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PubMed1979

Purification and properties of acetate kinase from Acholeplasma laidlawii.

Acetate kinase (EC 2.7.2.1) was purified from Acholeplasma laidlawii cytoplasm by a combination of ammonium sulfate fractionation, gel filtration, diethylaminoethyl-cellulose chromatography, and affinity chromatography on 8-(6-aminohexylamino)-adenosine 5'-triphosphate conjugated to Sepharose 4B. The enzyme was composed of polypeptide chains of about 50,000 molecular weight as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nondenaturating conditions, apparent molecular weights between 64,000 and 130,000 were obtained, depending upon mainly the ionic strength of the test solution. The enzyme had a narrow specificity for phosphate acceptor acids, whereas both purine and pyrimidine nucleoside triphosphates were suitable phosphate donors. Na(+) and K(+) inhibited both acetyl phosphate and adenosine 5'-triphosphate synthesis, and the latter was also inhibited by high concentrations of adenosine 5'-diphosphate and acetyl phosphate. This substrate inhibition was partially abolished by 0.5 M NaCl. The enzyme catalyzed the independent adenosine 5'-diphosphate<-->adenosine 5'-triphosphate and acetate<-->acetyl phosphate exchanges. The rate of the latter was enhanced by the addition of cosubstrate Mg(2+)-adenosine 5'-triphosphate. The high affinity for substrates, except for acetate, indicated that under physiological conditions the direction of the enzymic reaction favors adenosine 5'-triphosphate synthesis. Thus, a mechanism for adenosine 5'-triphosphate generation in mycoplasmas is suggested.

Acetate Kinase↗

PubMedDate not supplied

Continuous ATP regeneration process with stable acetate kinase.

Heat-stable acetate kinase (AK) from Bacillus stearothermophilus was successfully immobilized covalently to Sepharose resin by several conventional methods including carbodiimide, hydroxysuccinimide, cyanogen bromide, and glutaraldehyde and also by a new method which utilizes a bifunctional ADP derivative as a spacer. The latter method gave a higher yield in terms of enzyme activity than the conventional methods. The properties and kinetics of the immobilized AK were studied batchwise and in a column. The Michaelis-Menten equation could be applied to the immobilized AK column. The apparent Km values of ADP and acetyl phosphate for immobilized AK were not significantly different from those for free AK. The pH-activity profile of immobilized AK was similar to that of free AK. The heat stability of immobilized AK was markedly improved as compared with free AK. The immobilized AK retained more than 80% of the initial activity after continuous operation at 30 degrees C for 1 month. It was shown that the immobilized AK from B. stearothermophilus could be utilized as an ATP regeneration system in the bioreactor.

Acetate Kinase↗

PubMed1996

The acetate kinase of Clostridum acetobutylicum strain P262.

Clostridum acetobutylicum strain P262 fermented glucose, pyruvate, or lactate, and the butyrate production was substrate-dependent. Differences in butyrate yield could not be explained by changes in butyrate kinase activities, but the butyrate production was inversely related to acetate kinase activity. The acetate kinase had a pH optimum of 8.0, a Km for acetate of 160 mM, and a kcat of 16, 800 min-1. The enyzme had a native molecular mass of 78 kDa; the size of 42 kDa on SDS-PAGE indicated that the acetate kinase of strain P262 was a homodimer.

Acetate Kinase↗

PubMed1986

Phosphate transfer between acetate kinase and enzyme I of the bacterial phosphotransferase system.

Interactions between homogeneous acetate kinase and proteins of the phosphoenolpyruvate:glucose phosphotransferase system (PTS) were studied. The phosphorylation of D-glucose was followed spectrophotometrically using a coupled assay system, and acetate kinase and GTP were found to substitute for phosphoenolpyruvate provided that each of the PTS proteins was present in the mixture. To further define the phosphoryl transfer reaction pathway, the system was simplified to include only the homogeneous, soluble PTS proteins. 32P was transferred from [gamma-32P]ATP to the protein IIIGlc, but this transfer reaction required acetate kinase, and the PTS proteins Enzyme I and HPr. These results suggested that acetate kinase interacts with the first protein in the PTS sequence, Enzyme I. Acetate kinase was therefore incubated with [32P] phospho-Enzyme I, and a direct transfer of the phosphoryl group was observed without the addition of any other protein. These results show that there is a reversible transfer of the phosphoryl group between Enzyme I and acetate kinase. The possible role of this interaction in regulating sugar uptake by the Krebs cycle is discussed.

Acetate Kinase↗

PubMed1985

[Effect of the redox state of glutathione on acetate kinase activity in E. coli].

Oxidized glutathione inhibits acetate kinase (EC 2.7.2.1) of E. coli. The rate of inactivation depends on ATP concentration. The rate constant for the glutathione-induced inhibition is 0.17 min-1, Ki is 4.2 mM (pH 7.2, 25 degrees C). The inhibition of acetate kinase by glutathione is reversible, the equilibrium constant being equal to 4.4 or 0.09 at saturating concentrations of ATP (pH 8.0, 25 degrees C). The physiological level of reduced and oxidized glutathione can modulate the acetate kinase activity in vivo.

Acetate Kinase↗

PubMed1999

Cloning, sequence analysis, expression and inactivation of the Corynebacterium glutamicum pta-ack operon encoding phosphotransacetylase and acetate kinase.

The Corynebacterium glutamicum ack and pta genes encoding the acetate-activating enzymes acetate kinase and phosphotransacetylase were isolated, subcloned on a plasmid and re-introduced into Corynebacterium glutamicum. Relative to the wild-type, the recombinant strains showed about tenfold higher specific activities of both enzymes. Sequence analysis of a 3657 bp DNA fragment revealed that the ack and pta genes are contiguous in the corynebacterial chromosome, with pta upstream and the last nucleotide of the pta stop codon (TAA) overlapping the first of the ack start codon (ATG). The predicted gene product of pta consists of 329 amino acids (Mr 35242), that of ack consists of 397 amino acids (Mr 43098) and the amino acid sequences of the two polypeptides show up to 60 % (phosphotransacetylase) and 53% (acetate kinase) identity in comparison with respective enzymes from other organisms. Northern (RNA) blot hybridizations using pta- and ack-specific probes and transcriptional cat fusion experiments revealed that the two genes are transcribed as a 2.5 kb bicistronic mRNA and that the expression of this operon is induced when Corynebacterium glutamicum grows on acetate instead of glucose as a carbon source. Directed inactivation of the chromosomal pta and ack genes led to the absence of detectable phosphotransacetylase and acetate kinase activity in the respective mutants and to their inability to grow on acetate. These data indicate that no isoenzymes of acetate kinase and phosphotransacetylase are present in Corynebacterium glutamicum and that a functional acetate kinase/phosphotransacetylase pathway is essential for growth of this organism on acetate.

Acetate Kinase↗

PubMed1993

Regulation of the Bacillus subtilis acetate kinase gene by CcpA.

The Bacillus subtilis gene encoding acetate kinase was identified on the basis of sequence similarity to the Escherichia coli ackA gene and to a second E. coli gene closely related to ackA. Insertional inactivation of this region of the B. subtilis chromosome resulted in the disappearance of acetate kinase enzyme activity in cell extracts. The ackA gene was mapped to a site close to the ccpA gene, at 263 degrees. The transcriptional start site for B. subtilis ackA was located 90 bp upstream from the start of the coding region, and expression was increased by growth in the presence of excess glucose. Growth of the AckA- mutant was inhibited by glucose, suggesting that acetate kinase is important for excretion of excess carbohydrate. The stimulation of ackA expression by glucose was blocked in a CcpA- mutant, indicating that CcpA, which is required for glucose repression of certain carbon source utilization genes, including amyE, may also be involved in activation of carbon excretion pathways. Two sequences resembling the amyO operator site were identified upstream of the ackA promoter; removal of this region resulted in loss of glucose activation of ackA expression.

Acetate Kinase↗

PubMed1997

Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold.

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.

Acetate Kinase↗

PubMed1978

Purification and properties of acetate kinase from Bacillus stearothermophilus.

1. Acetate kinase [EC 2.7.2.1] from an thermophile, B. stearothermophilus, was purified and crystalized. 2. This enzyme was shown to be a tetramer of identical subunits which had a molecular weight of about 40,000. Amino acid analysis showed no SH group. By analyzing the CD spectrum it was deduced that this enzyme is composed of 36% beta-structure, 21% alpha-helix and 43% unordered structure. 3. This enzyme shared many common enzymatic properties with the counterpart from mesophiles, i.e. pH optimum, substrate specificity, requirement of metal ions and essential amino acid residues necessary for the catalytic activity. However, this enzyme was remarkably thermostable. 4. A plot of the reaction velocity against the concentration of acetate, ADP or acetyl phosphate gave a curve of the Michaelis-Menten type. However, such a plot against ATP gave a sigmoid curve, suggesting a homotropic allosteric nature of the enzyme. 5. From the results of chemical modification it was deduced that an amino group and an imidazole group, at least, are involved in the active site of the enzyme.

Acetate Kinase↗

PubMed1998

Both acetate kinase and acetyl coenzyme A synthetase are involved in acetate-stimulated change in the direction of flagellar rotation in Escherichia coli.

Escherichia coli strains overproducing the response regulator CheY respond to acetate by increasing their clockwise bias of flagellar rotation, even when they lack other chemotaxis proteins. With acetate metabolism mutants, we demonstrate that both acetate kinase and acetyl coenzyme A synthetase are involved in this response. Thus, a response was observed when one of these enzymes was missing but not when both were absent.

Acetate Kinase↗

PubMed2002

Cloning of a gene encoding acetate kinase from Methanosarcina mazei 2-P isolated from a Japanese paddy field soil.

The ack gene encoding acetate kinase from the mesophilic Methanosarcina mazei 2-P, isolated from a paddy field soil in Japan, was cloned, sequenced, and functionally expressed in Escherichia coli. The terminal region of the putative pta gene, probably encoding phosphotransacetylase, was found upstream of the ack gene. The deduced amino acid sequence of the acetate kinase is 86.5% identical to that of the Methanosarcina thermophila acetate kinase. The activity of the His(6)-tagged acetate kinase purified from E. coli JM109 was optimal at 35 degrees C.

Acetate Kinase↗

PubMed2000

The role of histidines in the acetate kinase from Methanosarcina thermophila.

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.

Acetate Kinase↗

PubMed1997

Acetate kinase from Clostridium acetobutylicum: a highly specific enzyme that is actively transcribed during acidogenesis and solventogenesis.

Acetate kinase (ATP:phosphotransferase, EC 2.7.2.1) has been purified 294-fold from acid-producing cells of Clostridium acetobutylicum DSM 1731 to a specific activity of 1087 U mg-1 (ADP-forming direction). The dimeric enzyme consisted of subunits with a molecular mass of 43 kDa. The molecular mass of the native acetate kinase was in the range 87-94 kDa as judged by gel filtration and native gel electrophoresis. The enzyme showed high specificity for the substrates acetate and ATP, and maximal activity was obtained with Mn2+ as divalent cation. The presence of mercury compounds such as HgCl2 and p-hydroxymercuribenzoate resulted in an essential loss of activity. The apparent K(m) values of acetate, Mg-ATP, acetyl phosphate, and Mg-ADP were 73, 0.37, 0.58 and 0.71 mM. An activity-staining procedure for detection of acetate kinase in crude cell extracts after separation on native polyacrylamide gels was developed. A DNA fragment encoding 246 bp of the acetate kinase gene of C. acetobutylicum DSM 792 was cloned by a PCR-based approach. Northern blot analysis revealed transcription of the gene under acid- and solvent-producing conditions with no significant differences at the level of transcription.

Acetate Kinase↗

PubMed2002

Evidence for a transition state analog, MgADP-aluminum fluoride-acetate, in acetate kinase from Methanosarcina thermophila.

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 gamma-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.

Acetate Kinase↗

PubMed2001

Purification and characterization of homo- and hetero-dimeric acetate kinases from the sulfate-reducing bacterium Desulfovibrio vulgaris.

Two distinct forms of acetate kinase were purified to homogeneity from a sulfate-reducing bacterium Desulfovibrio vulgaris Miyazaki F. The enzymes were separated from the soluble fraction of the cells on anion exchange columns. One acetate kinase (AK-I) was a homodimer (alpha(S)(2)) and the other (AK-II) was a heterodimer (alpha(S)alpha(L)). On SDS-PAGE, alpha(L) and alpha(S) subunits migrated as bands of 49.3 and 47.8 kDa, respectively, but they had an identical N-terminal amino acid sequence. A rapid HPLC method was developed to directly measure ADP and ATP in assay mixtures. Initial velocity data for AK-I and AK-II were collected by this method and analyzed based on a random sequential mechanism, assuming rapid equilibrium for the substrate binding steps. All kinetic parameters for both the forward acetyl phosphate formation and the reverse ATP formation catalyzed by AK-I and AK-II were successfully determined. The two enzymes showed similar kinetic properties in Mg(2+) requirement, pH-dependence and magnitude of kinetic parameters. These results suggest that two forms of acetate kinase are produced to finely regulate the enzyme function by post-translational modifications of a primary gene product in Desulfovibrio vulgaris.

Acetate Kinase↗

PubMed1980

Acetate kinase in the genus Veillonella: effect of succinate, serological cross-reactivity, and separation by electrophoresis.

Acetate kinases from the genus Veillonella were divided into two types: a succinate-stimulated enzyme and a succinate-independent enzyme. Three strains, V. parvula ATCC 17743 (antigenic group II), V. parvula ATCC 17744 (V), and V. parvula ATCC 10790 (VI), contained the succinate-stimulated enzyme. Among four types strains of V. alcalescens, three strains, ATCC 17747 (I), ATCC 17746 (III), and ATCC 17748 (VII), contained the succinate-independent enzyme, whereas only one strain, ATCC 17745 (IV), contained the succinate-stimulated enzyme. Small amounts of antiserum to the purified acetate kinase from V. alcalescens ATCC 17748 completely inhibited the purified and crude enzyme activity from the strain. Classification of the enzymes on the basis of stimulation by succinate was consistent with classification based on serological reactions using the antiserum as an independent parameter. The succinate-stimulated enzyme could be separated into two classes according to the degree of sensitivity to succinate: (i) enzymes from V. parvula ATCC 17744 and V. alcalescens ATCC 17745, which could be demonstrated on gel after electrophoresis by a histochemical method to be highly stimulated by the presence of succinate in the reaction mixture, and (ii) enzymes from V. parvula ATCC 10790 and V. parvula ATCC 17743, which could be easily demonstrated without succinate. Four groups of acetate kinases from the genus Veillonella were separated by gel electrophoretic mobility. The results showed that almost all enzymes from the seven type strains were heterogeneous at the molecular level.

Acetate Kinase↗

PubMed1999

Purification and characterization of two extremely thermostable enzymes, phosphate acetyltransferase and acetate kinase, from the hyperthermophilic eubacterium Thermotoga maritima.

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 -->/<-- acetyl phosphate + CoA) were determined at the pH optimum of pH 6.5. The apparent Km values for acetyl-CoA, Pi, acetyl phosphate, and coenzyme A (CoA) were 23, 110, 24, and 30 microM, respectively; the apparent Vmax values (at 55 degrees C) were 260 U/mg (acetyl phosphate formation) and 570 U/mg (acetyl-CoA formation). In addition to acetyl-CoA (100%), the enzyme accepted propionyl-CoA (60%) and butyryl-CoA (30%). The enzyme had a temperature optimum at 90 degrees C and was not inactivated by heat upon incubation at 80 degrees C for more than 2 h. AK had an apparent molecular mass of 90 kDa and consisted of one 44-kDa subunit, indicating a homodimer (alpha2) structure. The N-terminal amino acid sequence showed significant similarity to those of all known acetate kinases from eubacteria as well that of the archaeon Methanosarcina thermophila. The kinetic constants of the reversible enzyme reaction (acetyl phosphate + ADP -->/<-- 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.

Acetate Kinase↗

PubMed1978

Acetate kinase production by Escherichia coli during steady-state and transient growth in continuous culture.

The synthesis of acetate kinase by Escherichia coli ATCC 9637 was studied during growth in anaerobic continuous cultures under steady-state and transient conditions. During growth in anaerobic, glucose-limited chemostats, acetate kinase synthesis was linearly associated with growth. Two types of non-steady-state transients were studied: the perturbation in one was the addition of glucose alone, and, in the second, glucose plus Casamino Acids. During the nutritional shift-up in the second case, but not in the first, the instantaneous specific acetate kinase activities and specific synthesis rates exceeded pre- and postshift values. Trajectory curves demonstrated that the increase in specific activity remained within the bounds of values obtainable under steady-state conditions with minimal and Casamino Acids media. Specific synthesis rates, however, greatly exceeded steady-state values. Enzyme yield values on glucose after the transient nutritional shift-up increased up to fivefold. Active protein synthesis is shown to be necessary to achieve the enhanced specific synthesis rates and enzyme yields. The results from these transient responses are discussed in terms of a conceptful model for metabolic regulation.

Acetates↗