarify the substrate binding sites and support a direct in-line transfer mechanism in which AlF(3) mimics the meta-phosphate transition state. Monomers A of both structures contained ADP and sulfate, and the active site clefts were closed less than in monomers B, suggesting that domain movement contributes to catalysis. The finding that His(180) was in close proximity to AlF(3) is consistent with a role for stabilization of the meta-phosphate that is in agreement with a previous report indicating that this residue is essential for catalysis. Residue Arg(241) was also found adjacent to AlF(3), consistent with a role for stabilization of the transition state. Kinetic analyses of Arg(241) and Arg(91) replacement variants indicated that these residues are essential for catalysis and also indicated a role in binding acetate.

Acetate Kinase↗
PubMed1994

Identification and characterization of the ackA (acetate kinase A)-pta (phosphotransacetylase) operon and complementation analysis of acetate utilization by an ackA-pta deletion mutant of Escherichia coli.

The pta gene encoding phosphotransacetylase was cloned on a high copy plasmid with or without the ackA gene encoding acetate kinase in Escherichia coli. The acetate kinase and phosphotransacetylase were overproduced in cells harboring the plasmid possessing both genes. Nucleotide sequencing of the pta gene revealed that it is able to produce a polypeptide comprising 714 amino acid residues, which starts at 70 base pairs downstream from the stop codon of the ackA gene. The 77-kDa protein band of overproduced phosphotransacetylase was observed on SDS-polyacrylamide gel electrophoresis, of which the amino terminal sequence corresponds to that of the deduced polypeptide without the amino terminal methionine. Two transcripts of pta of different sizes were found in the cells. A 3,700 nucleotide transcript, which covers the ackA and pta genes, seemed to be produced by the first promoter in the operon and a 2,300 nucleotide transcript, which covers just pta, seemed to be produced by the second promoter. In a synthetic medium containing acetate as the sole carbon source, the growth of an ackA-pta double mutant was greatly impaired. Complementation analyses revealed that both the acetate kinase and phosphotransacetylase were required for the rapid growth in the acetate medium.

Acetate Kinase↗
PubMed1978

[Acetate kinase chromatography on agarose derivatives].

Acetate kinase from E. coli K-12 was studied chromatographically on omega-aminoalkyl polysacharide sorbents. The dependence of the protein sorption-desorption on ionic strength and the effect of pH on the acetate kinase sorption were studied. The increase in the ionic strength caused a decrease in the amount of protein sorbed on the hexamethylenediamine- and chlorotriasinehexamethylenediamine sepharoses. On hexamethylenediamine-, octamethylenediamine- and dimethylhexamethylenediamine agaroses acetate kinase was adsorbed within the pH range of 6.5-9.0, whereas on the chlorotriasinehexamethylenediamine sepharose--at pH 6.5-8.0. The active protein was eluted at ionic strength of 0.14-0.17 M. Acetate kinase was not adsorbed on the carboxypropyonylaminohexyl sepharose within the pH range studied, i.e. 5.0-9.0 and was not adsorbed on hexamethylenediamine agarose at pH 4.0 and on chlorotriasinehexamethylenediamine sepharose--at pH 9.0. The mechanism of the enzyme-adsorbent interaction is discussed.

Acetates↗
PubMed2001

Urkinase: structure of acetate kinase, a member of the ASKHA superfamily of phosphotransferases.

Acetate kinase, an enzyme widely distributed in the Bacteria and Archaea domains, catalyzes the phosphorylation of acetate. We have determined the three-dimensional structure of Methanosarcina thermophila acetate kinase bound to ADP through crystallography. As we previously predicted, acetate kinase contains a core fold that is topologically identical to that of the ADP-binding domains of glycerol kinase, hexokinase, the 70-kDa heat shock cognate (Hsc70), and actin. Numerous charged active-site residues are conserved within acetate kinases, but few are conserved within the phosphotransferase superfamily. The identity of the points of insertion of polypeptide segments into the core fold of the superfamily members indicates that the insertions existed in the common ancestor of the phosphotransferases. Another remarkable shared feature is the unusual, epsilon conformation of the residue that directly precedes a conserved glycine residue (Gly-331 in acetate kinase) that binds the alpha-phosphate of ADP. Structural, biochemical, and geochemical considerations indicate that an acetate kinase may be the ancestral enzyme of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases.

Acetate Kinase↗
PubMed1980

Inactivation of Escherichia coli acetate kinase by N-ethylmaleimide. Protection by substrates and products.

Acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) from Escherichia coli exhibited a time-dependent loss of activity when incubated with N-ethylmaleimide at micromolar concentrations. However, prolonged incubation did not eliminate all catalytic activity and generally about 15% of its initial activity remained. When incubated with 7.2 microM N-ethylmaleimide, acetate kinase was inactivated with a rate constant of 0.063 min-1. Adenine nucleotides, ATP, ADP and AMP, protected the enzyme against such inactivation, but acetate up to 3.0 M and in the presence of 0.2 M MgCl2 and acetyl phosphate at 24 mM did not interfere with the rate of inactivation. While both acetate and acetyl phosphate did not affect the protection rendered by AMP, the presence of acetyl phosphate altered ADP protection. However, both substrates prevented ATP from protecting the enzyme. These data suggest that the binding sites for acetate and acetyl phosphate are different from that of the adenosine binding domain, but are in close vicinity to the phosphoryl binding regions of the nucleotides.

Acetate Kinase↗
PubMed1986

Purification and properties of an acetate kinase from Rhodopseudomonas palustris.

An acetate kinase from the photolithoautotrophically grown purple bacterium Rhodopseudomonas palustris was purified to apparent homogeneity by use of high resolving liquid chromatography steps. The monomeric enzyme was characterized by a relative molecular mass of 46,500 and an isoelectric point of 4.9. There was an absolute requirement for divalent metal ions in the enzymatic reaction. Mg2+ and Mn2+ were the most activating cations. The acetate kinase used pyrimidine and purine nucleotides almost equally well as phosphoryl donors. The enzyme phosphorylated acetate, propionate, butyrate and isobutyrate. ATP and acetate revealed the lowest apparent Km values and seemed to act as the favoured substrates. The apparent Km values for ATP formation were considerable lower than those for the formation of acetyl phosphate. The activation energy Ea = 21 kJ/mol of the acetyl phosphate formation was determined by application of Arrhenius plots.

Acetate Kinase↗
PubMed1980

Acetate kinase: a triple-displacement enzyme.

Facts relating to the mechanism of phosphoryl transfer by acetate kinase (ATP:acetate phosphotransferase, EC 2.7.2.1) are reviewed. They point to the existence of at least one experimentally established phosphoenzyme (E-P) intermediate on the reaction pathway. Sterically, the phosphoryl transfer occurs with a net inversion of the configuration of the phosphorus atom. These facts are best in accord with a triple-displacement mode of action for acetate kinase, with two E-P intermediates and three steric inversions on phosphorus. It follows that a second E-P for acetate kinase must exist.

Acetate Kinase↗
PubMed1993

Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila.

The genes for the acetate-activating enzymes, acetate kinase and phosphotransacetylase (ack and pta), from Methanosarcina thermophila TM-1 were cloned and sequenced. Both genes are present in only one copy per genome, with the pta gene adjacent to and upstream of the ack gene. Consensus archaeal promoter sequences are found upstream of the pta coding region. The pta and ack genes encode predicted polypeptides with molecular masses of 35,198 and 44,482 Da, respectively. A hydropathy plot of the deduced phosphotransacetylase sequence indicates that it is a hydrophobic polypeptides; however, no membrane-spanning domains are evident. Comparison of the amino acid sequences deduced from the M. thermophila and Escherichia coli ack genes indicate similar subunit molecular weights and 44% identity (60% similarity). The comparison also revealed the presence of several conserved arginine, cysteine, and glutamic acid residues. Arginine, cysteine, and glutamic acid residues have previously been implicated at or near the active site of the E. coli acetate kinase. The pta and ack genes were hyperexpressed in E. coli, and the overproduced enzymes were purified to homogeneity with specific activities higher than those of the enzymes previously purified from M. thermophila. The overproduced phosphotransacetylase and acetate kinase migrated at molecular masses of 37,000 and 42,000 Da, respectively. The activity of the acetate kinase is optimal at 65 degrees C and is protected from thermal inactivation by ATP. Diethylpyrocarbonate and phenylglyoxal inhibited acetate kinase activity in a manner consistent with the presence of histidine and arginine residues at or near the active site; however, the thiol-directed reagents 5,5'-dithiobis (2-nitrobenzoic acid) and N-ethylmaleimide were ineffective.

Acetate Kinase↗
PubMed1981

Acetate kinase activity in mycoplasmas.

Acetate kinase activity was assayed in 13 mycoplasmas. Nine species exhibited the enzymic activity in the direction of either synthesis of acetylphosphate or adenosine triphosphate. On the other hand Mycoplasma orale, Mycoplasma arthritidis, Ureaplasma urealyticum (10 serotypes), and two strains of Anaeroplasma species exhibited only minimal levels of the enzymic activity. In these four species, the enzyme does not seem to play a key role in adenosine triphosphate formation.

Acetate Kinase↗