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1.
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. 相似文献
2.
Matthew L. Fowler Cheryl J. Ingram-Smith Kerry S. Smith 《Journal of visualized experiments : JoVE》2011,(58)
Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. 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 genus15,16.In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi. 相似文献
3.
Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PPi-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PPi were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PPi displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed. 相似文献
4.
Tarjani M. Thaker Mikio Tanabe Matthew L. Fowler Anita M. Preininger Cheryl Ingram-Smith Kerry S. Smith T.M. Iverson 《Journal of structural biology》2013,181(2):185-189
Acetate kinases (ACKs) are members of the acetate and sugar kinase/hsp70/actin (ASKHA) superfamily and catalyze the reversible phosphorylation of acetate, with ADP/ATP the most common phosphoryl acceptor/donor. While prokaryotic ACKs have been the subject of extensive biochemical and structural characterization, there is a comparative paucity of information on eukaryotic ACKs, and prior to this report, no structure of an ACK of eukaryotic origin was available. We determined the structures of ACKs from the eukaryotic pathogens Entamoeba histolytica and Cryptococcus neoformans. Each active site is located at an interdomain interface, and the acetate and phosphate binding pockets display sequence and structural conservation with their prokaryotic counterparts. Interestingly, the E. histolytica ACK has previously been shown to be pyrophosphate (PPi)-dependent, and is the first ACK demonstrated to have this property. Examination of its structure demonstrates how subtle amino acid substitutions within the active site have converted cosubstrate specificity from ATP to PPi while retaining a similar backbone conformation. Differences in the angle between domains surrounding the active site suggest that interdomain movement may accompany catalysis. Taken together, these structures are consistent with the eukaryotic ACKs following a similar reaction mechanism as is proposed for the prokaryotic homologs. 相似文献
5.
A microprocedure for the colorimetric determination of inorganic pyrophosphate (PPi) in the presence or absence of orthophosphate (Pi) has been developed. PPi is estimated quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and thiol reagent (monothioglycerol or 2-mercaptoethanol). The latter is obligatory for color formation. Pi is estimated without thiol reagent. The two chromophores differ in absorption spectra, the greatest difference being at 580 nm. For both, color develops fully by 10 min and is stable up to 1 hr. Just less than 0.4 μm PPi can be detemined. The extinction coefficients are 2.70 × 104 and 8.76 × 103 for PPi and Pi, respectively, both with thiol reagent present, and 2.77 × 103 for Pi with no thiol reagent.A ten-fold excess of Pi does not interfere with the determination of PPi and in fact can be estimated in the same mixture. A 15-fold excess, however, diminishes the accuracy of PPi estimations. Trichloroacetic acid and sodium fluoride inhibi color formation, but this inhibition is overcome by the addition of sodium acetate buffer, pH 4.0. Nucleoside triphosphates and adenosine 3′:5′-cyclic monophosphate are stable in the reaction mixture.The method was tested in assays of Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6). Progress curves measured by either the rate of PPi formation or the rate of synthesis of labeled RNA were very similar. Product PPi formed by as little as 0.6 unit of RNA polymerase in a 225-μl incubation medium could be measured.An automated version of the method was devised which allows accurate determination of PPi down to 1 μm (without range expander attachment) at a sampling rate of 20–40 tubes/hr. 相似文献
6.
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. 相似文献
7.
A generally applicable, inexpensive, and sensitive method for the determination of inorganic pyrophosphate (PPi) was developed. PPi was quantitatively separable from solution even in nanomolar concentrations by filtration through a membrane filter in the presence of CaCl2 and KF. The separated PPi was dissolved by immersing the filter in 0.5 n H2SO4. Inorganic phosphate (Pi) was removed by precipitating it as a phosphomolybdate-triethylamine complex and the PPi was measured as a green pyrophosphomolybdate in the presence of 2-mercaptoethanol. Nucleotides and phosphate esters do not react. PPi can be accurately assayed even when there is a 104-fold excess of Pi. Trimetaphosphate, tripolyphosphate, and tetrapolyphosphate also give this green color, but the rate of the color formation is 50 times slower than that with PPi. Thus this interference of the polyphosphates can be eliminated or the polyphosphates can be assayed simultaneously with the PPi in the same sample. 相似文献
8.
Enzyme activities conceivably involved in the activation of sulfate were studied with Desulfotomaculum ruminis, D. acetoxidans, D. nigrificans, D. orientis, and Desulfovibrio vulgaris. Cell lysates of these species revealed activities of at least 8 nkat/mg protein (i.e., 480 nmol per min and mg protein) of ATP sulfurylase, acetate kinase, phosphotransacetylase and adenylate kinase. ADP sulfurylase was not detected. Pyrophosphatase activity was high (73 to 97 nkat/mg protein) in Desulfotomaculum orientis and Desulfovibrio vulgaris. In these strains pyrophosphatase was activated by addition of a reductant (dithionite). In Desulfotomaculum ruminis, D. acetoxidans, and D. nigrificans, only low pyrophosphatase activity (2.5 to 6.3 nkat/mg protein) was measured, which was not reductant-activated. Some hints indicated a membrane association of the pyrophosphatase in D. ruminis, and possibly also in D. acetoxidans and D. nigrificans. Activities of a pyrophosphate-dependent acetate kinase (PPi:acetate kinase), a PPi:AMP kinase or a polyphosphate:AMP kinase were not detected or negligible. The results are not in favour of the assumption that pyrophosphate formed by ATP sulfurylase during sulfate activation might be utilized to form acetyl phosphate in Desulfotomaculum species. Contrary results of other authors were shown to be artefacts caused by chemical hydrolysis of acetyl phosphate in the molybdate-sulfuric acid reagent used for phosphate determination.Abbreviations Pi
orthophosphate
- PPi
pyrophosphate
- APS
adenosine phosphosulfate
- AP5A, P1
P5-di(adenosine-5-)pentaphosphate
- CTAB
cetyltrimethylammonium bromide
- MOPS
3-(N-morpholino)propanesulfonic acid
- HEPES
N(-2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid 相似文献
9.
A new method for the rapid analysis of inorganic pyrophosphate (PPi) which utilizes the enzyme ATP sulfurylase is described. All components of the assay system are commercially available and inexpensive. The assay is linear over the range of 0.5–50.0 nmol of PPi and is not affected by inorganic phosphate. ATP and PPi can both be analyzed using this method. 相似文献
10.
Flux into the glycolytic pathway of most cells is controlled via allosteric regulation of the irreversible, committing step catalyzed by ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC 2.7.1.11), the key enzyme of glycolysis. In some organisms, the step is catalyzed by PPi-dependent PFK (PPi-PFK; EC 2.7.1.90), which uses PPi instead of ATP as the phosphoryl donor, conserving ATP and rendering the reaction reversible under physiological conditions. We have determined the enzymic properties of PPi-PFK from the anaerobic, hyperthermophilic archaeon Thermoproteus tenax, purified the enzyme to homogeneity, and sequenced the gene. The ∼100-kDa PPi-PFK from T. tenax consists of 37-kDa subunits; is not regulated by classical effectors of ATP-PFKs such as ATP, ADP, fructose 2,6-bisphosphate, or metabolic intermediates; and shares 20 to 50% sequence identity with known PFK enzymes. Phylogenetic analyses of biochemically characterized PFKs grouped the enzymes into three monophyletic clusters: PFK group I represents only classical ATP-PFKs from Bacteria and Eucarya; PFK group II contains only PPi-PFKs from the genus Propionibacterium, plants, and amitochondriate protists; whereas group III consists of PFKs with either cosubstrate specificity, i.e., the PPi-dependent enzymes from T. tenax and Amycolatopsis methanolica and the ATP-PFK from Streptomyces coelicolor. Comparative analyses of the pattern of conserved active-site residues strongly suggest that the group III PFKs originally bound PPi as a cosubstrate.As first discovered in Entamoeba histolytica (27), in some members of all three domains of life (Bacteria, Eucarya, and Archaea), the first committing step of glycolysis, the phosphorylation of fructose 6-phosphate (Fru 6-P), is catalyzed not by common ATP-dependent phosphofructokinase (PFK) (ATP-PFK; EC 2.7.1.11) but by an enzyme which uses PPi as a phosphoryl donor (PPi-PFK; EC 2.7.1.90) (22–34). The only archaeal PPi-PFK described so far is the enzyme of Thermoproteus tenax, a hyperthermophilic, anaerobic archaeon which is able to grow chemolithotrophically with CO2, H2, and S0, as well as chemo-organothrophically in the presence of S0 and carbohydrates (11, 41). As shown by enzymatic and in vivo studies (pulse-labeling experiments), T. tenax metabolizes glucose mainly via a variation of the Embden-Meyerhof-Parnas pathway distinguished by the reversible PPi-PFK reaction (34, 35).In contrast to the virtually irreversible reaction catalyzed by the ATP-PFK, the phosphorylation by PPi is reversible. Thus, for thermodynamic reasons, the PPi-PFK should be able to replace the enzymes of both the forward (ATP-PFK) and reverse (fructose-bisphosphatase [FBPase]) reactions. Consistent with the presumed bivalent function of the PPi-dependent enzyme, in prokaryotes and parasitic protists which possess PPi-PFK, little, if any, ATP-PFK or FBPase activity is present. Strikingly, the PPi-PFKs of these organisms proved to be nonallosteric, suggesting that the control of the carbon flux through the pathway is no longer exerted by the PFK in these organisms. A considerably different situation has been described for higher plants and the green alga Euglena gracilis, showing comparable ATP-PFK, FBPase, and PPi-PFK activities and allosteric regulation of their PPi-dependent enzyme by fructose 2,6-bisphosphate (12, 22). However, in most cases it is not obvious which physiological role PPi-PFK performs: reversible catalysis of glycolysis/gluconeogenesis, increase of the energy yield of glycolysis under certain conditions in which the energy charge is low, or ATP-conservation in obligately fermentative organisms (22).Closely related to questions concerning the biological function of PPi-PFKs is the matter of their evolutionary origin: are these enzymes the result of a secondary adaptation from ATP-PFKs, or do they represent an original phenotype, as suggested by their specificity for PPi, which is thought to be an ancient source of metabolic energy (9, 18, 19, 26). Indicated by sequence similarity (3, 4), all known PPi- and ATP-PFKs are homologous and therefore originated from a common ancestral root. From more recent studies of Streptomyces coelicolor PFK (4), the previous assumption of a single event which separated PPi- and ATP-PFKs had to be revised in favor of a multiple differentiation, leaving open, however, the question of the primary or secondary origin of PPi-PFK.Understanding of PFK evolution has been impaired by a lack of knowledge concerning archaeal PFK, although the existence of ATP-PFK (31), PPi-PFK (34), and also ADP-dependent PFK (16, 31) in Archaea has been described. To address the evolution of PFK, we describe the PPi-PFK from T. tenax and compare its sequence and structure to those of known bacterial and eucaryal PFK enzymes. 相似文献
11.
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. 相似文献
12.
Acetate kinase catalyzes the reversible magnesium-dependent phosphoryl transfer from ATP to acetate to form acetyl phosphate and ADP. Here, we report functional and some structural properties of cold-adapted psychrotrophic enzyme; acetate kinase with those from mesophilic counterpart in Escherichia coli K-12. Recombinant acetate kinase from Shewanella sp. AS-11 (SAK) and E. coli K-12 (EAK) were purified to homogeneity following affinity chromatography and followed by Super Q column chromatography as reported before [44]. Both purified enzymes are shared some of the common properties such as (similar molecular mass, amino acid sequence and similar optimum pH), but characterized shift in the apparent optimum temperature of specific activity to lower temperature as well as by a lower thermal stability compared with EAK. The functional comparisons reveal that SAK is a cold adapted enzyme, having a higher affinity to acetate than EAK. In the acetyl phosphate and ADP-forming direction, the catalytic efficiency (k cat/K m) for acetate was 8.0 times higher for SAK than EAK at 10 °C. The activity ratio of SAK to EAK was increased with decreasing temperature in both of the forward and backward reactions. Furthermore, the activation energy, enthalpy and entropy in both reaction directions that catalyzed by SAK were lower than those catalyzed by EAK. The model structure of SAK showed the significantly reduced numbers of salt bridges and cation-pi interactions as compared with EAK. These results suggest that weakening of intramolecular electrostatic interactions of SAK is involved in a more flexible structure which is likely to be responsible for its cold adaptation. 相似文献
13.
Diel vertical migration by Heterosigma akashiwo (Hada) Hada (Raphidophyceae) was monitored in a 1.5 in tall microcosm. Vertical stratification, with low salinity and low orthophosphate (Pi) concentration in the upper layer and high salinity and high Pi concentration in the lower layer, was simulated in the tank, analogous to summer stratification in the Seto Inland Sea. The phosphate metabolism of H. akashiwo during this vertical migration was studied using 31P-NMR spectroscopy. At night this species migrated to the lower phosphate-rich layer and took up inorganic phosphate (Pi) which then was accumulated as polyphosphate (PPi) by an increase in the chain length of PPi During the daytime this species migrated to the phosphate-depleted surface water and utilized the accumulated PPi for photophosphorylation by decreasing the chain length of PPi During the first night after the phosphorus was introduced to the previously impoverished waters, the cells took up inorganic phosphate, accumulating the new phosphorus nutrient internally as Pi But the cells did not convert Pi to PPi presumably due to their lack of ATP. After the second day of the experiment, conversion of Pi to PPi at night was much more rapid than on the first day, presumably due to increased ATP availability. Then the cycle continued, with uptake of Pi and conversion to PPi at night at the bottom and its utilization during the day at the surface. These data suggest that the role of PPi in the metabolism of this species appears to be as a phosphate pool which regulates the level of Pi and ATP in the cell. Diel vertical migration allows this red tide species to shuttle between the phosphate-rich lower layer and the photic upper layer in stratified waters. 31P-NMR is shown to be a valuable tool in studying the phosphorus metabolism in migrating organisms. 相似文献
14.
Yoko Chiba ) Ryoma Kamikawa ) Kumiko Nakada-Tsukui ) Yumiko Saito-Nakano ) Tomoyoshi Nozaki ) 《The Journal of biological chemistry》2015,290(39):23960-23970
Phosphoenolpyruvate carboxykinase (PEPCK) is one of the pivotal enzymes that regulates the carbon flow of the central metabolism by fixing CO2 to phosphoenolpyruvate (PEP) to produce oxaloacetate or vice versa. Whereas ATP- and GTP-type PEPCKs have been well studied, and their protein identities are established, inorganic pyrophosphate (PPi)-type PEPCK (PPi-PEPCK) is poorly characterized. Despite extensive enzymological studies, its protein identity and encoding gene remain unknown. In this study, PPi-PEPCK has been identified for the first time from a eukaryotic human parasite, Entamoeba histolytica, by conventional purification and mass spectrometric identification of the native enzyme, followed by demonstration of its enzymatic activity. A homolog of the amebic PPi-PEPCK from an anaerobic bacterium Propionibacterium freudenreichii subsp. shermanii also exhibited PPi-PEPCK activity. The primary structure of PPi-PEPCK has no similarity to the functional homologs ATP/GTP-PEPCKs and PEP carboxylase, strongly suggesting that PPi-PEPCK arose independently from the other functional homologues and very likely has unique catalytic sites. PPi-PEPCK homologs were found in a variety of bacteria and some eukaryotes but not in archaea. The molecular identification of this long forgotten enzyme shows us the diversity and functional redundancy of enzymes involved in the central metabolism and can help us to understand the central metabolism more deeply. 相似文献
15.
The covalent derivative of the tryptophanyl-tRNA synthetase obtained under the action of32PPi contains one mole of the covalently bound pyrophosphate (or 2 moles of orthophosphate) per mole of dimeric enzyme. Dephosphorylation with alkaline phosphatase causes practically no changes of enzymatic activity although the enzyme looses its ability to bind PPi.Enzymes
tryptophanyl-tRNA synthetase (EC 6.1.1.2), alkaline phosphatase (EC 3.1.3.1), inorganic pyrophosphatase (EC 3.6.1.1) 相似文献
16.
The growth of an anaerobic, spore-forming rod we have isolated from the cockroach gut after enrichment on media containing PPi was stimulated by the presence of PPi. The doubling time decreased and cell yield increased proportionately to PPi concentrations of up to 0.35%. A similar stimulation of the growth of Desulfotomaculum sp. by PPi has been reported. The PPi-stimulated Clostridium sp. fermented a number of sugars with the production of hydrogen, acetate, and butyrate, with smaller amounts of ethanol and butanol being produced from some substrates. The fermentation products were not qualitatively changed by the presence of PPi, but significantly more hydrogen was produced. The organism contained several of the enzymes previously reported from Entamoeba sp. and Propionibacterium sp., in which PPi serves as a source of a high-energy bond in place of ATP. These include significant amounts of pyruvate-phosphate dikinase and phosphoenolpyruvate carboxytransphosphorylase. The activities of many of the catabolic enzymes of the organism, as well as of its phosphatases and pyrophosphatase, were similar whether it was grown in the presence or absence of PPi. The organism did not accumulate intracellular polyphosphate granules but stored large amounts of glycogen. 相似文献
17.
Inactivation of Escherichia coli acetate kinase by N-ethylmaleimide. Protection by substrates and products 总被引:2,自引:0,他引:2
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. 相似文献
18.
《BBA》1986,851(2):276-282
Photosynthetic formation of inorganic pyrophosphate (PPi) in Rhodospirillum rubrum chromatophores has been studied utilizing a new and sensitive method for continuous monitoring of PPi synthesis. Studies of the reaction kinetics under a variety of conditions, e.g., at different substrate concentrations and different electron-transport rates, have been performed. At very low light intensities the rate of PPi synthesis is twice the rate of ATP synthesis. Antimycin A, at a concentration which strongly inhibited the photosynthetic ATP formation, inhibited the PPi synthesis much less. Even at low rates of electron transport a significant rate of PPi synthesis is obtained. The rate of photosynthetic ATP formation is stimulated up to 20% when PPi synthesis is inhibited. It is shown that PPi synthesis and ATP synthesis compete with each other. No inhibition of pyrophosphatase activity is observed at high carbonyl cyanide p-trifluoromethoxyhydrazone concentration while ATPase activity is strongly inhibited under the same conditions. 相似文献
19.
G E Edwards S P Robinson N J Tyler D A Walker 《Archives of biochemistry and biophysics》1978,190(2):421-433
In studying conditions for obtaining photosynthetically functional chloroplasts from mesophyll protoplasts of sunflower and wheat, a strong requirement for chelation was found. The concentration of chelator, either EDTA or pyrophosphate (PPi), required for maximum activation depended on the pH, the concentration of orthophosphate (Pi) in the assay, and the chelator used. Studies with EDTA indicate that including the chelator in the isolation, resuspension, and assay media, in the absence of divalent cations, was most effective. Increased concentration of EDTA from 1 to 10 mm broadened the pH response curve for photosynthesis, inasmuch as a higher concentration of chelator was required for activation of photosynthesis at lower pH.Either EDTA, PPi, or citrate could activate photosynthesis of sunflower chloroplasts isolated and assayed at pH 8.4. At pH 7.6, PPi and EDTA were equally effective at low Pi concentrations but PPi was particularly effective in shortening the induction period at high concentrations of Pi (2.5 mm) in the assay medium. Including 1 mm 3-phosphoglycerate in the assay medium with or without Pi could not replace the need for chelation. However, 3-phosphoglycerate + EDTA in the assay medium with 0.5 mm Pi, pH 7.6, gave a short induction period and rates of photosynthesis similar to those with 10 mm PPi. The results suggest that PPi can have a dual effect at the lower pH through chelation and inhibition of the phosphate transporter.Photosynthesis by sunflower chloroplasts isolated and assayed at pH 8.4 with 0.2 mm EDTA (+ 0.5 mm Pi in the assays) was severely inhibited by 2 mM CaCl2, MgCl2, or MnCl2. Wheat chloroplasts isolated and assayed at pH 8.4 without chelation, and assayed with 0.2 mm Pi, had low rates of photosynthesis (25 μmol O2 evolved mg?1 chlorophyll h?1) which were strongly inhibited by 2 to 4 mm MgCl2, MnCl2, or CaCl2. With inclusion of EDTA and Pi at optimum levels, isolated chloroplasts of sunflower and wheat have high rates of photosynthesis and PPi or divalent cations are not of benefit. 相似文献
20.
Shoji Shirota Shiro Watanabe Isao Takeda 《Bioscience, biotechnology, and biochemistry》2013,77(3):325-332
It was found that CDP-choline was formed with good yield from 5′-CMP and choline phosphate or choline chloride by yeast cells. The effects of pyrophosphate (PPi) on the formation of UDPG, GDPM and CDP-choline from respective nucleoside monophosphate by yeast cells were studied. By the addition of PPi to the reaction mixture, the phosphorylation of G-6-P from glucose was inhibited and then the phosphorylation of nucleoside monophosphates was restrained. Such inhibition was reversed by the decomposition of PPi by inorganic pyrophosphatase of yeast cells. The addition of PPi after the formation of nucleotide derivatives caused the accumulation of UTP and GTP and molar yields from nucleotide as substrate was about 80%. But that of CTP was a little in the reaction system of CDP-choline synthesis. Further, this method seems to be suitable for the accumulation of sugar-1-phosphates. 相似文献