Acetate Kinase

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James G. Ferry - One of the best experts on this subject based on the ideXlab platform.

  • Acetate Kinase and phosphotransacetylase
    Methods in Enzymology, 2011
    Co-Authors: James G. Ferry
    Abstract:

    Most of the methane produced in nature derives from the methyl group of Acetate, the major end product of anaerobes decomposing complex plant material. The Acetate is derived from the metabolic intermediate acetyl-CoA via the combined activities of phosphotransacetylase and Acetate Kinase. In Methanosarcina species, the enzymes function in the reverse direction to activate Acetate to acetyl-CoA prior to cleavage into a methyl and carbonyl group of which the latter is oxidized providing electrons for reduction of the former to methane. Thus, phosphotransacetylase and Acetate Kinase have a central role in the conversion of complex organic matter to methane by anaerobic microbial food chains. Both enzymes have been purified from Methanosarcina thermophila and characterized. Both enzymes from M. thermophila have also been produced in Escherichia coli permitting crystal structures and amino acid variants, the kinetic and biochemical studies of which have lead to proposals for catalytic mechanisms. The high identity of both enzymes to paralogs in the domain Bacteria suggests ancient origins and common mechanisms.

  • crystallization of Acetate Kinase from methanosarcina thermophila and prediction of its fold
    Protein Science, 2008
    Co-Authors: Kathryn A. Buss, James G. Ferry, David Avram Sanders, Cheryl Ingramsmith, Miriam S. Hasson
    Abstract:

    The unique biochemical properties of Acetate Kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of Acetate Kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of Acetate Kinase from several organisms, we hypothesize that Acetate Kinase is a member of the sugar Kinase/actin/hsp70 structural family.

  • investigation of the methanosarcina thermophila Acetate Kinase mechanism by fluorescence quenching
    Biochemistry, 2007
    Co-Authors: Andrea Gorrell, James G. Ferry
    Abstract:

    Acetate Kinase [E.C. 2.7.2.1] is a homodimer which catalyzes the reversible magnesium-dependent transfer of the γ-phosphoryl group from ATP to Acetate, yielding ADP and acetyl phosphate (CH3COO- + ATP ⇔ CH3CO2PO3-2 + ADP). The enzyme is widely distributed among fermentative prokaryotes in the Bacteria domain where it functions together with phosphotransacetylase (CH3CO2PO3-2 + HS-CoA ⇔ CH3COSCoA + HPO4-2) to convert acetyl-CoA to Acetate and synthesize ATP. These enzymes also function to activate Acetate to acetyl-CoA in the first step of the pathway for conversion of the methyl group to methane by Methanosarcina species from the Archaea domain (1). Though Acetate Kinase was one of the earliest phosphoryl transfer enzymes to be identified (2) and investigated, a debate persisted in the literature until recently whether the catalytic mechanism is a triple displacement of the phosphate involving two covalent enzyme intermediates (3) or a direct in-line phosphoryl transfer (4, 5). Support for the direct in-line mechanism accrues from studies with the Acetate Kinase from Methanosarcina thermophila (6-12). The enzyme co-crystallized with ADP, Al+3, F-, and Acetate, shows a linear array of ADP-AlF3-Acetate in the active site cleft wherein the AlF3 is proposed to mimic the meta-phosphate transition state (11). Kinetic analyses of Arg241 and Arg91 replacement variants indicate these active site residues are essential for catalysis and important for binding Acetate (11). In a direct in-line phosphoryl transfer, the hypothesized transition state is a trigonal bipyramidal structure of the γ-phosphoryl group, with the axial positions occupied by β-phosphoryl and acetyl oxygens. The guanidino groups of Arg241 and Arg91 are proposed to stabilize this structure by interacting with oxygen atoms of the γ-phosphoryl and carboxyl groups of Acetate (11). The guanidino group of Arg241 is adjacent to AlF3 consistent with the proposed role. However, the guanidino group of Arg91 is displaced 7A from AlF3 placing doubt on the proposed role, although the structure may not accurately represent the position of Arg91 during catalysis (11). Based upon structural similarities of the M. thermophila enzyme, Acetate Kinase was identified as a member of the Acetate and sugar Kinase/Hsc70/actin (ASKHA) structural superfamily (12). Each monomer of the homodimeric Acetate Kinase is characterized by a duplicated central β-sheet surrounded by α-helices (βββαβαβα core) and consisting of two domains (Fig. 1). The sugar Kinase and actin family members are all known to undergo a catalytically essential domain closure upon ligand binding (13-18). Although there is no experimental evidence to support domain closure during catalysis of Acetate Kinase from M. thermophila, domain closure has been suggested to participate in stabilization of the transition state based upon the architecture of the crystal structure (12). Figure 1 A. Structure of the M. thermophila Acetate Kinase (1TUY, (11)). The dimer is shown with monomer A (ribbon representation) in rainbow hue from the blue N-terminus to the red C-terminus. Monomer B (wire representation) with domain I (residues 1-148 and ... The M. thermophila Acetate Kinase has no tryptophan residues which has precluded intrinsic fluorescence measurements as an approach to investigate the catalytic mechanism. Here we report Kd values for ADP:Mg and ATP:Mg for the M. thermophila Acetate Kinase enabled by fluorescence quenching of the Gln43Trp variant. Double variants, with either Arg241 or Arg91 replaced in the Gln43Trp variant, had Kd values consistent with previously proposed roles for these arginines in catalysis and substrate binding. The Gln43Trp variant has also provided evidence for domain motion with implications for the catalytic mechanism. The results indicate that catalysis is not as two independent active sites, but that the active site activities are coordinated in a half-the-sites manner.

  • Characterization of the Acetate Binding Pocket in the Methanosarcina thermophila Acetate Kinase
    Journal of Bacteriology, 2005
    Co-Authors: Cheryl Ingram-smith, Kerry S. Smith, Andrea Gorrell, Sarah H. Lawrence, Prabha Iyer, James G. Ferry
    Abstract:

    Acetate Kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP γ-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 Val93, Leu122, Phe179, and Pro232 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 Km 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 Phe179 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.

  • structural and kinetic analyses of arginine residues in the active site of the Acetate Kinase from methanosarcina thermophila
    Journal of Biological Chemistry, 2005
    Co-Authors: Andrea Gorrell, Sarah H. Lawrence, James G. Ferry
    Abstract:

    Abstract Acetate Kinase catalyzes transfer of the γ-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′-[γ-thio]triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B. The enzyme co-crystallized with ADP, Acetate, Al3+, and F- contained a linear array of ADP-AlF3-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 AlF3 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 His180 was in close proximity to AlF3 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 Arg241 was also found adjacent to AlF3, consistent with a role for stabilization of the transition state. Kinetic analyses of Arg241 and Arg91 replacement variants indicated that these residues are essential for catalysis and also indicated a role in binding Acetate.

Peter Schonheit - One of the best experts on this subject based on the ideXlab platform.

  • 14 phosphate acetyltransferase and Acetate Kinase from thermotoga maritima
    Methods in Enzymology, 2001
    Co-Authors: Peter Schonheit
    Abstract:

    Publisher Summary Acetate is an important end-product of energy yielding fermentation processes of many anaerobic and facultative procaryotes. Generally, Acetate is formed from acetyl-coenzyme A (acetyl-CoA), a central intermediate of metabolism. The mechanism of actetate formation from acetyl-CoA in prokaryotes appears to be dependent on the phylogenetic domain that the organisms belong to. In all eubacteria analyzed, acetyl-CoA is converted to Acetate by the long known classic mechanism involving two enzymes, phosphate acetyltransferase (PTA) and Acetate Kinase (AK). Acetate Kinases and phosphate acetyltransferases have been purified from mesophilic and moderate thermophilic bacteria and the archaeon Methanosarcina thermophila . This chapter describes the purification and characterization of Acetate Kinase and phosphate acetyltransferase from a hyperthermophile, the eubacterium Thermotoga maritima , which grows at temperatures up to 90°, with an optimum around 800. This anaerobic organism ferments various organic compounds, incuding starch and glucose, to Acetate as the main product.

  • purification and characterization of two extremely thermostable enzymes phosphate acetyltransferase and Acetate Kinase from the hyperthermophilic eubacterium thermotoga maritima
    Journal of Bacteriology, 1999
    Co-Authors: Annekatrin Bock, Jurgen Glasemacher, Roland Schmidt, Peter Schonheit
    Abstract:

    Phosphate acetyltransferase (PTA) and Acetate Kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (alpha4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from Clostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi -->/ /<-- Acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, Acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent Vmax values (at 50 degrees C) were 2,600 U/mg (Acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to Acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90 degrees C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4 (1 M), which was most effective, the enzyme did not lose activity upon incubation at 100 degrees C for 3 h. The temperature optimum at 90 degrees C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.

Roland Schmidt - One of the best experts on this subject based on the ideXlab platform.

  • purification and characterization of two extremely thermostable enzymes phosphate acetyltransferase and Acetate Kinase from the hyperthermophilic eubacterium thermotoga maritima
    Journal of Bacteriology, 1999
    Co-Authors: Annekatrin Bock, Jurgen Glasemacher, Roland Schmidt
    Abstract:

    Acetate is an important end product of energy-yielding fermentation processes of many anaerobic and facultative procaryotes. Generally Acetate is formed from acetyl coenzyme A (acetyl-CoA), a central intermediate of metabolism. The mechanism of conversion of acetyl-CoA to Acetate in prokaryotes, which is coupled with ATP formation, has recently been shown to be dependent on the phylogenetic domain to which the organisms belong (33, 34). (i) In all eubacteria analyzed, acetyl-CoA is converted to Acetate by the “classical” mechanism involving two enzymes, phosphate acetyltransferase (PTA) (EC 2.3.1.8) and Acetate Kinase (AK) (EC 2.7.2.1). ATP is formed in the Acetate Kinase reaction by the mechanism of substrate-level phosphorylation. Acetyl-CoA + Pi ⇌ acetyl phosphate + CoA (PTA) Acetyl phosphate + ADP ⇌ Acetate + ATP (AK) (ii) In all Acetate forming archaea studied so far, including anaerobic hyperthermophiles and aerobic mesophilic halophiles, the conversion of acetyl-CoA to Acetate and the formation of ATP from ADP and phosphate is catalyzed by only one enzyme, an acetyl-CoA synthetase (ADP forming) (33, 34). Acetyl-CoA + ADP + Pi ⇌ Acetate + ATP + CoA This unusual synthetase, which was first discovered in the anaeobic eukaryote Entamoeba histolytica (23, 30), is part of a novel mechanism of Acetate formation and energy conservation in prokaryotes. Acetate also serves as substrate of catabolism and anabolism in several aerobic and anaerobic prokaryotes. The activation of Acetate to acetyl-CoA, which is the first step prior to its utilization in metabolism, is catalyzed either by a single enzyme, an AMP-forming acetyl-CoA synthetase (EC 6.2.1.1) (Acetate + CoA + ATP ⇌ acetyl-CoA + AMP + PPi) or by the AK-PTA couple operating in the reverse direction as described above (12, 33, 36, 40). Besides their function in Acetate metabolism, PTA and AK play a role, via acetyl phosphate, in various other processes. For example, in Escherichia coli, acetyl phosphate functions as the phosphoryl donor of response regulator proteins of two-component systems, and a function as a global regulatory signal has therefore been proposed (22, 44). To date, Acetate Kinases and phosphate acetyltransferases have been purified from various bacteria and from the archaeon Methanosarcina thermophila. However, these enzymes have not yet been isolated and characterized from hyperthermophilic prokaryotes, which are considered to represent the most ancient living organisms (39). We have recently studied the glucose metabolism of the hyperthermophilic Thermotoga maritima, (Toptimum = 80°C), which belongs to the deepest branches in the phylogenetic tree within the bacterial domain. The organism ferments glucose to Acetate, CO2, H2, and various amounts of lactate (15, 35). Glucose degradation to pyruvate proceeds via the classical Embden-Meyerhof pathway, and pyruvate oxidation to acetyl-CoA involves pyruvate:ferredoxin oxidoreductase. The conversion of acetyl-CoA to Acetate and ATP is catalyzed by PTA and AK (34, 35), which is the mechanism of Acetate formation typical of bacteria (see above). In this communication we report on the purification and characterization of AK and PTA from the hyperthermophilic eubacterium Thermotoga maritima.

  • purification and characterization of two extremely thermostable enzymes phosphate acetyltransferase and Acetate Kinase from the hyperthermophilic eubacterium thermotoga maritima
    Journal of Bacteriology, 1999
    Co-Authors: Annekatrin Bock, Jurgen Glasemacher, Roland Schmidt, Peter Schonheit
    Abstract:

    Phosphate acetyltransferase (PTA) and Acetate Kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (alpha4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from Clostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi -->/ /<-- Acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, Acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent Vmax values (at 50 degrees C) were 2,600 U/mg (Acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to Acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90 degrees C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4 (1 M), which was most effective, the enzyme did not lose activity upon incubation at 100 degrees C for 3 h. The temperature optimum at 90 degrees C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.

Cheryl Ingramsmith - One of the best experts on this subject based on the ideXlab platform.

  • novel pyrophosphate forming Acetate Kinase from the protist entamoeba histolytica
    Eukaryotic Cell, 2012
    Co-Authors: Matthew L Fowler, Cheryl Ingramsmith, Kerry S. Smith
    Abstract:

    ABSTRACT Acetate Kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-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 (PPi)/inorganic phosphate (Pi) as the sole phosphoryl donor/acceptor. Detection of ACK activity in E. histolytica cell extracts in the direction of Acetate/PPi formation but not in the direction of acetyl phosphate/Pi formation suggests that the physiological direction of the reaction is toward Acetate/PPi production. Kinetic parameters determined for each direction of the reaction are consistent with this observation. The E. histolytica PPi-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.

  • crystallization of Acetate Kinase from methanosarcina thermophila and prediction of its fold
    Protein Science, 2008
    Co-Authors: Kathryn A. Buss, James G. Ferry, David Avram Sanders, Cheryl Ingramsmith, Miriam S. Hasson
    Abstract:

    The unique biochemical properties of Acetate Kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of Acetate Kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of Acetate Kinase from several organisms, we hypothesize that Acetate Kinase is a member of the sugar Kinase/actin/hsp70 structural family.

  • the role of histidines in the Acetate Kinase frommethanosarcina thermophila
    Journal of Biological Chemistry, 2000
    Co-Authors: Cheryl Ingramsmith, Robert D Barber, James G. Ferry
    Abstract:

    The role of histidine in the catalytic mechanism of Acetate Kinase from Methanosarcina thermophila was investigated by diethylpyrocarbonate inactivation and site-directed mutagenesis. Inactivation was accompanied by an increase in absorbance at 240 nm with no change in absorbance at 280 nm, and treatment of the inactivated enzyme with hydroxylamine restored 95% activity, results that indicated diethylpyrocarbonate inactivates the enzyme by the specific modification of histidine. The substrates ATP, ADP, Acetate, and acetyl phosphate protected against inactivation suggesting at least one active site where histidine is modified. Correlation of residual activity with the number of histidines modified, as determined by absorbance at 240 nm, indicated that a maximum of three histidines are modified per subunit, two of which are essential for full inactivation. Comparison of the M. thermophila Acetate Kinase sequence with 56 putative Acetate Kinase sequences revealed eight highly conserved histidines, three of which (His-123, His-180, and His-208) are perfectly conserved. Diethylpyrocarbonate inactivation of the eight histidine → alanine variants indicated that His-180 and His-123 are in the active site and that the modification of both is necessary for full inactivation. Kinetic analyses of the eight variants showed that no other histidines are important for activity. Analysis of additional His-180 variants indicated that phosphorylation of His-180 is not essential for catalysis. Possible functions of His-180 are discussed.

  • identification of essential arginines in the Acetate Kinase from methanosarcina thermophila
    Biochemistry, 2000
    Co-Authors: Kavita Singhwissmann, Rebecca D. Miles, Cheryl Ingramsmith, James G. Ferry
    Abstract:

    Site-directed mutagenesis is a powerful tool for identifying active-site residues essential for catalysis; however, this approach has only recently become available for Acetate Kinase. The enzyme from Methanosarcina thermophila has been cloned and hyper-produced in a highly active form in Escherichia coli (recombinant wild-type). The role of arginines in this Acetate Kinase was investigated. Five arginines (R91, R175, R241, R285, and R340) in the M. thermophila enzyme were selected for individual replacement based on their high conservation among sequences of Acetate Kinase homologues. Replacement of R91 or R241 with alanine or leucine produced variants with specific activities less than 0.1% of the recombinant wild-type enzyme. The circular dichroism spectra and other properties of these variants were comparable to those of recombinant wild-type, indicating no global conformational changes. These results indicate that R91 and R241 are essential for activity, consistent with roles in catalysis. The variant produced by conservative replacement of R91 with lysine had approximately 2% of recombinant wild-type activity, suggesting a positive charge is important in this position. The K(m) value for Acetate of the R91K variant increased greater than 10-fold relative to recombinant wild-type, suggesting an additional role for R91 in binding this substrate. Activities of both the R91A and R241A variants were rescued 20-fold when guanidine or derivatives were added to the reaction mixture. The K(m) values for ATP of the rescued variants were similar to those of recombinant wild-type, suggesting that the rescued activities are the consequence of replacement of important functional groups and not changes in the catalytic mechanism. These results further support roles for R91 and R241 in catalysis. Replacement of R285 with alanine, leucine, or lysine had no significant effect on activity; however, the K(m) values for Acetate increased 6-10-fold, suggesting R285 influences the binding of this substrate. Phenylglyoxal inhibition and substrate protection experiments with the recombinant wild-type enzyme and variants were consistent with the presence of one or more essential arginine residues in the active site as well as with roles for R91 and R241 in catalysis. It is proposed that R91 and R241 function to stabilize the previously proposed pentacoordinate transition state during direct in-line transfer of the gamma-phosphate of ATP to Acetate. The kinetic characterization of variants produced by replacement of R175 and R340 with alanine, leucine, or lysine indicated that these residues are not involved in catalysis but fulfill important structural roles.

  • identification of essential glutamates in the Acetate Kinase from methanosarcina thermophila
    Journal of Bacteriology, 1998
    Co-Authors: Rebecca D. Miles, James G. Ferry, Cheryl Ingramsmith, Kavita Singhwissmann
    Abstract:

    Acetate Kinase catalyzes the reversible phosphorylation of Acetate (CH3COO 2 1 ATP^CH3CO2PO3 22 1 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 kcat, 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 is an end product of most fermentative microbes and is the major growth substrate for the methanoarchaea (22); thus, carbon flow through Acetate is of primary importance in anaerobic microbial consortia and the global carbon cycle. Although the metabolisms of fermentatives and acetotrophic methanoarchaea represent the extremes of biochemical divergence in energy-yielding pathways, these microbes have in common the enzymes Acetate Kinase (reaction 1) and phosphotransacetylase (reaction 2).

Annekatrin Bock - One of the best experts on this subject based on the ideXlab platform.

  • purification and characterization of two extremely thermostable enzymes phosphate acetyltransferase and Acetate Kinase from the hyperthermophilic eubacterium thermotoga maritima
    Journal of Bacteriology, 1999
    Co-Authors: Annekatrin Bock, Jurgen Glasemacher, Roland Schmidt
    Abstract:

    Acetate is an important end product of energy-yielding fermentation processes of many anaerobic and facultative procaryotes. Generally Acetate is formed from acetyl coenzyme A (acetyl-CoA), a central intermediate of metabolism. The mechanism of conversion of acetyl-CoA to Acetate in prokaryotes, which is coupled with ATP formation, has recently been shown to be dependent on the phylogenetic domain to which the organisms belong (33, 34). (i) In all eubacteria analyzed, acetyl-CoA is converted to Acetate by the “classical” mechanism involving two enzymes, phosphate acetyltransferase (PTA) (EC 2.3.1.8) and Acetate Kinase (AK) (EC 2.7.2.1). ATP is formed in the Acetate Kinase reaction by the mechanism of substrate-level phosphorylation. Acetyl-CoA + Pi ⇌ acetyl phosphate + CoA (PTA) Acetyl phosphate + ADP ⇌ Acetate + ATP (AK) (ii) In all Acetate forming archaea studied so far, including anaerobic hyperthermophiles and aerobic mesophilic halophiles, the conversion of acetyl-CoA to Acetate and the formation of ATP from ADP and phosphate is catalyzed by only one enzyme, an acetyl-CoA synthetase (ADP forming) (33, 34). Acetyl-CoA + ADP + Pi ⇌ Acetate + ATP + CoA This unusual synthetase, which was first discovered in the anaeobic eukaryote Entamoeba histolytica (23, 30), is part of a novel mechanism of Acetate formation and energy conservation in prokaryotes. Acetate also serves as substrate of catabolism and anabolism in several aerobic and anaerobic prokaryotes. The activation of Acetate to acetyl-CoA, which is the first step prior to its utilization in metabolism, is catalyzed either by a single enzyme, an AMP-forming acetyl-CoA synthetase (EC 6.2.1.1) (Acetate + CoA + ATP ⇌ acetyl-CoA + AMP + PPi) or by the AK-PTA couple operating in the reverse direction as described above (12, 33, 36, 40). Besides their function in Acetate metabolism, PTA and AK play a role, via acetyl phosphate, in various other processes. For example, in Escherichia coli, acetyl phosphate functions as the phosphoryl donor of response regulator proteins of two-component systems, and a function as a global regulatory signal has therefore been proposed (22, 44). To date, Acetate Kinases and phosphate acetyltransferases have been purified from various bacteria and from the archaeon Methanosarcina thermophila. However, these enzymes have not yet been isolated and characterized from hyperthermophilic prokaryotes, which are considered to represent the most ancient living organisms (39). We have recently studied the glucose metabolism of the hyperthermophilic Thermotoga maritima, (Toptimum = 80°C), which belongs to the deepest branches in the phylogenetic tree within the bacterial domain. The organism ferments glucose to Acetate, CO2, H2, and various amounts of lactate (15, 35). Glucose degradation to pyruvate proceeds via the classical Embden-Meyerhof pathway, and pyruvate oxidation to acetyl-CoA involves pyruvate:ferredoxin oxidoreductase. The conversion of acetyl-CoA to Acetate and ATP is catalyzed by PTA and AK (34, 35), which is the mechanism of Acetate formation typical of bacteria (see above). In this communication we report on the purification and characterization of AK and PTA from the hyperthermophilic eubacterium Thermotoga maritima.

  • purification and characterization of two extremely thermostable enzymes phosphate acetyltransferase and Acetate Kinase from the hyperthermophilic eubacterium thermotoga maritima
    Journal of Bacteriology, 1999
    Co-Authors: Annekatrin Bock, Jurgen Glasemacher, Roland Schmidt, Peter Schonheit
    Abstract:

    Phosphate acetyltransferase (PTA) and Acetate Kinase (AK) of the hyperthermophilic eubacterium Thermotoga maritima have been purified 1,500- and 250-fold, respectively, to apparent homogeneity. PTA had an apparent molecular mass of 170 kDa and was composed of one subunit with a molecular mass of 34 kDa, suggesting a homotetramer (alpha4) structure. The N-terminal amino acid sequence showed significant identity to that of phosphate butyryltransferases from Clostridium acetobutylicum rather than to those of known phosphate acetyltransferases. The kinetic constants of the reversible enzyme reaction (acetyl-CoA + Pi -->/ /<-- Acetate + ATP) were determined at the pH optimum of pH 7.0. The apparent Km values for acetyl phosphate, ADP, Acetate, and ATP were 0.44, 3, 40, and 0.7 mM, respectively; the apparent Vmax values (at 50 degrees C) were 2,600 U/mg (Acetate formation) and 1,800 U/mg (acetyl phosphate formation). AK phosphorylated propionate (54%) in addition to Acetate (100%) and used GTP (100%), ITP (163%), UTP (56%), and CTP (21%) as phosphoryl donors in addition to ATP (100%). Divalent cations were required for activity, with Mn2+ and Mg2+ being most effective. The enzyme had a temperature optimum at 90 degrees C and was stabilized against heat inactivation by salts. In the presence of (NH4)2SO4 (1 M), which was most effective, the enzyme did not lose activity upon incubation at 100 degrees C for 3 h. The temperature optimum at 90 degrees C and the high thermostability of both PTA and AK are in accordance with their physiological function under hyperthermophilic conditions.