SODYUM ASETAT PROFOSFAT (SODIUM ACETATE PYROPHOSPHATE)

SODYUM ASETAT PROFOSFAT (SODIUM ACETATE PYROPHOSPHATE)

SODIUM ACID PYROPHOSPHATE

SYNONYMS: Technetium (99mTc) pyrophosphate; Technetium TC 99M Pyrophosphate; soda lime; sod disease; sodium; sodium acetate; sodium acid carbonate; sodium acid phosphate; sodium acid pyrophosphate; Disodium pyrophosphate; Disodium dihydrogen diphosphate Other names; Diphosphoric acid, disodium salt; Disodium dihydrogen pyrophosphate; Disodium diphosphate; Sodium acid pyrophosphate, SAPP; TSPP;pyro;Nsc56751;phosphotex;victortspp;SODIUM DI;pyrophosphate;SODIUM DIPHOSPHATE;natriumpyrophosphat;SODIUM PYROPHOSPHAYE; Sodium phosphate pyro, sodium pyrophosphate decahydrate, E450, sodium pyrophosphate, sodium diphosphate; sodium tert-pentoxide, sodium 2-methylbutan-2-olate, sodium tert-amylate, sodium t-amylate, sodium-t-amylate, unii-51z39py5z8, sodium-tert-pentoxide, sodium tert-; monosodium phosphate, sodium dihydrogen phosphate, sodium dihydrogenorthophosphate, sodium phosphate monobasic, sodium acid phosphate, sodium phosphate,NIRAN Thailand; Citric Acid; Monosodium Citrate; Trisodium Citrate; Other citrates; Flavors and Food Ingredients; Aspartame; Disodium Ribotide; Gibberellic Acid; L Cysteine Hydrochloride Monohydrate; Malic Acid; Methyl Paraben; Monosodium Glutamate; Potassium Sorbate; Propyl Paraben; Sodium Benzoate; Sodium Erythorbate; Sodium Saccharin; Sodium Tripolyphosphate; Sorbic Acid; Titanium Dioxide; Tricalcium Phosphate; Trisodium Phosphate; Xanthan Gum; Xylitol; Xylitol DC; Vitamins and Amino Acids; Ascorbic Acid; Calcium Ascorbate; Sodium Ascorbate; Vitamin B; Vitamin E Oil

 

 

From Wikipedia, the free encyclopedia Jump to navigationJump to search Disodium pyrophosphate Disodium pyrophosphate Names IUPAC name Disodium dihydrogen diphosphate Other names Diphosphoric acid, disodium salt Disodium dihydrogen pyrophosphate Disodium diphosphate Sodium acid pyrophosphate, SAPP Identifiers CAS Number 7758-16-9 ? 3D model (JSmol) Interactive image ChemSpider 22859 ? ECHA InfoCard 100.028.941 EC Number 231-835-0 E number E450(i) (thickeners, …) PubChem CID 24451 UNII H5WVD9LZUD ? InChI SMILES Properties Chemical formula Na2H2P2O7 Molar mass 221.94 g/mol Appearance White odorless powder Density 2.31 g/cm3 Melting point >600 °C Solubility in water 11.9 g/100 mL (20 °C) Refractive index (nD) 1.4645 (hexahydrate) Hazards Flash point Non-flammable Lethal dose or concentration (LD, LC): LD50 (median dose) 2650 mg/kg (mouse, oral) Related compounds Other anions Disodium phosphate Pentasodium triphosphate

Sodium hexametaphosphate Other cations Calcium pyrophosphate

 

SODIUM ACETATE PYROPHOSPHATE

Dipotassium pyrophosphate Related compounds Tetrasodium pyrophosphate Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa). ? verify (what is ?? ?) Infobox references Disodium pyrophosphate or sodium acid pyrophosphate (SAPP)[1] is an inorganic compound consisting of sodium cations and pyrophosphate anion. It is a white, water-soluble solid that serves as a buffering and chelating agent, with many applications in the food industry. When crystallised from water, it forms a hexahydrate, but it dehydrates above room temperature. Pyrophosphate is a polyvalent anion with a high affinity for polyvalent cations, e.g. Ca2+. Disodium pyrophosphate is produced by heating sodium dihydrogen phosphate: 2 NaH2PO4 › Na2H2P2O7 + H2;Contents 1 Food use 2 Other uses 3 References 4 External links Food uses Disodium pyrophosphate is a popular leavening agent found in baking powders. It combines with sodium bicarbonate to release carbon dioxide: Na2H2P2O7 + NaHCO3 › Na3HP2O7 + CO2 + H2O It is available in a variety of grades that affect the speed of its action. Because the resulting phosphate residue has an off-taste, SAPP is usually used in very sweet cakes which mask the off-taste.[2] Disodium pyrophosphate and other sodium and potassium polyphosphates are widely used in food processing; in the E number scheme, they are collectively designated as E450, with the disodium form designated as E450(a). In the United States, it is classified as generally recognized as safe (GRAS) for food use. In canned seafood, it is used to maintain color and reduce purge[clarification needed] during retorting. Retorting achieves microbial stability with heat.[3] It is an acid source for reaction with baking soda to leaven baked goods.[4] In baking powder, it is often labeled as food additive E450.[5] In cured meats, it speeds the conversion of sodium nitrite to nitrite (NO2-) by forming the nitrous acid (HONO) intermediate,[clarification needed] and can improve water-holding capacity. Disodium pyrophosphate is also found in frozen hash browns and other potato products, where it is used to keep the color of the potatoes from darkening.[4] Disodium pyrophosphate can leave a slightly bitter aftertaste in some products, but “the SAPP taste can be masked by using sufficient baking soda and by adding a source of calcium ions, sugar, or flavorings.”[1] Other uses In leather treatment, it can be used to remove iron stains on hides during processing. It can stabilize hydrogen peroxide solutions against reduction. It can be used with sulfamic acid in some dairy applications for cleaning, especially to remove soapstone. When added to scalding water, it facilitates removal of hair and scurf in hog slaughter and feathers and scurf in poultry slaughter. In petroleum production, it can be used as a dispersant in oil well drilling muds.[citation needed] It is used in cat foods as a palatability additive.[6] Disodium pyrophosphate is used as a tartar control agent in toothpastes. References “Lallemand Baking Update: Chemical Leaveners Volume 1 / Number 12” (PDF). www.lallemand.com. Lallemand Inc. 1996. Retrieved 6 January 2018. John Brodie, John Godber “Bakery Processes, Chemical Leavening Agents” in Kirk-Othmer Encyclopedia of Chemical Technology 2001, John Wiley & Sons. doi:10.1002/0471238961.0308051303082114.a01.pub2 [1] -Retorting, Accessed 2010-11-27 Ellinger, R.H. (1972). “Phosphates in Food Processing”. Handbook of Food Additives (2nd ed.). Cleveland: CRC Press. pp. 617-780. [2] Roach, Mary (2013-03-25). “The Chemistry of Kibble”. Popular Science. Retrieved 2016-02-16. Pyrophosphates have been described to me as “cat crack.” Coat some kibble with it, and the pet food manufacturer can make up for a whole host of gustatory shortcomings. Effects of the food additives sodium acid pyrophosphate, sodium acetate, and citric acid on hemato-immunological pathological biomarkers in rats: Relation to PPAR-?, PPAR-? and tnf? signaling pathway. Abd-Elhakim YM1, Hashem MM2, Anwar A3, El-Metwally AE4, Abo-El-Sooud K2, Moustafa GG5, Mouneir SM2, Ali HA6 Author information

 

 

Abstract

The food additives sodium acid pyrophosphate (SAPP), sodium acetate (SA), and citric acid (CA) were evaluated for their hemato-immunotoxic effects. Forty adult Sprague-Dawley rats were distributed into four groups and were orally administered water, SAPP (12.6?mg/kg), CA (180?mg/kg), or SA (13.5?mg /kg) daily for 90 days. Erythrogram and leukogram profiles were evaluated. The levels of lysozyme, nitric oxide, immunoglobulin, and phagocytic activity were measured. Histologic and immunohistochemical evaluations of splenic tissues were performed. Changes in the mRNA expression levels of peroxisome proliferator-activated receptor ? and ? (PPAR-? and PPAR-?), and tumor necrosis factor ? (TNF-?) genes were assessed. A significant leukopenic condition was observed with SAPP, while CA induced marked leukocytosis, and SA showed a lymphocytosis condition. Both the innate and humoral parameters were significantly depressed. Various pathological lesions were observed, including diffuse hyperplasia of the red pulp, depletion of the white pulp, and capsular and parenchymal fibrosis. A marked decrease in CD3 T-lymphocyte and CD20 B-lymphocyte immunolabeling in rats treated with SAPP and SA was evident. Marked downregulation of PPAR-? and PPAR-? together with upregulation of TNF-? was recorded. These results indicate that high doses of SAPP, SA and CA exert hematotoxic and immunotoxic effects with long-term exposure.

Copyright © 2018 Elsevier B.V. All rights reserved.

 

 

 

KEYWORDS:

Citric acid; Food additives; Immune function; Sodium acetate; Sodium acid pyrophosphate PMID: 29986283 DOI: 10.1016/j.etap.2018.07.002 [Indexed for MEDLINE] Share on FacebookShare on TwitterShare on Google+ Sodium Acetate, Sodium Acid Pyrophosphate, and Citric Acid Impacts on Isolated Peripheral Lymphocyte Viability, Proliferation, and DNA Damage. Abd-Elhakim YM1, Anwar A2, Hashem MM3, Moustafa GG1, Abo-El-Sooud K3. Author information Abstract The present study examined the impacts of sodium acetate (SA), sodium acid pyrophosphate (SAPP), and citric acid (CA) on the viability, proliferation, and DNA damage of isolated lymphocytes in vitro. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays were adopted to evaluate cell viability, while comet assay was employed to assess the genotoxic effects. The cells were incubated with different levels of SA (50, 100, and 200 mM), SAPP (25, 50, and 100 mM/L), or CA (100, 200, and 300 µg/mL). The lymphocytes treated with the tested food additives showed concentration-dependent decreases in both cell viability and proliferation. A concentration-dependent increase in LDH release was also observed. The comet assay results indicated that SA, SAPP, and CA increased DNA damage percentage, tail DNA percentage, tail length, and tail moment in a concentration-dependent manner. The current results showed that SA, SAPP, and CA are cytotoxic and genotoxic to isolated lymphocytes in vitro. © 2018 Wiley Periodicals, Inc. Effects of the food additives sodium acid pyrophosphate, sodium acetate, and citric acid on hemato-immunological pathological biomarkers in rats: Relation to PPAR-?, PPAR-? and tnf? signaling pathway Author links open overlay panelYasmina M.Abd-ElhakimaMohamed M.HashembAbeerAnwarcAbeer E.El-MetwallydKhaledAbo-El-SooudbGihan G.MoustafaaSamar M.MouneirbHaytham A.Alief Show more https://doi.org/10.1016/j.etap.2018.07.002Get rights and content Highlights Hemato-mmunotoxic effect of sodium acid pyrophosphate, sodium acetate, and citric acid was studied. The innate and humoral parameters were significantly depressed. A marked decrease in CD3 T-lymphocyte and CD20 B-lymphocyte immunolabeling was noted. A significant downregulation of PPAR-? and PPAR-? and upregulation of TNF-? was recorded.

 

 

 

Abstract

The food additives sodium acid pyrophosphate (SAPP), sodium acetate (SA), and citric acid (CA) were evaluated for their hemato-immunotoxic effects. Forty adult Sprague-Dawley rats were distributed into four groups and were orally administered water, SAPP (12.6?mg/kg), CA (180?mg/kg), or SA (13.5?mg /kg) daily for 90 days. Erythrogram and leukogram profiles were evaluated. The levels of lysozyme, nitric oxide, immunoglobulin, and phagocytic activity were measured. Histologic and immunohistochemical evaluations of splenic tissues were performed. Changes in the mRNA expression levels of peroxisome proliferator-activated receptor ? and ? (PPAR-? and PPAR-?), and tumor necrosis factor ? (TNF-?) genes were assessed. A significant leukopenic condition was observed with SAPP, while CA induced marked leukocytosis, and SA showed a lymphocytosis condition. Both the innate and humoral parameters were significantly depressed. Various pathological lesions were observed, including diffuse hyperplasia of the red pulp, depletion of the white pulp, and capsular and parenchymal fibrosis. A marked decrease in CD3 T-lymphocyte and CD20 B-lymphocyte immunolabeling in rats treated with SAPP and SA was evident. Marked downregulation of PPAR-? and PPAR-? together with upregulation of TNF-? was recorded. These results indicate that high doses of SAPP, SA and CA exert hematotoxic and immunotoxic effects with long-term exposure. Previous article in issueNext article in issue Keywords Food additivesSodium acid pyrophosphateSodium acetateCitric acidImmune function Disodium pyrophosphate From Wikipedia, the free encyclopedia Jump to navigationJump to search Disodium pyrophosphate Disodium pyrophosphate Names IUPAC name Disodium dihydrogen diphosphate Other names Diphosphoric acid, disodium salt; Disodium dihydrogen pyrophosphate; Disodium diphosphate; Sodium acid pyrophosphate, SAPP Identifiers CAS Number 7758-16-9 ? 3D model (JSmol) Interactive image ChemSpider 22859 ? ECHA InfoCard 100.028.941 EC Number 231-835-0 E number E450(i) (thickeners, …) PubChem CID 24451 UNII H5WVD9LZUD ? InChI SMILES Properties Chemical formula Na2H2P2O7 Molar mass 221.94 g/mol Appearance White odorless powder Density 2.31 g/cm3 Melting point >600 °C Solubility in water 11.9 g/100 mL (20 °C) Refractive index (nD) 1.4645 (hexahydrate) Hazards Flash point Non-flammable Lethal dose or concentration (LD, LC): LD50 (median dose) 2650 mg/kg (mouse, oral) Related compounds Other anions Disodium phosphate Pentasodium triphosphate Sodium hexametaphosphate Other cations Calcium pyrophosphate Dipotassium pyrophosphate Related compounds Tetrasodium pyrophosphate Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa). ? verify (what is ?? ?) Infobox references Disodium pyrophosphate or sodium acid pyrophosphate (SAPP)[1] is an inorganic compound consisting of sodium cations and pyrophosphate anion. It is a white, water-soluble solid that serves as a buffering and chelating agent, with many applications in the food industry. When crystallised from water, it forms a hexahydrate, but it dehydrates above room temperature. Pyrophosphate is a polyvalent anion with a high affinity for polyvalent cations, e.g. Ca2+. Disodium pyrophosphate is produced by heating sodium dihydrogen phosphate:

 

 

 

2 NaH2PO4 › Na2H2P2O7 + H2O Contents 1 Food uses 2 Other uses 3 References 4 External links Food uses Disodium pyrophosphate is a popular leavening agent found in baking powders. It combines with sodium bicarbonate to release carbon dioxide: Na2H2P2O7 + NaHCO3 › Na3HP2O7 + CO2 + H2O It is available in a variety of grades that affect the speed of its action. Because the resulting phosphate residue has an off-taste, SAPP is usually used in very sweet cakes which mask the off-taste.[2] Disodium pyrophosphate and other sodium and potassium polyphosphates are widely used in food processing; in the E number scheme, they are collectively designated as E450, with the disodium form designated as E450(a). In the United States, it is classified as generally recognized as safe (GRAS) for food use. In canned seafood, it is used to maintain color and reduce purge[clarification needed] during retorting. Retorting achieves microbial stability with heat.[3] It is an acid source for reaction with baking soda to leaven baked goods.[4] In baking powder, it is often labeled as food additive E450.[5] In cured meats, it speeds the conversion of sodium nitrite to nitrite (NO2-) by forming the nitrous acid (HONO) intermediate,[clarification needed] and can improve water-holding capacity. Disodium pyrophosphate is also found in frozen hash browns and other potato products, where it is used to keep the color of the potatoes from darkening.[4] Disodium pyrophosphate can leave a slightly bitter aftertaste in some products, but “the SAPP taste can be masked by using sufficient baking soda and by adding a source of calcium ions, sugar, or flavorings.”[1]

Other uses

In leather treatment, it can be used to remove iron stains on hides during processing. It can stabilize hydrogen peroxide solutions against reduction. It can be used with sulfamic acid in some dairy applications for cleaning, especially to remove soapstone. When added to scalding water, it facilitates removal of hair and scurf in hog slaughter and feathers and scurf in poultry slaughter. In petroleum production, it can be used as a dispersant in oil well drilling muds.[citation needed] It is used in cat foods as a palatability additive.[6] Disodium pyrophosphate is used as a tartar control agent in toothpastes.

References

“Lallemand Baking Update: Chemical Leaveners Volume 1 / Number 12” (PDF). www.lallemand.com. Lallemand Inc. 1996. Retrieved 6 January 2018. John Brodie, John Godber “Bakery Processes, Chemical Leavening Agents” in Kirk-Othmer Encyclopedia of Chemical Technology 2001, John Wiley & Sons. doi:10.1002/0471238961.0308051303082114.a01.pub2 [1] -Retorting, Accessed 2010-11-27 Ellinger, R.H. (1972). “Phosphates in Food Processing”. Handbook of Food Additives (2nd ed.). Cleveland: CRC Press. pp. 617-780. [2] Roach, Mary (2013-03-25). “The Chemistry of Kibble”. Popular Science. Retrieved 2016-02-16. Pyrophosphates have been described to me as “cat crack.” Coat some kibble with it, and the pet food manufacturer can make up for a whole host of gustatory shortcomings. Sodium Acetate, Sodium Acid Pyrophosphate, and Citric Acid Impacts on Isolated Peripheral Lymphocyte Viability, Proliferation, and DNA Damage.

Yasmina Mohammed Abd-Elhakim, Abeer Anwar, +2 authors Khaled Abo-El-SooudPublished in Journal of biochemical and molecular toxicology 2018 DOI:10.1002/jbt.22171 The present study examined the impacts of sodium acetate (SA), sodium acid pyrophosphate (SAPP), and citric acid (CA) on the viability, proliferation, and DNA damage of isolated lymphocytes in vitro. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays were adopted to evaluate cell viability, while comet assay was employed to assess the genotoxic effects. The cells were incubated with different levels of SA (50, 100, and 200 mM), SAPP (25, 50, and 100 mM/L), or CA (100, 200, and 300 µg/mL). The lymphocytes treated with the tested food additives showed concentration-dependent decreases in both cell viability and proliferation. A concentration-dependent increase in LDH release was also observed. The comet assay results indicated that SA, SAPP, and CA increased DNA damage percentage, tail DNA percentage, tail length, and tail moment in a concentration-dependent manner. The current results showed that SA, SAPP, and CA are cytotoxic and genotoxic to isolated lymphocytes in vitro. LESS Investigation of pyrophosphate versus ATP substrate selection in the Entamoeba histolytica acetate kinase Thanh Dang & Cheryl Ingram-Smith Scientific Reportsvolume 7, Article number: 5912 (2017) | Download Citation

 

 

 

Abstract

Acetate kinase (ACK; E.C. 2.7.2.1), which catalyzes the interconversion of acetate and acetyl phosphate, is nearly ubiquitous in bacteria but is present only in one genus of archaea and certain eukaryotic microbes. All ACKs utilize ATP/ADP as the phosphoryl donor/acceptor in the respective directions of the reaction (acetate?+?ATP?[Math Processing Error]?acetyl phosphate?+?ADP), with the exception of the Entamoeba histolytica ACK (EhACK) which uses pyrophosphate (PPi)/inorganic phosphate (Pi) (acetyl phosphate?+?Pi? [Math Processing Error]?acetate?+?PPi). Structural analysis and modeling of EhACK indicated steric hindrance by active site residues constricts entry to the adenosine pocket as compared to ATP-utilizing Methanosarcina thermophila ACK (MtACK). Reciprocal alterations were made to enlarge the adenosine pocket of EhACK and reduce that of MtACK. The EhACK variants showed a step-wise increase in ADP and ATP binding but were still unable to use these as substrates, and enzymatic activity with Pi/PPi was negatively impacted. Consistent with this, ATP utilization by MtACK variants was negatively affected but the alterations were not sufficient to convert this enzyme to Pi/PPi utilization. Our results suggest that controlling access to the adenosine pocket can contribute to substrate specificity but is not the sole determinant.

Introduction

Acetate kinase (ACK; E.C. 2.7.2.1) catalyzes the reversible transfer of phosphate from acetyl phosphate to a phosphoryl acceptor (S), yielding acetate and a phosphorylated product (S-P) [Eq. 1]. ACK, nearly ubiquitous in bacteria, has been identified in just a single genus of archaea, Methanosarcina. In recent years, ACK has also been identified in certain eukaryotic microbes including the green algae Chlamydomonas, euascomycete and basidiomycete fungi, and certain protists, namely Entamoeba histolytica 1.

[Math Processing Error] (1) This enzyme was discovered in 19442 and the first kinetic characterization was reported in 19543. In 2001, Buss et al. solved the structure for the Methanosarcina thermophila ACK (MtACK), and subsequent studies with this archaeal enzyme determined that ACK proceeds through a direct in-line mechanism of phosphoryl transfer4,5,6. Acyl substrate selection in ACK has been studied in the Methanosarcina enzyme. Four key residues, Val93, Leu122, Phe179, and Pro232, have been shown to form a hydrophobic pocket for acetate binding7 implicated in acyl substrate selection in this enzyme. In particular, Val93 appears to play an important role in limiting substrate length.

Ordinarily, ACK utilizes ATP/ADP as phosphoryl donor/acceptor; however, the E. histolytica enzyme is unusual in that it is PPi-dependent. Instead of using ATP/ADP as the phosphoryl donor/acceptor [Eq. 2], E. histolytica ACK (EhACK) can only use pyrophosphate (PPi)/inorganic phosphate (Pi) as the phosphoryl donor/acceptor [Eq. 3]8,9,10.

[Math Processing Error] (2) [Math Processing Error] (3) whereas most ATP-dependent ACKs function in both directions of the reaction, the E. histolytica enzyme strongly prefers the acetate-forming direction8,9,10. Double reciprocal plots of substrate concentration versus enzyme activity indicated EhACK follows a ternary-complex mechanism8,9,10, supporting a direct in-line mechanism of phosphoryl transfer as seen for MtACK4,5,6. Currently, EhACK is the only known ACK to utilize pyrophosphate or inorganic phosphate as a phosphoryl donor or acceptor instead of ATP.

ACK belongs to the ASKHA (acetate, sugar kinase, heat shock and actin) enzyme superfamily. In 1992, Bork et al. identified PHOSPHATE1, PHOSPHATE2 and ADENOSINE as three signature ATPase motifs shared by members of this superfamily11. These three conserved motifs form part of the adenosine binding pocket and are directly involved in ATP binding. Thaker et al. solved the EhACK structure and noted two amino acids substitutions in the ADENOSINE motif versus MtACK that may sterically hinder ATP binding12.

Here, we investigated the role of residues in the ADENOSINE and PHOSPHATE2 motifs in phosphoryl substrate selection and utilization in ACK. Our results indicated that the adenosine pocket and the ADENOSINE motif play a critical role in ATP binding. However, ATP binding alone did not lead to utilization. Thus, although EhACK shares strong similarities with ATP-dependent ACKs, subtle differences have dramatically shaped its identity and function.

Results Structures have been solved for six bacterial (four of which are from Mycobacterium), one archaeal, and two eukaryotic ACKs4, 12,13,14. Although the global structures are similar, the percent identity and similarity between these ACK sequences showed that the eukaryotic ACKs are less related to the bacterial and archaeal enzymes (Supplemental Table S1). Previous phylogenetic analysis revealed that fungal ACKs belong to a distinct clade but the E. histolytica and other eukaryotic sequences group with the bacterial and archaeal ACKs1. Thus, the unique Pi/PPi-dependence of EhACK must be due to localized differences in the active site.

Structural differences between the adenosine binding pocket of PPi-and ATP-ACKs In addition to the PHOSPHATE1, PHOSPHATE2, and ADENOSINE sequence motifs, Ingram-Smith et al.15 defined two other regions designated as LOOP3 and LOOP4 that also influence ATP binding in ACK. Inspection of the active site in the MtACK structure showed that these regions surround the ATP binding site, with ADENOSINE forming a hydrophobic pocket for the adenosine moiety of ADP/ATP4, 7, 10, 12, 15. ConSurf analysis (http://consurf.tau.ac.il), which estimates the evolutionary conservation at each position based on phylogenetic and structural analysis, indicated that the central positions in ADENOSINE have the highest conservation level (Fig. 1). Positions 322-327 of EhACK are of particular note as this region of the ADENOSINE motif is strongly conserved in other ACKs but not EhACK.

Figure 1 figure1Partial alignment of ACK amino acid sequences. Sequences of ACKs for which the structure have been solved were aligned and ConSurf analysis was performed to examine sequence conservation. The PHOSPHATE2 and ADENOSINE motifs are shown. The full alignment is provided in the Supplemental Information (Supplemental Figure S2). Abbreviations and PDB accession numbers: Ehist, E. histolytica, PDB ID 4H0O; Methanosarcina thermophila, PDB ID 1TUY; Cneo, Cryptococcus neoformans, PDB ID 4H0P; Styph, Salmonella typhimurium, PDB ID 3SLC; Tmari, Thermotoga maritima, PDB ID 2IIR; Msmeg, Mycobacterium smegmatis, PDB ID 4IJN; Mavium, Mycobacterium avium, PDB ID 3P4I; Mpara, Mycobacterium paratuberculosis, PDB ID 3R9P; Mmari, Mycobacterium marinum PDB ID 4DQ8. 

Full size image ADP/ATP-utilizing ACKs have a highly conserved Gly residue and an adjacent Ile/Val within the ADENOSINE motif (positions 331 and 332 in MtACK). Inspection of the MtACK structure revealed a large opening to the adenosine binding pocket (Fig. 2A) that is also evident in the structures of other ATP-ACKs. Occlusion of the adenosine pocket is evident in the surface representation of EhACK (Fig. 2B). Thaker et al.12 postulated that Gln323Met324 within the ADENOSINE motif of EhACK may sterically prevent ADP/ATP binding.

Figure 2 figure2 The ACK adenosine binding pocket. (A) Surface representation of MtACK with bound ADP. (B) Surface representation of EhACK. The constricted opening to the adenosine pocket is circled. The position of bound ADP in MtACK was superimposed into the EhACK structure. (C) Superimposition of the adenosine pocket from MtACK (yellow) and EhACK (cyan) showing the positions of targeted residues. (D) Superimposed PHOSPHATE2 motifs from MtACK (yellow) and EhACK (cyan), showing the position of the additional Gly in the EhACK PHOSPHATE2 motif.

Full size image Role of the ADENOSINE motif in ATP/ADP versus PPi/Pi utilization To investigate the role of the ADENOSINE motif in determining substrate selection, EhACK variants were created that simulate the open adenosine pocket observed in ATP-dependent ACKs. Gln323 and Met324 were altered to Gly and Ile, respectively, to mimic the residues found at equivalent positions in MtACK. These positions were also both altered to Ala to minimize side chain intrusions into the opening of the adenosine pocket. The reverse alterations were made in MtACK, converting Gly331-Ile332 to Gln-Met, respectively, to determine the effect of closing the entry to the adenosine pocket. The EhACK and MtACK variants were purified (Supplemental Figure S1) and kinetic parameters were determined in both directions of the reaction (Table 1).

Table 1 Apparent kinetic parameters for wild-type and variant EhACKs and MtACKs. Full size table The Q323G-M324I and Q323A-M324A EhACK variants displayed similar K m values for acetyl phosphate and slightly decreased K m values for phosphate as the unaltered enzyme but the k cat values were decreased 8.3-fold for the Q323G-M324I variant and 19-fold for Q323A-M324A variant, resulting in ~7 and 11-fold reduced catalytic efficiency, respectively. In the direction of acetyl phosphate formation, these variants displayed slightly increased K m for acetate but no increase in K m for PPi and only mild decrease in k cat. No activity was observed with either variant using ATP or ADP as substrate in the respective direction of the reaction.

The G331Q-I332M alteration in MtACK resulted in substantial reductions in k cat (Table 1). In the acetate-forming direction, catalysis was reduced over 100-fold, and in the acetyl phosphate-forming direction, k cat was reduced ~50-fold. This alteration resulted in ~5-fold increase in K m for ADP and ATP, and a 15-fold increase in K m for acetate but no substantial change in the K m for acetyl phosphate. As for wild-type MtACK, no activity was observed with Pi or PPi as substrate in the respective directions of the reaction.

Additional structural elements may contribute to occlusion of the ATP/ADP binding pocket A salt bridge between Arg274 and Asp272 on LOOP4 of EhACK may cause further constriction of the adenosine pocket by positioning the Arg side chain in toward the pocket12. These two residues are conserved in MtACK but the side chain of Arg is positioned away from the adenosine pocket. These residues are not conserved in among all ACKs though and LOOP4 does not impinge upon the adenosine pocket. The PHOSPHATE2 motif, which interacts with the ß phosphate of ATP and was suggested to have a role in substrate positioning4, 11, 15, is longer in EhACK and protrudes farther into the active site than in the ATP-dependent enzymes (Fig. 2C). Sequence alignment and structural superposition of the PHOSPHATE 2 motif illustrate that this difference arises from addition of a single residue, Gly203 (Figs 1 and 2D).

To examine whether this salt bridge and the extended PHOSPHATE2 motif influence substrate selection, EhACK Q323G-M324I variants in which the salt bridge has been eliminated (D272A-R274A-Q323G-M324I) or in which the PHOSPHATE2 motif has been shortened (?G203- Q323G-M324I) were analyzed. The D272A-R274A-Q323G-M324I replacement decreased k cat in the acetate-forming direction by ~2,500-5,000 fold but had little effect on K m for either substrate (Table 1). This variant had no detectable activity in the acetyl phosphate-forming direction (Table 1). The ?G203- Q323G-M324I variant was inactive in either direction of the reaction, and thus the effect of the Gly203 deletion compounded onto the D272A-R274A-Q323G-M324I alteration was not examined. No enzymatic activity was observed with either of these variants using ATP or ADP as the substrate in place of PPi or Pi.

Inhibition of EhACK and MtACK by alternative phosphoryl donors and acceptors Since wild-type EhACK and MtACK cannot utilize ATP/ADP or PPi/Pi, respectively, as alternative phosphoryl donor/acceptor, inhibition assays were performed to determine whether these compounds can bind and inhibit activity even if they cannot be used productively as substrate. EhACK activity was measured in the favored acetate-forming direction in the presence or absence of 10?mM AMP, ADP, or ATP (Fig. 3A). Although AMP had no effect, both ADP and ATP were found to inhibit EhACK activity but to differing extents. The presence of ATP resulted in nearly 80% inhibition versus ~30% inhibition by ADP. The presence of Pi or PPi with AMP was not sufficient to mimic the effect of inhibition by ADP or ATP, respectively (data not shown). For MtACK, Pi had no effect on enzymatic activity. PPi inhibited the enzyme in both directions of the reaction to differing extents, producing ~70% inhibition in the acetate-forming direction and ~90% inhibition in the acetyl phosphate-forming direction (Fig. 3B and C).

Figure 3 figure3 Inhibition of EhACK and MtACK by alternative phosphoryl donors and acceptors. (A) Inhibition of wild-type EhACK in the presence of 10?mM AMP, ADP, or ATP in the acetate-forming direction of the reaction. (B) Inhibition of wild-type MtACK in the presence of 20?mM Pi or PPi in the acetate-forming direction. (C) Inhibition of wild-type MtACK in the presence of 20?mM Pi or PPi in the acetyl phosphate-forming direction. Significant difference between inhibitions compared to wild-type enzyme activity is tested using an unpaired Welch t-test with R. *p-value?Full size image The mode of inhibition of EhACK by ATP was determined by kinetic analysis using a matrix of reactions in which the ATP concentration was varied versus Pi concentration with the acetate concentration held constant. ATP was found to be a competitive inhibitor of EhACK, as demonstrated by the results in Fig. 4. Further examination of ATP inhibition of the EhACK variants in the favored acetate-forming direction of the reaction revealed that a similar final level of inhibition of ~85-90% was achieved for the variants and wild-type enzyme by 15?mM ATP (Fig. 5A).

Figure 4 figure4 ATP is a competitive inhibitor of EhACK. Double reciprocal plot of EhACK activity versus Pi concentration in the absence (0) or presence of 2.5?mM (•), 5?mM (?), or 7.5?mM ATP (¦). Activities are the mean?±?SD of three replicates.

Full size image Figure 5 figure5 Inhibition curves for EhACK and MtACK wild-type and variant enzymes. Enzymatic activity was determined for each enzyme in the presence of the indicated concentration of ATP or PPi. Activities were plotted as a percentage of the activity observed for the wild-type enzyme in the absence of inhibitor. Activities are the mean?±?SD of three replicates. (A) ATP inhibition of EhACK and its variants in the acetate-forming direction. EhACK wild-type, (0); EhACK Q323G-M324I variant (•); EhACK Q323A-M324A variant, (?); EhACK D272A-R274A-Q323G-M324I variant (¦). (B and C) PPi inhibition of MtACK and its variants in the acetate-forming (B) and acetyl phosphate-forming (C) directions. MtACK wild-type, (0); MtACK G331Q-I332M variant (•).

 

 

 

Full size image

The IC50 value for ATP, defined as the concentration of ATP required to cause 50% inhibition of enzymatic activity, was reduced for the EhACK variants versus the wild-type enzyme (Table 2). The IC50 values were reduced 20-30% for the two Q323-M324 variants versus the wild-type enzyme, whereas the quadruple variant in which both the Q323-M324 residues and the D272-R274 salt bridge were altered had an IC50 value that was reduced by over 60%. Since ATP is a competitive inhibitor of EhACK activity, this increase in inhibition suggests that the D272A-R274A-Q323G-M324I variant binds ATP more efficiently than wild-type enzyme even though it cannot use it as a substrate.

Table 2 IC50 values for ATP inhibition of EhACK and PPi inhibition of MtACK. Full size table For MtACK, inhibition by PPi in the acetate-forming direction was similar for the wild-type enzyme and the G331Q-I332M variant, with ~70% maximum inhibition observed (Fig. 5B). The IC50 values for PPi were similar for both enzymes (Table 2). PPi inhibition in the acetyl phosphate-forming direction was much stronger, reaching greater than 90% for both the wild-type and variant enzymes. However, maximum inhibition for the wild-type enzyme was achieved at lower ATP concentration (20-25?mM versus 40?mM for the variant). This is reflected in the IC50 value, which is nearly two-fold higher for the G331Q-I332M variant than for the wild-type.

 

 

 

Discussion Substrate selection in ATP-utilizing ACKs Thaker et al.12, in analysis of the MtACK and EhACK structures, predicted that Pi/PPi binding does not involve the adenosine pocket and PPi likely binds in a position corresponding to the position of the ß- and ?-phosphates of ATP in MtACK. Our inspection of structures for the four Mycobacterium ACKs and the S. enterica ACKs in addition to those for MtACK and C. neoformans ACK showed that the opening to the adenosine pocket is not occluded in ATP-utilizing ACKs, only in the EhACK structure. Thus, we investigated whether phosphoryl donor selection by ACK is based primarily on accessibility of the adenosine pocket.

Alterations were made to MtACK to determine if the substrate specificity could be changed from ATP to PPi if the adenosine pocket was occluded. Catalysis was greatly reduced (~50-150 fold) in the enzyme variants and this was accompanied by increases in the K m values for both acetate and ATP in the acetyl phosphate-forming direction of the reaction. Gorrell et al.16, using tryptophan fluorescence quenching, found that domain closure occurs upon nucleotide binding. Thus, the effects of these alterations on MtACK activity may be complicated to interpret as reduced catalysis could be due to inefficient utilization of ATP and to an influence in domain closure. Notably though, substrate specificity did not change and the MtACK variant was unable to utilize PPi as a substrate. Thus, conversion of ATP-dependent MtACK to a Pi/PPi-dependent enzyme could not be achieved by simple closure of the adenosine pocket.

MtACK has a broad NTP substrate range15, 17 with a preference for ATP and is highly active in both the acetate- and acetyl phosphate-forming directions. Ingram-Smith et al.15 examined the roles of conserved active site residues in NTP substrate selection in MtACK, and found that Gly331 in the ADENOSINE motif exerted a strong influence. Asn211 in the PHOSPHATE2 motif and Gly239 in the LOOP3 motif were found to be important for enzymatic activity but did not play a substantial role in NTP preference.

Yoshioka et al.10 studied four residues in the ADENOSINE motif and one residue in the PHOSPHATE2 motif of E. coli ACK for their role in ATP versus PPi substrate determination. The candidate residues Asn213, Gly332, Gly333, Ile334 and Asn337 were altered to the respective residues present in EhACK (Thr, Asp, Gln, Met, and Glu, respectively) and the ability of the enzyme variants to utilize PPi in place of ATP was examined. All five variants displayed increased K m for ATP and decreased catalysis but none was able to utilize PPi.

Yoshioka et al.10 also examined the distribution of the E. coli ACK candidate residues and the corresponding residues in EhACK among 2625 ACK homologs. They suggested that Asn337 (Glu327 of EhACK) is most important in determining substrate selection as it is present in the ten ACK sequences most closely related to EhACK. However, their kinetic results with the Asn337 variants are inconclusive in this regard, although the kinetic results for this and other variants do strongly support a major role in ATP binding for the ADENOSINE motif but do not delineate specific residues responsible for determining ATP versus PPi utilization.

Substrate selection in PPi-dependent EhACK As a converse to our experiments with MtACK, we altered residues blocking the opening to the adenosine pocket in EhACK to reduce the occlusion and evaluated the enzyme’s ability to utilize Pi/PPi versus ATP/ADP. The EhACK variants exhibited decreased activity with Pi and PPi, much as we expected. Although catalysis was reduced for the Q323G-M324I and Q323A-M324A variants, further opening of the entrance to the adenosine pocket in the D272A-R274A-Q323G-M324I variant almost completely eliminated activity. This suggested that as the opening to the adenosine pocket increases, Pi and PPi may still bind but their positioning may be suboptimal.

Although the enzyme variants were still unable to utilize ATP as a substrate, ATP and ADP did inhibit enzyme activity. The level of inhibition increased as the opening to the adenosine pocket was expanded, especially for the D272A-R274A-Q323G-M324I variant. This suggested that ATP and ADP could now enter the adenosine pocket and interfere with Pi binding. Such an interpretation of these results is supported by the observation that ATP inhibition is competitive versus Pi.

The similar behavior of the Q323G-M324I and Q323A-M324A variants with respect to inhibition by ATP indicated that the increased binding (as judged by IC50 values) must be due to expanding the entrance to the adenosine pocket rather than a specific interaction between the altered residues and ATP. Models of the enzyme variants indicated that these alterations to the adenosine pocket would result in decreased impairment of ATP binding (Fig. 6). In particular, deletion of G203 to shorten the PHOSPHATE2 loop combined with alteration of the ADENOSINE motif and removal of the D272-R274 salt bridge should allow the pocket to accommodate ATP well (Fig. 6C). Although the alterations made to EhACK appeared to increase the enzyme’s ability to bind ATP, as indicated by the inhibition results, ATP was still not an effective substrate.

 

 

 

Figure 6 figure6 In silico modeling of the adenosine binding pocket of EhACK variants. Models were built using Accelrys Discovery Studio version 3.5 (Biovia). ADP binding in MtACK was superimposed into the EhACK structure models. (A) Q323G-M324I variant. (B) D272A-R274A-Q323G-M324I variant. (C) ?G203- D272A-R274A-Q323G-M324I variant.

Full size image Other possible PPi-dependent ACKs Using a BLASTp search of the non-redundant protein sequence database at NCBI with EhACK as the query sequence, we identified a small number of putative ACK sequences that may also be PPi-dependent or require a substrate other than ATP or PPi. Several of these putative ACK sequences came from metagenome analyses of anaerobic digestors. However, there were four putative ACK deduced amino acid sequences that came from draft genomes for the bacteria Ornatilinea apprima, Longilinea arvoryzae, Flexilinea flocculi, and Leptolinea tardivitalis 18,19,20,21. These bacteria are all obligate anaerobes from the phylum Chloroflexi within the family Anaerolineaceae. Three of the four produce acetate as a main product from glucose fermentation; L. arvoryzae also produces acetate as a primary fermentation product but from growth on sucrose instead of glucose. Little else is known about these bacteria beyond their initial characterization for recognition as new species.

Alignment of these putative ACK sequences with those of the four Entamoeba ACK sequences (those from E. histolytica, Entamoeba nuttalli, Entamoeba dispar, and Entamoeba invadens) revealed two key findings. Within the PHOSPHATE2 motif, all eight of these sequences have the extended loop containing the second Gly residue (Fig. 7). These are the only putative ACK sequences identified to have this extended PHOSPHATE2 loop (see Supplemental Figure S1 for comparison). Within the ADENOSINE motif, all of these sequences have a conserved Asp residue (immediately adjacent to Q323-M324 of EhACK) that is not conserved in any other ACK sequences (all of which have Ala or Gly at the equivalent position, as shown in Fig. 1). Interestingly, these bacterial ACKs have a conserved Asp at the equivalent position to Gln323 of PPi-dependent EhACK and the other Entamoeba ACKs. A completely conserved Gly resides at this position (Gly331 of MtACK) in all ATP-dependent ACKs. Whether this indicates that these enzymes are neither ATP-dependent nor PPi-dependent, or whether there is some flexibility in the identity of the residue at this position in PPi-dependent ACKs is unknown. Figure 7 figure7

Partial alignment of putative PPi-ACK amino acid sequences. Sequences were aligned using Clustal Omega. The PHOSPHATE2 and ADENOSINE motifs are shown. The full alignment is provided in the Supplemental Information (Supplemental Figure S3). Abbreviations and sequence accession numbers: Ehist, E. histolytica, XP_655990.1; Enut, Entamoeba nuttalli, XP_008860710.1; Edis, Entamoeba dispar, XP_001741606.1; Einv, Entamoeba invadens, XP_004254504.1; Oapp, Ornatilinea apprima, WP_075061087.1; Larv, Longilinea arvoryzae, WP_075074878.1; Fflo, Flexilinea flocculi, WP_062279690.1; Ltar, Leptolinea tardivitalis; WP_062422928.1.

Full size image Conclusions Our results demonstrate that phosphoryl donor specificity in ACK is mediated not just by access to the adenosine binding pocket but by other elements as well, as simple opening or occlusion of the entrance to this pocket was not sufficient to alter substrate specificity. This suggests that the active sites of the ADP/ATP-dependent and Pi/PPi-dependent enzymes have evolved to optimize utilization of their preferred substrate at the expense of the ability to use alternative substrates, and thus better suit their biological function.

Materials and Methods Materials Chemicals were purchased from Sigma-Aldrich, VWR International, Gold Biotechnology, Fisher Scientific, and Life Technologies. Oligonucleotide primers were purchased from Integrated DNA Technologies.

Site-directed mutagenesis Site-directed mutagenesis of the E. histolytica ack (ehack) and M. thermophila ack (mtack) genes was performed according to manufacturer’s instructions with the QuikChange II kit (Agilent Technologies, CA, USA). The altered sequences were confirmed by sequencing at the Clemson University Genomics Institute. Mutagenesis primers used are shown in Supplemental Table S2.

 

 

 

Recombinant protein production EhACK and its variants were produced in Escherichia coli strain YBS121 ?ack ?pta carrying the pREP4 plasmid containing the lacI gene and purified as described in Fowler et al.9. MtACK and its variants were produced in E. coli Rosetta2 (DE3) pLysS and purified as described in Fowler et al.9. Purified enzymes were dialyzed overnight in 25?mM Tris-HCl, 150?mM NaCl, and 10% glycerol (pH 7.4), aliquoted, and stored at -80?°C. Recombinant enzymes were examined by SDS-PAGE and estimated to be greater than 95% pure. Protein concentration was measured by absorbance at 280?nm using Take3 micro-volume plate (Biotek, VT, USA).

Determination of kinetic parameters Kinetic parameters for the EhACK enzymes were determined using the colorimetric hydroxamate assay for the acetyl phosphate-forming direction2, 3, 17 and the reverse modified hydroxamate assay9, 22 for the acetate-forming direction as previously described9. In the acetyl phosphate-forming direction, activities for EhACK and its variants were assayed in a mixture containing 100?mM morpholinoethanesulfonic acid (pH 5.5), 5?mM MgCl2, and 600?mM hydroxylamine hydrochloride (pH 7.5) with varying concentrations of acetate and sodium pyrophosphate. Reactions were performed at 45?°C. For the acetate-forming direction, kinetic parameters were determined in a mixture of 100?mM Tris-HCl (pH 7.0) and 10?mM MgCl2 with varying concentrations of sodium phosphate and acetyl phosphate. Enzymatic reactions were performed at 37?°C.

The acetyl phosphate produced (acetyl phosphate-forming direction) or remaining (acetate-forming direction) was reacted with hydroxylamine to produced acetyl hydroxamate, which was then was converted to a ferric hydroxamate complex by reaction with an acidic ferric chloride solution, making the solution change from a yellow to brownish red color that can detected spectrophotometrically at 540?nm. Acetyl phosphate formation or depletion was determined by measuring the absorbance at 540?nm with an Epoch microplate spectrophotometer (Biotek) and comparison to an acetyl phosphate standard curve. Kinetic data were fit to the Michaelis-Menten equation by nonlinear regression using KaleidaGraph (Synergy Software) for determination of apparent kinetic parameters.

Similarly, kinetic parameters for MtACK and its variants were determined using the hydroxamate assay2, 3, 17 and the reverse modified hydroxamate assay9, 22 as previously described. In the acetate-forming direction, enzyme activities were determined in 100?mM Tris (pH 7.5) with varying concentrations of MgADP and acetyl phosphate. For the acetyl phosphate-forming direction, kinetic parameters were determined in 100?mM Tris (pH 7.5) and 600?mM hydroxylamine (pH 7.5) with varying concentrations of acetate and MgATP. Enzymatic reactions were performed at 37?°C.

Determination of inhibition parameters Inhibition of EhACK by ATP and ADP and of MtACK by PPi was determined using the hydroxamate and reverse modified hydroxamate assays as described above. All inhibition assays were performed using substrates at their K m concentrations, with the exception of acetyl phosphate which was used at a concentration of 2?mM for all reactions in the acetate-forming direction. The half maximal inhibitory concentrations (IC50 values) were determined using PRISM 5 (Graphpad Software). ATP’s mode of inhibition of wild-type EhACK was determined by measuring enzymatic activity in the acetate forming direction in a four by seven matrix of varied ATP and Pi concentrations, with other substrate concentrations kept constant.

ACK sequence alignment, ConSurf analysis, and structural modeling ACK sequences were obtained from NCBI. Accession numbers are as follows: E. histolytica, PDB ID 4H0O; Methanosarcina thermophila, PDB ID 1TUY; Cryptococcus neoformans, PDB ID 4H0P; Salmonella typhimurium, PDB ID 3SLC; Thermotoga maritima, PDB ID 2IIR; Mycobacterium smegmatis, PDB ID 4IJN; Mycobacterium avium, PDB ID 3P4I; Mycobacterium paratuberculosis, PDB ID 3R9P; Mycobacterium marinum PDB ID 4DQ8. Sequences alignments were performed using Clustal Omega23,24,25. ACK structures were downloaded from Protein Data Bank (PDB): 4H0O (Entamoeba histolytica), 1TUY (M. thermophila), 4H0P (C. neoformans), and 3SLC (S. typhimurium). Structure superposition and modeling were performed using Accelrys Discovery Studio 3.5 (Biovia). ConSurf analysis26,27,28 (http://consurf.tau.ac.il) was used to examine evolutionary conservation of ACK sequence and identify amino acids likely to play important structural and functional roles.

 

 

 

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Chemical Properties white crystalline powder or colourless crystals Uses Tetrasodium Pyrophosphate is a coagulant, emulsifier, and sequestrant that is mildly alkaline, with a ph of 10. it is moderately soluble in water, with a solubility of 0.8 g/100 ml at 25°c. it is used as a coagulant in noncooked instant puddings to provide thicken- ing. it functions in cheese to reduce the meltability and fat separa- tion. it is used as a dispersant in malted milk and chocolate drink powders. it prevents crystal formation in tuna. it is also termed sodium pyrophosphate, tetrasodium diphosphate, and tspp.

Uses Pharmaceutic aid. General Description Odorless, white powder or granules. Mp: 995°C. Density: 2.53 g cm-3. Solubility in water: 3.16 g / 100 mL (cold water); 40.26 g / 100 mL boiling water. Used as a wool de-fatting agent, in bleaching operations, as a food additive. The related substance Tetrasodium pyrophosphate decahydrate (Na4P2O7 0H2O) occurs as colorless transparent crystals. Loses its water when heated to 93.8°C.

Reactivity Profile Tetrasodium pyrophosphate is basic. Reacts exothermically with acids. Incompatible with strong oxidizing agents. Decomposes in ethyl alcohol.

Hazard Toxic by inhalation. Safety Profile Poison by ingestion, intraperitoneal, intravenous, and subcutaneous routes. It is not a cholinesterase inhibitor. When heated to decomposition it emits toxic fumes of POx and Na2O.

Enzyme inhibitor This conjugate base (also called diphosphate and previously inorganic pyrophosphate; abbreviated PPi) of pyrophosphoric acid (FW = 177.98 g/mol; pK1 = 0.85, pK2 = 1.96, pK3 = 6.60, and pK4 = 9.41 (at 25°C); pKa values sensitive to changes in ionic strength) is commonly supplied as its crystalline sodium salts that are very soluble in water. Pyrophosphate anion hydrolyzes to orthophosphate, but much more slowly pyrophosphoric acid. Pyrophosphate binds many metal ions quite tightly. This property alone often accounts for its inhibition of numerous enzymes. The Mg2+-HP2O7 3- stability constant is reported to be 1200 M-1.1 Values for Ni2+, Cu2+, Co2+, and Zn2+ are higher. 

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Raw materials Sodium hydroxide–>Sodium carbonate–>Phosphorous acid–>N-(Isoxazol-5-yl)sulphanilamide Identification of a pyrophosphate-dependent kinase and its donor selectivity determinants

Ryuhei Nagata, Masahiro Fujihashi, Takaaki Sato, Haruyuki Atomi & Kunio Miki Nature Communicationsvolume 9, Article number: 1765 (2018) | Download Citation

 

 

 

Abstract

Almost all kinases utilize ATP as their phosphate donor, while a few kinases utilize pyrophosphate (PPi) instead. PPi-dependent kinases are often homologous to their ATP-dependent counterparts, but determinants of their different donor specificities remain unclear. We identify a PPi-dependent member of the ribokinase family, which differs from known PPi-dependent kinases, and elucidate its PPi-binding mode based on the crystal structures. Structural comparison and sequence alignment reveal five important residues: three basic residues specifically recognizing PPi and two large hydrophobic residues occluding a part of the ATP-binding pocket. Two of the three basic residues adapt a conserved motif of the ribokinase family for the PPi binding. Using these five key residues as a signature pattern, we discover additional PPi-specific members of the ribokinase family, and thus conclude that these residues are the determinants of PPi-specific binding. Introduction of these residues may enable transformation of ATP-dependent ribokinase family members into PPi-dependent enzymes. Introduction Kinases transfer a phosphate group from a phosphate donor to an acceptor. Various compounds are known as phosphate acceptors (e.g., proteins, lipids, and carbohydrates), while the donor for most kinases is ATP. A few kinases utilize atypical donors other than ATP. One utilizes ADP as the donor1,2,3,4, another uses inorganic pyrophosphate (PPi)5,6,7, and enzymes that utilize inorganic polyphosphate are also known8,9. The mechanism governing donor specificity and the evolutional trajectory of these unique enzymes have been widely discussed but are still unclear1,5,6,8,9,10,11,12.

Only three types of enzyme have been identified as PPi-dependent kinases: PPi-dependent phosphofructokinase (PPi-PFK), PPi-dependent pyruvate phosphate dikinase (PPi-PPDK), and PPi-dependent acetate kinase (PPi-ACK). PPi-PFK catalyzes the phosphorylation of D-fructose 6-phosphate using PPi to produce D-fructose 1,6-bisphosphate, and also catalyzes the reverse reaction11,13. PPi-PPDK catalyzes the reversible conversion of phospho(enol)pyruvate, PPi, and AMP into pyruvate, orthophosphate (Pi), and ATP13. PPi-ACK, which has been found only in Entamoeba histolytica, catalyzes the reversible phosphorylation of acetate with PPi as the phosphate donor. However, PPi-ACK from E. histolytica is thought to primarily produce PPi and acetate from Pi and acetyl phosphate under physiological conditions because the kcat value of the PPi-producing reaction is 1000-fold higher than that of the PPi-consuming reaction7. The former two reactions are involved in glycolysis/gluconeogenesis13, while PPi-ACK is presumed to provide PPi for PPi-PFK and PPi-PPDK during glycolysis in E. histolytica7,14. The reason why the PPi-dependent kinases prefer PPi to ATP as the phosphate donor remains unclear. The mechanisms of the donor specificity in PPi-PFK and PPi-ACK have been discussed without reference to their PPi-complex structures, but based only on structural comparisons with their ATP-dependent homologs. For example, the aspartate residue at the phosphate-donor-binding site in PPi-PFK prevents the ATP binding6,15,16. In PPi-ACK from E. histolytica, the five residues in the donor-binding site occlude the ATP-binding cleft17,18. Thus, only mechanisms for interfering with ATP binding have been suggested, while the residues that specifically recognize PPi remain unclear.

Here, we identify a PPi-dependent kinase belonging to the ribokinase family, which is distinct from the families of the previously reported PPi-dependent kinases. The crystal structure complexed with a PPi analog reveals the PPi-binding mode of this enzyme. Structural comparison and sequence alignment with ATP-dependent or ADP-dependent members of the ribokinase family reveal the importance of five residues: two large hydrophobic residues occluding a part of the ATP-binding pocket and three basic residues specifically involved in PPi recognition. The five residues are used collectively as a signature pattern and enable us to newly identify PPi-specific members of the ribokinase family.

Results Identification of a PPi-dependent kinase A PPi-dependent kinase belonging to the ribokinase family was identified based on structural similarity to a myo-inositol 3-kinase from the hyperthermophilic archaeon Thermococcus kodakarensis (MI3K_TK), which is an ATP-dependent member of the ribokinase family19,20. A Dali search21 with the substrate-complex structure of MI3K_TK (Protein Data Bank (PDB) ID 4XF7) showed that the structure is the most similar to the unliganded structure of TM0415 from the hyperthermophilic bacterium Thermotoga maritima (PDB ID 1VK4; root-mean-square (RMS) distance 2.2?Å for 254 C? atoms out of 283 C? atoms of TM0415; Fig. 1a). This enzyme has been annotated as a carbohydrate kinase belonging to the ribokinase family and is thought to be involved in myo-inositol metabolism because its gene is located in a myo-inositol catabolic gene cluster22,23. However, this enzyme exhibited no ATP-dependent kinase activity toward various carbohydrates, including myo-inositol22,23. Consistent with this result, a part of the potential ATP-binding cleft in TM0415 is occluded by three large residues (F221, R232, and M266; Fig. 1b). In contrast, comparison of the acceptor-binding site between TM0415 and MI3K_TK indicated that the five residues interacting with myo-inositol in MI3K_TK are conserved in TM0415 (Fig. 1c), raising the possibility that myo-inositol is the phosphate acceptor of TM0415. Accordingly, the phosphate donor specificity of TM0415 was investigated using myo-inositol as the acceptor. This analysis demonstrated that TM0415 utilizes PPi but neither ATP nor ADP to generate myo-inositol monophosphate (Fig. 2a). Although the ribokinase family includes various ATP-dependent or ADP-dependent kinases, including MI3K_TK, no member has been shown to be PPi-dependent until now.

Fig. 1 figure1 Structural comparison between TM0415 and MI3K_TK. The structures of the unliganded TM0415 (PDB ID 1VK4) and the substrate complex of MI3K_TK (4XF7) are shown in pink and green, respectively. a Superposition of the C? traces of the overall structures. b,c Comparison of the phosphate-donor- or acceptor-binding site. Green dotted lines represent the interactions between myo-inositol and the residues in MI3K_TK. The ligands and residues in the ligand-binding site are shown as sticks

Full size image Fig. 2 figure2 Analyses of the phosphate donor and the product. LC-MS analyses on the myo-inositol monophosphate produced from the reactions with TM0415 (a), homolog No. 7 (b), and homolog No. 49 (c). Ins(1)P and Ins(3)P represent the authentic compounds, and “No” means that the reaction was performed without phosphate donors. Eluted myo-inositol monophosphate was detected by MS with a mass range of m/z 259.0211-259.0237

Full size image Next, we performed kinetic analyses of TM0415 toward PPi and myo-inositol (Supplementary Fig. 1a). The initial velocity of the TM0415 reaction was almost constant (~17?µmol?min-1?mg-1) at a PPi concentration ranging from 15 to 500?µM. At lower concentrations (<15?µM), the experimental signal to determine the initial velocity was lower than the detectable limit. From the results, the values of Km and kcat toward PPi were estimated to be <15?µM and ~9.5?s-1, respectively. The estimated Km value is comparable to those of previously reported PPi-dependent kinases (1.2-200?µM)6,7,12,24,25,26,27,28,29,30. On the other hand, the values of Km and kcat toward myo-inositol are 12?±?2?mM and 10.8?±?0.3?s-1, respectively. The Km value is 40 times higher than that of MI3K_TK (0.30?±?0.03?mM)19. This raises the possibility that the genuine phosphate acceptor of TM0415 is a compound other than myo-inositol: for example, compounds related with the myo-inositol metabolic pathway that is composed of enzymes encoded by the TM0411-TM0416 operon in T. maritima, such as 2-keto-myo-inositol, diketo-inositol, 5-keto-L-gluconate, and D-tagaturonate (Supplementary Fig. 1b)23. These compounds have many hydroxyl and carbonyl groups, and thus possibly make hydrogen bonds with residues in the acceptor-binding site of TM0415. The kcat value toward myo-inositol is approximately the same as that toward PPi, verifying that the kinetic analyses of the two substrates were carried out accurately.

The effect of a potassium ion on the TM0415 activity was investigated because the activation of some ribokinase family enzymes by monovalent cations has been reported31,32,33. However, activation of the TM0415 reaction was not observed while adding KCl up to 100?mM (Supplementary Fig. 1a). The lack of activation of TM0415 by the monovalent cation is consistent with one of its structural features. That is, structural comparison between TM0415 and ribokinase from Escherichia coli (RK_EC, PDB ID 1GQT), which is activated by potassium or cesium ions31, showed that the amino group of K265 in TM0415 occupies the position of the monovalent cation in RK_EC (Supplementary Fig. 2). MI3K_TK, which is not activated by the potassium ion, also possesses R252, which occupies the corresponding position20. The presence of these positive residues probably prevents the monovalent cations from binding and results in the insensitivity of TM0415 to the monovalent cations.

Phosphate-donor recognition In order to elucidate the PPi-binding mode of TM0415, we determined its crystal structure complexed with a PPi analog (methylenediphosphonic acid: PCP), which has a carbon atom instead of an oxygen atom in the bridge position of PPi. In an asymmetric unit, two protein molecules were found: chains A and B (Supplementary Fig. 3a). The dimer structure probably does not reflect the physiological form of the protein, because size exclusion chromatography showed that TM0415 is a monomeric protein in solution (Supplementary Fig. 3b). Chain A was complexed with PCP, a magnesium ion, and myo-inositol, while chain B possessed a sulfate ion, which is derived from a crystallization precipitant, instead of PCP without any magnesium ions (Supplementary Fig. 3a,c). The structures of chains A and B were very similar to each other (RMS distance 0.6?Å for 274?C? atoms out of 275 chain B C? atoms) and also similar to the unliganded structure (RMS distances 0.9?Å for 274?C? atoms and 1.0?Å for 275?C? atoms, respectively, out of 283?C? atoms of the unliganded structure; Supplementary Fig. 3d). Slight differences were found only around the ligand-binding site.

In chain A, the phosphoryl group of PCP near myo-inositol (the proximal phosphoryl group) interacts with the main-chain nitrogen atoms of G231 and D234, while the other (the distal phosphoryl group) is surrounded by K171, T204, and R232 (Fig. 3a). The two phosphoryl groups and four water molecules octahedrally coordinate with the magnesium ion, which is essential for the kinase activity (Fig. 3b). R229 is also located near PCP, but its side chain was disordered in the structure (Fig. 3a). We thus also determined the SO42–complex structure, which possesses only the sulfate ions and myo-inositol in both molecules in an asymmetric unit. The sulfate ion is well superimposed on the distal phosphoryl group of PCP in a protein molecule that crystallographically corresponds to the PCP-bound chain described above (Fig. 3a). This suggests that its binding mode mimics the true binding mode of the distal phosphate group of PPi because the sulfate ion has no methylene group. This sulfate ion interacts with the side chain of R229 and the main-chain nitrogen atom of G233 in addition to K171, T204, and R232 (Fig. 3a). This strongly suggests that R229 and G233 recognize PPi in the reaction. The contribution of R229 to the reaction was confirmed by the fact that an R229A mutant exhibited only 7% of the level of the wild-type activity (Fig. 3b). The disorder of R229 in the PCP-complex structure is thought to result from the inability of the methylene group in PCP to form hydrogen bonds with the guanidine head of R229. The probable PPi-binding mode is depicted in Fig. 3c.

Fig. 3 figure3 PPi recognition in TM0415. a The PPi-binding sites of the PCP complex (left, cyan) and the SO42- complex (right, yellow) of TM0415. Superposition of the two complexes is shown in a center panel. Dotted lines represent the interactions involving PCP, the sulfate ion, and the magnesium ion. The three panels are drawn from the same viewpoint. b Specific activities of TM0415 (wild-type and its mutants). WT, wild-type; WT?+?EDTA, wild-type in the presence of 1?mM EDTA. ND means no detectable activity. Activity measurements were performed in triplicate, and standard deviations are represented as error bars. c A schematic diagram depicting the probable PPi-recognition mode. Gray dotted lines show the interactions involving PPi (magenta) and the magnesium ion. d Sequence alignment of the ribokinase family enzymes based on 3D-structure superpositions. The sequences around the GXGD motif in AGK_PF and AGK_TL are displayed in a green box under the alignment, because the order of the secondary structures around the GXGD motif in the two AGKs is different from those in the other enzymes, although the regions around the motif are superimposable in 3D-structure. Details are explained in Supplementary Fig. 7. Red circles indicate the residues interacting with PPi in panel c. Yellow bars highlight the characteristic residues of TM0415 (K171, R229, and R232) and the corresponding residues. Green and blue triangles indicate the NXXE motif and GXGD motif, respectively. Abbreviations of the ribokinase family enzymes are shown in Supplementary Table 7

Full size image The conservation of the residues contributing to PPi recognition (Fig. 3c) was investigated in the ribokinase family enzymes. Sequence alignment based on structural superposition showed that K171, R229, and R232 of TM0415 are not conserved in the ATP-dependent and ADP-dependent members (Fig. 3d). We then thought that it might be possible to actually predict PPi-dependent kinases in the ribokinase family using these three characteristic residues as a part of a signature pattern. R232 is also one of the three large residues occluding the ATP-binding pocket, as described above (Fig. 1b). Mutation of each characteristic residue in TM0415 into alanine led to a drastic decrease in the specific activity (3-11% of the wild type; Fig. 3b), confirming that the three basic residues contribute to the reaction.

Phosphate-acceptor binding The PCP-complex structure of TM0415 also revealed that its myo-inositol-binding mode is different from that of MI3K_TK, although the residues interacting with myo-inositol in MI3K_TK are conserved in TM0415 (Fig. 1c). D11, N78, Q141, and R145 in TM0415 interact with four of the six hydroxyl groups in myo-inositol (Fig. 4a), whereas D12, G26, Q136, R140, and D219 in MI3K_TK interact with all six hydroxyl groups (Fig. 4b)19. As shown in Fig. 4c, structural superposition of the two enzymes showed that the six-membered ring of myo-inositol tilts 30-40° and rotates ~120° from each other. One of the possible reasons for this shift is the difference of the hydrophobic residues in the myo-inositol-binding pocket: namely, L77, L87, and V112 in MI3K_TK are replaced with N78, S89, and L116 in TM0415, respectively (Fig. 4a,b).

Fig. 4 figure4 myo-inositol-binding mode. a,b The myo-inositol-binding sites in the PCP-complex of TM0415 (cyan) and the substrate complex of MI3K_TK (green, PDB ID 4XF719). Dotted lines represent the interactions between myo-inositol and the residues. The two panels are drawn from the same viewpoint. c Structural superposition of the two myo-inositol molecules bound to TM0415 (light orange) and MI3K_TK (pale blue). Double-headed arrows indicate the rotation of myo-inositol. The two panels are drawn from two different viewpoints. The atom numbering of myo-inositol carbons is shown in the same color in panels a and b

Full size image The difference of the binding mode led to a difference of the phosphorylated position of the produced myo-inositol monophosphate. In the TM0415 structure, the probable catalytic residue D234 and the proximal phosphoryl group of PCP are nearest to the 1-hydroxyl group of myo-inositol (3.7 and 3.4?Å, respectively, Fig. 4a). In addition, HPLC analysis using a chiral column showed that the elution time of the TM0415 product coincided with that of 1D-myo-inositol 1-phosphate (Ins(1)P) but not that of 1D-myo-inositol 3-phosphate (Ins(3)P), which is the product of MI3K_TK19 (Fig. 2a). The structural and chromatographic analyses led us to conclude that TM0415 phosphorylates the 1-hydroxyl group of myo-inositol and produces Ins(1)P.

Seeking unidentified PPi-dependent kinases The three basic residues recognizing PPi (K171, R229, and R232; Fig. 3a,c) and the two large hydrophobic residues occluding the ATP-binding pocket (F221 and M266, which together with one of the three basic residues, R232, are the three large residues shown in Fig. 1b) in TM0415 are expected to be the key residues for discovering various unidentified PPi-dependent kinases from the ribokinase family. An initial BLAST search using the overall TM0415 sequence as the query found only 24 homologs possessing the five key residues. The hit numbers were increased to 52 homologs by submitting the PPi-binding domain (residues 169-286) of TM0415 as the query sequence. In general, the overall structure of the ribokinase family enzymes is divided into two domains: the phosphate-acceptor-binding domain (the N-terminal half) and the donor-binding domain (the C-terminal half; Supplementary Fig. 3e). The increase in the hit number may result from elimination of the noise from the acceptor-binding domain in the BLAST search. Among the 52 homologs, two were TM0415 itself, and the other 50 homologs were presumed as candidate PPi-dependent kinases. Twenty of the candidate PPi-dependent kinases displayed no significant difference in the acceptor-binding site compared to TM0415 (Supplementary Fig. 4; Supplementary Table 1), suggesting that their phosphate acceptors are the same as that of TM0415. The other 32 homologs exhibited differences in the acceptor-binding site (Supplementary Table 2), raising the possibility that their acceptors are different from that of TM0415.

In order to examine whether the candidate PPi-dependent kinases exhibit PPi-specific kinase activity, recombinant proteins of five homologs (Nos. 7, 48, 49, 92, and 111) among the 32 homologs in Supplementary Table 2 were prepared by heterologous expression in E. coli. There were some differences in the sequences of the acceptor-binding sites among the five homologs. The expression of homolog No. 92 was poor (Supplementary Fig. 5a), and thus the activities of the other four homologs were investigated. Fourteen candidates for acceptors were tested: pentose (D-ribose and D-xylose), hexose (D-fructose and D-glucose), amino sugar (D-glucosamine), sugar alcohol (glycerol, meso-erythritol, and myo-inositol), disaccharide (sucrose and maltose), nucleoside (inosine, adenosine, and cytidine), and 2-keto-3-deoxygluconate. The activities were assayed using malachite green, which allows photometric determination of the Pi concentrations. The assay showed that homolog No. 49, in addition to TM0415, produced Pi upon incubation with PPi and myo-inositol (Supplementary Fig. 5b). In contrast, low levels of Pi production were observed with homolog No. 7 under the same conditions, and no activity was observed with the other two homologs, Nos. 48 and 111, at least with the phosphate acceptors examined here (Supplementary Fig. 5b). We further carried out incubation for longer periods of time with these three homologs, and an apparent production of Pi by homolog No. 7 was observed when myo-inositol was used as the acceptor (Supplementary Fig. 5c). No significant activity of the other two homologs was detected even with overnight (17?h) incubation (Supplementary Fig. 5c). LC-MS analysis confirmed that homologs No. 7 and 49 produce myo-inositol monophosphate, utilizing PPi but not ATP or ADP (Fig. 2b,c), strongly suggesting that these two homologs are PPi-dependent kinases. We presume that the absence of activities in homologs No. 48 and 111 may simply be due to the fact that the proteins mainly recognize phosphate acceptors that differ from those applied in our experiments. The fact that two of the examined proteins actually exhibited PPi-dependent activity suggests that the five residues (K171, F221, R229, R232, and M266) in the donor-binding site can be used as signatures to predict and discover unidentified PPi-dependent kinases. Identification of the true substrates of homologs No. 48 and 111 and confirmation of PPi-dependent activity should greatly strengthen our proposal.

LC-MS analysis also provided insight into the phosphorylated position of the produced myo-inositol monophosphate by the homologs. The results clearly showed that the product of homolog No. 7 is not Ins(1)P (Fig. 2b). This change of product may arise from the replacement of I76 and S89 in TM0415 with cysteine and glutamine, respectively, in the acceptor-binding site (Supplementary Table 2). Homolog No. 49 seemed to produce three distinct myo-inositol monophosphates that exclude Ins(1)P (Fig. 2c). The product change may result from the replacement of N78, S89, and L116 in TM0415 with leucine, leucine, and serine, respectively, in the acceptor-binding site (Supplementary Table 2). The product diversity probably resulted from the ambiguous acceptor recognition. The non-specific recognition implies that the genuine acceptor of homolog No. 49 is a compound other than myo-inositol.

 

 

 

Discussion

We discovered a PPi-dependent member of the ribokinase family, TM0415, and structurally elucidated the reason why the enzyme prefers PPi to ATP as the phosphate donor. The structural comparison between TM0415 and MI3K_TK showed that F221, R232, and M266 in TM0415 occupy a part of the ATP-binding cleft (Fig. 1b), suggesting that these three large residues prevent ATP from binding. The determined structures of TM0415 revealed the residues contributing to PPi binding (Fig. 3a,c). Three of them (K171, R229, and R232) are characteristic of TM0415 in the ribokinase family (Fig. 3d), and R232 is noteworthy for its contribution to both hindering ATP binding and interacting with PPi. The identification of characteristic residues of PPi recognition in PPi-dependent kinases has not been reported until now. In PPi-PFK and PPi-ACK, the PPi-recognition mode is unclear, and only residues obstructing ATP binding have been reported so far. In PPi-PFK, the conserved aspartate residue (e.g., D175 in the enzyme from E. histolytica) is suggested to prevent ATP binding, because its replacement with glycine, which is conserved in ATP-PFK, led to an 18-fold better Km value toward ATP6. In PPi-ACK from E. histolytica, the five residues (T201, D322, Q323, M324, and E327) occlude the ATP-binding cleft of ATP-ACK17,18. These residues were introduced to ATP-dependent kinases in order to transform them into PPi-dependent ones, but these attempts were unsuccessful6,17,18. Our identification of the three basic residues of TM0415 may enable such transformation on ATP-dependent ribokinase family members, which phosphorylate various acceptors. This engineering will reduce costs for the production of a variety of phosphorylated compounds, because PPi is 1000-fold cheaper than ATP.

TM0415 and the 50-candidate PPi-dependent kinases may be members of a PPi-dependent group of the ribokinase family, which is the third subclass of this family in addition to those of the ATP-dependent and ADP-dependent enzymes. This is a unique example of a kinase family that contains ATP-, ADP-, and PPi-dependent enzymes. Thirty-two of the fifty enzymes exhibited some differences in the acceptor-binding site when compared to TM0415 (Supplementary Table 2). Two of them, homologs No. 7 and 49, phosphorylate myo-inositol utilizing PPi in the manner of TM0415, but the phosphorylated hydroxyl group is different from that of TM0415 (Fig. 2b,c). This change may result from the different binding orientation of the acceptor caused by substitutions in the acceptor-binding site. The other homologs in Supplementary Table 2 also display various substitutions in the acceptor-binding site, and thus the PPi-dependent kinases in the ribokinase family possibly include a variety of enzymes exhibiting different acceptor specificity. Among the homologs examined in this study, homolog No. 49, which exhibited PPi-dependent kinase activity but no activity with ATP/ADP, is from Levilinea saccharolytica, a member of the phylum Chloroflexi in bacteria. Homolog No. 7, from Acanthamoeba castellanii, is a eukaryotic enzyme that also displayed activity with PPi. This suggests that the PPi-dependent ribokinase family members are not confined to the Thermotogae or bacteria. The source organisms of the 50-candidate PPi-dependent kinases are members of diverse bacterial phyla (e.g., Thermotogae, Proteobacteria, Spirochetes, and Chloroflexi) and three were from eukaryotic organisms, suggesting that the PPi-dependent ribokinase family enzymes may be widely distributed in nature.

R229 and R232 of the three basic residues in TM0415 are located around the GXGD motif. R229 is positioned two residues before the motif, and R232 corresponds to the second residue in the motif (Fig. 3d). This motif is conserved in the ribokinase family enzymes33,34. The aspartate residue is the catalytic residue, and the two glycine residues recognize the phosphate groups of the phosphate donor. The second residue is a small one (Ala, Cys, Val, Ser, or Thr) in ATP-dependent members to make room for the adenine and ribose groups of the nucleotide (Fig. 3d). In the ADP-dependent members, the residue is replaced with Ile or Leu to fill the small space created by the size difference between ADP and ATP. In the PPi-dependent kinase TM0415, the large residue R232 is situated in the corresponding position in order to recognize a smaller phosphate donor molecule. In addition, the two arginine residues are conserved in the 50-candidate PPi-dependent kinases (Supplementary Table 1, 2). Therefore, we propose an RXGRGD motif (the residues GRGD correspond to the GXGD motif) for the PPi-dependent kinases belonging to the ribokinase family. The ribokinase family enzymes also possess an NXXE motif near the phosphate-donor-binding site, which is involved in magnesium binding33,35. In the PCP-complex structure, the magnesium-coordinated waters are surrounded by D139, D173, and E176 (Fig. 3a). The latter two residues correspond to the first and last residues of the NXXE motif. The asparagine residue in the motif is replaced with aspartic acid in TM0415. This substitution is also observed in some ATP-dependent enzymes in the ribokinase family (Fig. 3d). Thus, the substitution of Asp for Asn in the NXXE motif may be unrelated to the specificity toward PPi.

As already described, three kinds of PPi-dependent kinases (PPi-PFK, PPi-PPDK, and PPi-ACK) have been identified thus far. We investigated whether the RXGRGD motif and/or the five key residues for the PPi-dependent members of the ribokinase family are found in the three kinds of enzymes. No signature patterns corresponding to the motif and the key residues were observed in PPi-PPDK and PPi-ACK. In the phosphate-donor-binding site of PPi-PFK, two basic residues were found (e.g., K148 and H384 in PPi-PFK from Borrelia burgdorferi), which are not conserved in ATP-PFK. However, their contributions to PPi binding remain to be elucidated. Further investigations including determination of the PPi-complex structure are necessary for identifying signature patterns for the three kinds of PPi-dependent kinases.

The evolutional trajectory of the PPi-dependent kinases is a controversial topic. For example, three hypotheses of the evolutional relationship between PPi-PFK and ATP-PFK have been proposed: PPi-PFK evolved into or from ATP-PFK5,6,11 or emerged from a common ancestor independently of ATP-PFK36. In the ribokinase family, the evolutional trace has been discussed based on the size of the lid domain37, which covers the ligand-binding site. A kinase without a lid domain is thought to be the ancient type of kinase. All kinases without the lid domain are ATP-dependent, suggesting that this ribokinase family originated from an ATP-dependent enzyme without a lid. As the enzyme evolved, the lid domain occurred and became larger to protect its substrates from the solvent (Supplementary Fig. 6a). The size of the lid varies widely, ranging from those consisting of only loops (smallest) to those with five strands and four helices (largest). All reported ADP-dependent ribokinases harbor the largest lid domain1, and are thus considered to have evolved from ATP-dependent enzymes with large, but slightly smaller lids with five strands and two helices (Supplementary Fig. 6a). TM0415 possesses a medium-sized lid domain with four ß strands (Supplementary Fig. 6a). According to the hypothesis stated above, TM0415 is not at the root of the evolutional tree of the ribokinase family enzymes. Primary sequences suggest that lid domains of similar size are found in most of the 50-candidate PPi-dependent kinases (Supplementary Fig. 6b). It should be noted that the relationship between PPi dependency and lid size is free from any query sequence bias. The query was composed of only the phosphate-donor-binding domain (the C-terminal half), whereas the lid domain is positioned in the N-terminal half. The conserved medium-sized lid domain implies that the PPi-dependent members of the ribokinase family emerged from the ancient ATP-dependent enzymes during the evolutional process.

 

 

 

Methods

Plasmid preparation

Enzymes selected for activity measurements were prepared as N-terminal His-tag fusion proteins, while those for crystallization were produced without a His-tag. The TM0415 gene was synthesized to produce a His-tag fusion protein (Supplementary Table 3) and inserted into the NcoI site of pET-15b by GenScript. The plasmids for expressing its mutants (K171A, R229A, and R232A) were prepared by inverse PCR using the TM0415 plasmid and the oligonucleotides K171A-F4, K171A-R4, R229A-F, R229A-R, R232A-F, and R232A-R (Supplementary Table 4) as a template and primers, respectively. In preparation of the enzymes for crystallization, the oligonucleotides (tag-rm-F and tag-rm-R; Supplementary Table 4) were used as primers for inverse PCR in order to remove the His-tag. The genes of the TM0415 homologs (No. 7, 48, 49, 92, and 111) were synthesized by GENEWIZ (Supplementary Table 5) and inserted into the NdeI and BamHI sites of the pCold II vector. The sequences of the resultant plasmids were confirmed by DNA sequencing (Hokkaido System Science or Macrogen Japan). Protein expression and purification

E. coli strain BL21(DE3)pLysS (Novagen) cells were transformed with the plasmids described above. The transformants were cultured at 37?°C in lysogeny broth medium containing 100?µg/mL ampicillin and 100?µg/mL chloramphenicol. For gene expression using the pET-15b vector, isopropyl-ß-D-1-thiogalactopyranoside (IPTG) was added (final concentration 0.2?mM) at a cell density of 0.4 (optical density at 600?nm) to induce gene expression. After a further culture for 4?h, the cells were harvested by centrifugation (5,000×g for 15?min at 4?°C). In the expression with pCold II vector, the medium was cooled on ice for 30?min before the addition of IPTG. After the addition of IPTG and a further culture at 15?°C for 24?h, the cells were harvested by centrifugation. 

The His-tagged TM0415 enzymes for the analysis of phosphate donor specificity were purified by Ni affinity chromatography. Cells were resuspended in buffer A (20?mM Tris-HCl (pH 7.4), 150?mM NaCl, 0.25?mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl)) containing an EDTA-free protease inhibitor cocktail (Nacalai Tesque) and disrupted by sonication. The sonicate was centrifuged (20,000×g for 30?min at 4?°C), and imidazole-HCl (pH 7.4) was added (final concentration 10?mM) into the supernatant. The supernatant was then mixed with Ni-NTA Superflow resin (QIAGEN) for 30-45?min at room temperature (RT). This mixture was loaded onto the column, and the flow-through fraction was collected. The resin was washed with buffer A supplemented with 50?mM imidazole-HCl (pH 7.4) for three column volumes (CVs), and the sample was eluted by buffer A supplemented with 300?mM imidazole-HCl (pH 7.4) for three CVs. The buffer of the eluate was exchanged with buffer A by ultrafiltration with an Amicon Ultra centrifugal filter unit (molecular weight cut off 10,000; Millipore). The His-tagged TM0415 enzymes for kinetic analysis were purified by Ni affinity, anion exchange, and size exclusion chromatography. Cells were resuspended in buffer B (50?mM Tris-HCl (pH 7.9), 50?mM NaCl, 1?mM MgCl2, 0.25?mM TCEP-HCl) and disrupted by sonication. The sonicate was centrifuged, and imidazole-HCl (pH 7.9) was added (final concentration 10?mM) into the supernatant. Affinity chromatography was performed as described above using the following buffers: wash buffer (50?mM potassium phosphate buffer (pH 7.8), 40?mM imidazole-HCl (pH 7.9), 300?mM NaCl, 10% (v/v) glycerol, 0.25?mM TCEP-HCl) and elution buffer (20?mM Tris-HCl (pH 7.9), 300?mM imidazole-HCl (pH 7.9), 10% (v/v) glycerol, 0.25?mM TCEP). The buffer of the eluted fractions was exchanged with buffer C (20?mM Tris-HCl (pH 8.1), 0.25?mM TCEP-HCl) by ultrafiltration. The sample was applied to a 1?mL MonoQ anion exchange column (GE Healthcare) equilibrated with buffer C and eluted with a linear gradient of 0-250?mM NaCl within 10 CVs. The eluted fractions at NaCl concentrations of 30-80?mM were concentrated by ultrafiltration. The sample was applied to a Superdex 200 Increase size exclusion column (GE Healthcare) equilibrated with buffer C supplemented with 150?mM NaCl and separated with the same buffer. The buffer of the relevant fractions was exchanged with 50?mM MES-NaOH (pH 6.1) for kinetic analysis.

Purification of the non-tagged TM0415 for crystallization was performed by heat treatment, anion exchange, and size exclusion chromatography. Cells were resuspended in buffer B supplemented with 10?mM MgCl2 and disrupted by sonication. The sonicate was heat-treated at 80?°C for 15?min and centrifuged. In order to remove nucleic acid, the supernatant was treated with ~130?µg/mL deoxyribonuclease I from bovine pancreas (Sigma-Aldrich) and ~13?µg/mL ribonuclease A from bovine pancreas (Nacalai Tesque) for 90?min at RT. The buffer of this sample was exchanged with buffer C. The sample was further purified by anion exchange and size exclusion chromatography as described for the kinetic analysis, except that the eluted sample of the size exclusion column was just concentrated for crystallization.

TM0415 for acceptor screening was purified by Ni affinity chromatography alone. Cells were resuspended in buffer B and disrupted by sonication. The suspension was centrifuged, and the supernatant was purified by affinity chromatography. The chromatography was performed using the procedures applied for the purification of enzymes for kinetic analysis as described above. The buffer of the eluted samples of the affinity column was exchanged with buffer D (50?mM Tris-HCl (pH 7.9), 100?mM NaCl, 0.25?mM TCEP-HCl).

The homolog proteins were purified in the same way as TM0415 used for the acceptor screening, but disruption was performed by BugBuster Protein Extract Reagent (Novagen). After purification with the Ni-NTA column, the buffer of the sample was exchanged with buffer D or 100?mM ammonium acetate buffer (pH 6.6) by ultrafiltration for the acceptor screening or LC-MS analysis of the donor specificity, respectively.

Acceptor screening of the TM0415 homologs Acceptor screening of homologs No. 7, 48, 49, and 111 was performed by the malachite green assay. The enzymatic reaction mixture (100?µL) was composed of 0.2?µg enzyme (TM0415 or its homologs), 350 or 7?mM phosphate acceptor, 500?µM PPi, 500?µM MgCl2, 100?mM KCl, 100?mM NaCl, 0.25?mM TCEP-HCl, and 50?mM Tris-HCl (pH 7.9). The following phosphate acceptors were tested: D-ribose, D-xylose, D-fructose, D-glucose, D-glucosamine, glycerol, meso-erythritol, myo-inositol, sucrose, maltose, inosine, adenosine, cytidine, and 2-keto-3-deoxygluconate (the former 10 compounds were used at 350?mM and the others at 7?mM). These compounds were the representative acceptors of the ribokinase family enzymes except for the compounds affecting the malachite green coloring. After preincubation at 37?°C (or 70?°C for TM0415) for 3?min, the enzymatic reaction was initiated by adding PPi. The reaction was carried out for 10?min, 8?h, or 17?h and was terminated by cooling on ice for 5?min. An aliquot (50?µL) of the reaction mixture was blended with 100?µL of the BIOMOL GREEN reagent (Enzo Life Science) and incubated at RT for 20?min. The coloration was checked visually.

LC-MS analysis on donor specificity and products Confirmation of the activities and analyses of the phosphate-donor specificities were performed by LC-MS. The reaction mixture with TM0415 (100?µL) was composed of 5?µg TM0415, 6?mM myo-inositol, 5?mM phosphate donor (ATP, ADP, or PPi), 10?mM MgCl2, and 100?mM ammonium acetate buffer (pH 6.65). The reaction was carried out for 5?min at 85?°C. For the homologs, the reaction mixture (100?µL) was composed of 3?µg enzyme (homolog Nos. 7 and 49), 50?mM myo-inositol, 500?µM PPi, 500?µM MgCl2, and 100?mM ammonium acetate buffer (pH 6.6). The reaction was performed for 17?h for 37?°C. The enzymes in the mixtures were removed by ultrafiltration, and the filtrate was analyzed by LC-MS. The methods used for the LC-MS analysis were reported previously19. Authentic Ins(1)P and Ins(3)P were purchased from Cayman Chemical Company.

 

 

 

Kinetic analysis of TM0415

Kinetic analysis of TM0415 was performed by detecting the produced Pi using a malachite green assay. The enzymatic reaction mixture (100?µL) was composed of 27?ng His-tagged TM0415, 15-500?µM PPi, 2-200?mM myo-inositol, 500?µM MgCl2, and 50?mM MES-NaOH (pH 6.1). After preincubation at 70?°C for 3?min, the kinase reaction was initiated by adding PPi. The reaction was carried out at 70?°C for 1, 2, or 3?min and terminated by cooling on ice for 5?min. An aliquot (50?µL) of the reaction mixture was blended with 100?µL of the BIOMOL GREEN reagent and incubated at RT for 20?min. The absorbance of the mixture at 620?nm was measured using a V-630 spectrophotometer (JASCO Corporation). The amount of Pi produced in the kinase reaction was calculated from the absorbance based on the calibration curve. The kinetic parameters were determined using the program SigmaPlot (HULINKS Inc.).

Specific activities of the TM0415 mutants (K171A, R229A, and R232A) and wild type were also measured with the malachite green assay. The enzymatic reaction mixture (100?µL) was composed of 0.02-1?µg His-tagged enzymes, 500?µM PPi, 200?mM myo-inositol, 500?µM MgCl2, and 50?mM MES-NaOH (pH 6.1). In these conditions, the enzymes seemed to exhibit Vmax from preliminary analysis. In analysis of the magnesium dependence, the mixture contained 1?mM EDTA instead of MgCl2. The further procedure is the same as that of kinetic analysis described above.

Crystallization and structure determination Crystallization was performed with the sitting-drop vapor diffusion method. The protein solution was composed of 10?mg/mL TM0415 (no His-tag, purified for crystallization), 500?mM myo-inositol, 10?mM PCP, 10?mM MgCl2, 150?mM NaCl, 0.25?mM TCEP-HCl, and 20?mM Tris-HCl (pH 8.1). The solution was incubated at RT for 1?h and centrifuged (15,400×g for 5?min at RT) in order to remove the salt precipitate of PCP and magnesium. The supernatant was blended with an equal amount of the precipitant solution composed of 21-29% (w/v) poly(ethylene glycol) 4000, 2 or 20?mM ammonium sulfate (A/S), and 100?mM sodium acetate buffer (pH 4.6), and then equilibrated at 20?°C. The precipitant including 2 or 20?mM?A/S was used for the co-crystallization with PCP or SO42-, respectively. The crystals were obtained within 1 month.

The crystals were soaked in cryo-protectant solution composed of 35% (w/v) poly(ethylene glycol) 4000, 2 or 20?mM?A/S, 50?mM myo-inositol, and 100?mM sodium acetate buffer (pH 4.6) and flash-frozen in a nitrogen stream at 100?K. The concentration of A/S was the same as that of the precipitant solution of the crystals. Diffraction data sets were collected at the beamline BL41XU of SPring-8 at a wavelength of 1.000?Å. The data sets were integrated and scaled with the program HKL200038. The phases were determined by the molecular replacement method with the atomic coordinates of the unliganded TM0415 (PDB ID 1VK4) using the program Molrep39. The structures were constructed using the program COOT40 and refined using the program REFMAC541,42 with the Translation Libration Screw refinement technique. The statistics for data collections and refinements are summarized in Supplementary Table 6.

Data availability The structural coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5YSP (the PCP-complex) and 5YSQ (the SO42–complex). Other data are available from the corresponding authors upon reasonable request.

Mubychem Group, established in 1976, is the pioneer manufacturer of Sodium Acetate and various other chemicals in India. Mubychem Group has manufacturing facilities spread across Western India. We know Sodium Acetate better than any other vendor in the world.

 

GRADES AND SPECIFICATIONS OF SODIUM ACETATE TRI-HYDRATE CRYSTALS

 

 

SPECIFICATIONS SODIUM ACETATE TRIHYDRATE PURE

Assay 99% Sodium Acetate Trihydrate minimum

Appearance White Transparent Crystalline

Test Solution A 10% Solution is clear & colourless

pH 7.5-9

Arsenic Less than 1 ppm

Calcium & Magnesium Less than 50 ppm

Heavy Metals Less than 1 ppm

Iron Less than 5ppm

Chlorides Less than 0.03%

Sulphates Less than 0.02%

Packing As required

SPECIFICATIONS SODIUM ACETATE TRIHYDRATE TECHNICAL

Assay 98% Sodium Acetate Trihydrate minimum

Appearance Off-White Crystalline

Test Solution A 10% Solution is clear & colourless

pH 7.5-9

Arsenic Less than 1 ppm

Calcium & Magnesium Less than 100 ppm

Heavy Metals Less than 10 ppm

Iron Less than 10 ppm

Chlorides Less than 0.5%

Sulphates Less than 0.5%

Packing As required

GRADES AND SPECIFICATIONS OF SODIUM ACETATE ANHYDROUS POWDER.

 

 

 

SPECIFICATIONS SODIUM ACETATE ANHYDROUS PURE

Purity 99 % minimum purity as CH3COONa

Appearance Snow-White Powder

Clarity of 10% Solution A 10% Solution w/v is clear and colourless

pH (10% in water) pH between 7.5-9

Arsenic Arsenic < 1ppm.

Calcium and Magnesium To pass the test 50 ppm

Heavy Metals Heavy Metals < 1ppm

Iron Iron < 5ppm

Chloride Chloride < 100ppm

Sulphate < 225ppm Sulphate < 20ppm

Packing 25kg(55 lbs)-50kg bags or pellets or as required

SPECIFICATIONS SODIUM ACETATE ANHYDROUS TECHNICAL

Purity 98.5% minimum purity as CH3COONa

Appearance White to Off-White Powder

Clarity of 10% Solution A 10% Solution w/v is clear

pH (10% in water) pH between 7.5-9

Arsenic Arsenic < 1ppm.

Calcium and Magnesium To pass the test 100 ppm

Heavy Metals Heavy Metals < 10ppm

Iron Iron < 10ppm

Chloride Chloride <0.5%

Sulphate < 225ppm Sulphate <0.5%

25kg(55 lbs)-50kg bags or pellets or as required

We offer Sodium Acetate IP Sodium Acetate BP Sodium Acetate USP Sodium Acetate Ph. Eur. and Extra Pure from a world class FDA approved, ISO-9001-2008 Certified facility

 

SODIUM ACETATE IP PHARMA GRADE

 

 

PARTICULARS SODIUM ACETATE IP GRADE

Dry Basis Assay 99 to 101%

Characteristics Colourless Crystals

Solubility Soluble in 0.8 part of water and in 19 parts of Ethanol (96%)

Clarity and colour of 10% w/v solution Clear and Colourless

Alkalinity (pH of 5% solution) 7.5-9.0

Arsenic 2 ppm

Calcium and Magnesium (calculated as Ca) 50 ppm

Heavy Metals as Lead 10 ppm

Iron 10 ppm

Chloride 200 ppm

Sulphate 200 ppm

Reducing Substances Passes test

Packing In 50 Kg HDPE bag with HMHDPE liner

Sodium Acetate Trihydrate & Anhydrous AR Analytical Reagent GR Guaranteed Reagent

Sodium Acetate Anhydrous ACS Analytical Reagent specifications

American Chemical Society Reagent Grade

Sodium Acetate Anhydrous

CH3COONa

Formula Wt 82.03

CAS Number 127-09-3

 

 

 

REQUIREMENTS

Assay: 99.0% C2H3O2Na

pH of a 5% solution: 7.0-9.2 at 25C

 

 

 

MAXIMUM ALLOWABLE

Insoluble matter: 0.01%

Loss on drying at 120C: 1.0%

Chloride (Cl): 0.002%

Phosphate (PO4): 0.001%

Sulfate (SO4): 0.003%

Calcium (Ca): 0.005%

Magnesium (Mg): 0.002%

Heavy metals (as Pb): 0.001%

Iron (Fe): 0.001%

 

 

 

Sodium Acetate Trihydrate ACS Analytical Reagent specifications

American Chemical Society Reagent Grade

NaC2H3O2 . 3H2O

Formula Wt 136.08

CAS Number 6131-90-4

 

 

 

REQUIREMENTS

Assay: 99.0-101% NaC2H3O2 . 3H2O

pH of a 5% solution: 7.5-9.2 at 25C

Substances reducing permanganate: Passes test

 

 

 

MAXIMUM ALLOWABLE

Insoluble matter: 0.005%

Chloride (Cl): 0.001%

Phosphate (PO4): 5 ppm

Sulfate (SO4): 0.002%

Heavy metals (as Pb): 5 ppm

Iron (Fe): 5 ppm

Calcium (Ca): 0.005%

Magnesium (Mg): 0.002%

Potassium (K): 0.005%

 

Applications or Uses:

Sodium acetate is used in the textile industry to neutralize sulfuric acid waste streams, and as a photo resist while using aniline dyes. It is also a pickling agent in chrome tanning, and it helps to retard vulcanization of chloroprene in synthetic rubber production. It is also used as “hot-ice” in hand warmer.

It is the chemical that gives salt and vinegar chips (crisps) their flavor. It may also be added to foods as a preservative; in this application it is usually written as “sodium diacetate” and labeled E262. It is also used for pH control or buffer.

As the conjugate base of a weak acid, a solution of sodium acetate and acetic acid can act as a buffer to keep a relatively constant pH. This is useful especially in biochemical applications where reactions are pH dependent. The FOOD industry relies on it as a buffer in controlling pH of food items during various stages of processing as well as for the finished consumable item. It is also used as a flavor enhancer in meat and poultry

It is also used in consumer HEATING PADS or hand warmers and is also used in hot ice. Sodium acetate trihydrate crystals melt at 58 °C, dissolving in their water of crystallization. When they are heated to around 100°C, and subsequently allowed to cool, the aqueous solution becomes supersaturated. This solution is capable of super cooling to room temperature without forming crystals. By clicking on a metal disc in the heating pad, a nucleation center is formed which causes the solution to crystallize into solid trihydrate crystals again. The bond-forming process of crystallization is exothermic, hence heat is emitted. The latent heat of fusion is about 264-289 kJ/kg.

The MEDICAL & PHARMACEUTICAL industry uses it in formula for diuretic expectorants and systemic alkalizers. It is commonly used in dry blends for renal dialysis. The heat of crystallization generated by sodium acetate is widely and effectively used in the heat pack industry.

Manufacturers Suppliers:

 

 

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List of All the IP BP USP FCC ACS Grades of Chemicals

Best Seller Fast Moving IP BP USP FCC ACS Grades of Chemicals Ex-stock Items

Aluminum Chloride Hexahydrate Aluminium Potassium Sulphate

Ammonium Bicarbonate Ammonium Bromide

Ammonium Carbonate Ammonium Chloride

Benzyl Alcohol Benzoic Acid

Borax; Sodium Borate Boric Acid

Calcium Acetate Calcium Carbonate

Calcium Chloride Calcium Hydroxide

Calcium Oxide Calcium Saccharate

Calcium Sulfate Carbamide Peroxide

Chromic Chloride Citric Acid

Copper Sulfate, Cupric Sulphate

 

 

 

Cupric or Copper Chloride

Ferric Chloride Hexahydrate Fumaric Acid

Gentian Violet Glacial acetic Acid

Magnesium Chloride Magnesium Sulfate

Manganese Chloride Manganese Sulfate

Octyldodecanol 

Potassium Bromide Potassium Carbonate

Potassium Chloride Potassium Hydroxide Pellets

Potassium Nitrate Propylene Carbonate

Selenious acid Sodium Acetate

Sodium Bicarbonate Sodium Butyrate

Sodium Molybdate Sodium Perborate

Sodium Sulfate Sodium Tartrate Dihydrate

Sodium Thiosulfate Stannous Chloride Dihydrate

Zinc Carbonate Zinc Chloride

 

 

 

Ammonium Persulfate Sodium Bromate

Sodium Diacetate Sodium Formaldehyde Bisulfite

Zinc Chloride

 

 

 

ISO/R 852:1968

Sodium tripolyphosphate and sodium pyrophosphate for industrial use — Determination of iron content — 2,2′- Bipyridyl spectrophotometric method

Applicable to products having Fe contents of more than 0.001 % (m/m). Preliminary hydrolysis of polyphophates by prolonged boiling in the presence of hydrochloric acid. Reduction of iron(III) by means of hydroxylammonium chloride. Formation of the iron(II)-2,2′-dipyridyl complex in the presence of ammonium acetate at pH of 3.1 and a temperature of 75 °C (under the test conditions the phosphate ions do not interfere). Spectrophotometric measurement of the coloured complex at a wavelength of about 522 nm.

 

 

 

Pyrophosphate Metabolism in Liver Mitochondria*

GALE W. RAFTER

From the Department of Microbiology, The Johns Hopkins School of Medicine and School of Hygiene and Public Health,

and the Department of Biochemistry, The Johns Hopkins School of Hygiene and Public Health, Baltimore 5, Maryland

(Received for publication, March 9, 1960)

Certain enzyme-catalyzed reactions occurring in various subcellular fractions produce pyrophosphate as one of their products;

for example, fatty acid activation in mitochondria (I), amino

acid activation in the soluble fraction (2), and adenosine 5′-triphosphate hydrolysis in microsomes (3). Consequently, the

disposal of pyrophosphate by cells is a matter of some importance.

At least two routes are possible for its metabolism: (a) conversion

to orthophosphate by hydrolysis and (b) nonhydrolytic cleavage

to form a new phosphate ester and orthophosphate. The second

alternative is an attractive one, since a portion of the pyrophosphate energy is preserved in the new phosphate ester. The present report deals in part with a pyrophosphate-dependent formation of glucose 6-phosphate in liver mitochondria.

Simultaneously with the formation of glucose 6-phosphate,

hydrolysis of pyrophosphate occurs. Evidence is presented (4)

which suggests that the glucose 6-phosphate-forming system is

closely related to a previously described inorganic pyrophosphatase of mitochondria. The phosphoryl-transferring activity of

certain phosphatases has been reviewed by Morton (5). An

interesting aspect of the system described here is its rapid inactivation upon incubating mitochondria at pH 5.0. This inactivation is prevented by pyrophosphate or by reagents which prevent the enzymatic hydrolysis of pyrophosphate.

METHODS

The preparation of mouse liver mitochondria and the method

of assay for PPiase’ have been described previously (4). The

glucose-6-P assays were carried out by utilization of the reaction

catalyzed by glucose-6-P dehydrogenase. The samples were

first centrifuged at 0″ to remove the mitochondria, then adjusted

to pH 7.6 with 0.1 M Tris buffer, and finally brought to a volume

of 3 ml. The reaction was initiated by adding 0.6 pmole of TPN.

Sufficient enzyme was used so that the reduction of TPN, measured at 340 rnF in the Beckman DU spectrophotometer, was

completed in about 2 minutes. The enzyme source was a yeast

autolysate which had been acid-precipitated and fractionated

with (NH&S04.2 The molar extinction coefficient 6.22 X lo6

moles+ cm2 (6) was used to convert absorbancy readings to

pmoles of TPNH. With known amounts of substrate, it was

found that 2 moles of TPN were reduced per mole of glucose-6-P.

* The author gratefully acknowledges the financial assistance

of the United States Public Health Service through research grant

A-1037. and the technical assistance of Carolvn Varner and Bettv

Hume.’

1 The abbreviations used are: PPiase, inorganic pyrophosphatase: and EDTA. ethvlenediaminetetraacetic acid.

2 Adapted from a preparation of yeast hexokinase in an unpublished description by R. Darrow and S. P. Colowick.

RESULTS

Requirement of PPi for Glucose-6-P Formation in Liver Mitochondriu-The formation of glucose-6-P by mitochondria required the presence of both glucose and PPi. The amount of

glucose-6-P formed depended on the time of incubation. These

results are summarized in Table I. The rate of the reaction fell

off with time. The reason for this is unknown; glucose-6-P,

7 X 10T5 M, did not inhibit the reaction, nor did mitochondria

metabolize added glucose-6-P as measured by the glucose-6-P

dehydrogenase assay or by the production of Pi.

The involvement of mitochondrial adenosine polyphosphates

with PPi in the phosphorylation of glucose was ruled out, since

washed liver mitochondria do not contain hexokinase (7).

The PPi-dependent phosphorylation of ribose and fructose,

catalyzed by mitochondria, was also investigated. With the

same concentrations of sugars, glucose formed approximately 8

times more organic P than ribose and 5 times more organic P

than fructose. From 2 to 3 times more organic P was formed

with glucose than anticipated by the glucose-6-P assay, indicating the presence of phosphorylated hexose other than glucose-6-P.

Thus it appears that the phosphoryl acceptor site does not exhibit a high degree of specificity.

E$ect of Glucose and PPi Concentration, and pH and Inhibitors

on Glucose-6-P Formation-The effect of different glucose and

PPi concentrations on the rate of glucose-6-P formation is shown

in Table II. The pH optimum for glucose-6-P formation was

5.1. The rate of the reaction was decreased 60% at pH values

5.6 and 4.5. A number of reagents were found to inhibit the

activity (Table III). The inhibition of glucose-6-P formation

by low concentrations of Mood- and its reversal by EDTA is

worthy of note. This inhibition was demonstrable only at low

substrate concentrations. A metal- and sulfhydryl-dependent

phosphoprotein phosphatase of liver is also inhibited by low concentrations of MoOh= (8). This inhibition is reversed by EDTA.3

Inactivation of GlucoseB-P-forming System and Effect of PPi

and Mood- on Inactivation-The glucose-B-P-forming activity of

mitochondria rapidly disappeared if the mitochondria were incubated at pH 5.0 and 37″. The activity was protected if small

amounts of Mood- were included in reaction mixtures, and the

protection was abolished by addition of EDTA. The addition

of PPi alone to reaction mixtures prevented the inactivation.

These results are presented in Table IV.

The Mood- effect on inactivation is interpretable in terms of

PPi protection of the glucose-B-P-forming system. As reported

previously (4), mitochondria possess a PPiase with a pH optimum

3 G. W. Rafter, unpublished observation.

2475

by guest on July 22, 2019 http://www.jbc.org/ Downloaded from 

Liver Pyrophosphate Vol. 235, No. 8

TABLE I TABLE IV

Requirement of PPi for glucose-6-P formation

All reaction mixtures contained mitochondria, 0.12 mg of protein, in 2.5 ml of 0.08 M sodium acetate buffer, pH 5.2. The glucose concentration in reaction mixtures No. 2, 3, and 4 was 0.036

M, and the PPi concentration in reaction mixtures No. 1, 3, and 4

was 0.008 M. An aliquot of the reaction mixture was assayed for

Effect of MoOh on inactivation of glucose-&P-forming system

All the reaction mixtures contained mitochondria, 0.25 mg of

protein, in 2.5 ml of 0.08 M sodium acetate buffer, pH 5.2. The

tubes were preincubated for 5 minutes at 37″. Then the additions

of 90 rmoles of glucose to all mixtures and 20 rmoles of PPi to

the first three mixtures were made. After incubation for 10 minglucose-6-P. utes at 37″, an aliquot was removed for the glucose-6-P assay.

Mixture No. Reaction mixture

(incl. mitochondria)

PP,

Glucose

PPi, glucose

PPi, glucose

Incubf;:ion at 0

min

20

20

10

20

(

–

;lucose.e~;P per

pmoles

0

0

0.051

0.098

Additions before preincubation

None

MoOh, 10 mpmoles

Mood-, 10 mrmoles + EDTA, 40 pmoles

PP i, 20 rmoles

TABLE II TABLE V

Effect of glucose and PPi concentration on glucose-6-P formation

All reaction mixtures contained mitochondria, 0.12 mg of protein, in 2.5 ml of 0.08 M sodium acetate buffer, pH 5.2. The PPi

concentration in reaction mixtures with different glucose concentrations was 0.0045 M, and the glucose concentration in reaction

mixtures with different PPi concentrations was 0.036 M. After

incubation for 15 minutes at 37″, an aliquot was removed from

each mixture for the glucose-6-P assay.

Glucose PPi Glucose-6-P per

tube

–

M pnwlcs M pmoles

0.004 0.009 0.0005 0.010

0.010 0.025 0.0011 0.023

0.024 0.043 0.0018 0.033

0.036 0.077 0.0036 0.067

0.048 0.081 0.0045 0.082

0.072 0.085 0.0072 0.089

TABLE III

Effect of inhibitors on glucose-6-P formation

Reaction mixtures No. 1 and 2 contained mitochondria, 0.25

mg of protein, 90 rmoles of glucose, and 20 pmoles of PPi in 2.5

ml of 0.08 M sodium acetate buffer, pH 5.2. Reaction mixtures

No. 3 and 4 contained only 4 pmoles of PPi. After incubation

at 37″ for 15 minutes, an aliquot was removed for the glucose-6-P

assay. Controls were performed with no additions.

Additions Inhibition

p-Mercuribenzoate, 0.7 pmole

I?, 50 rmoles

Mood, 40 mpmoles

Moor-, 40 mpmoles + EDTA, 40pmoles

%

85

50

60

0

at 5.2. Subsequent to the latter report, it was found that the

PPiase was inhibited by low concentrations of MoOh-. The inhibition was reversed by EDTA. For example, 1.9 X 10e5 M

Moor- inhibited PPi hydrolysis 50%; the inhibition was reduced

to approximately 10% in the presence of 2 X 10m3 M EDTA.

The concentration of PPi in the reaction mixtures was 4 X 10e3

M. Thus it appears that Mood- acts by protecting mitochondrial PPi from hydrolysis by the PPiase.

Formation ot Pi from PP; in Liver Mitochondria-As shown in

YG ;lucose-6-P

per tube

pmoles

0

0.046

0.005

0.048

Simultaneous formation of Pi and glucose-6-P from PPi in

liver mitochondria

All reaction mixtures contained mitochondria, 0.25 mg of protein, 90 pmoles of glucose, and 20 rmoles of PP i, in 2.5 ml of 0.08

M sodium acetate buffer, pH 5.2. For the PP iase assay, 0.5 ml of

11% trichloroacetic acid was added to each mixture. A Pi analysis was performed on an aliquot of the protein-free filtrate. Aliquots were removed from separate mixtures