Formation Of Pyrophosphate Research Articles

Phosphoenolpyruvate Carboxytransphosphorylase: II. CRYSTALLIZATION AND PROPERTIES

Abstract Phosphoenolpyruvate carboxytransphosphorylase catalyzes reversibly the formation of oxalacetate and pyrophosphate from phosphoenolpyruvate, bicarbonate, and orthophosphate, and also catalyzes in the absence of bicarbonate the irreversible conversion of phosphoenolpyruvate and inorganic orthophosphate to pyruvate and inorganic pyrophosphate. Conditions have been investigated which influence the yield of the enzyme from Propionibacterium shermanii. The yield is higher with glycerol than with glucose as a substrate and increases with time of fermentation, 17 to 50 days being optimum. The enzyme has been purified by ammonium phosphate or sulfate fractionation, and chromatography on cellulose-phosphate and diethylaminoethyl cellulose. The enzyme has been crystallized in the form of very small needles. The resulting enzyme is nearly homogenous on sedimentation, and the molecular weight is 430,000 ± 30,000. The electrophoretic mobility (microns) descending was 10.3 x 10-5 cm2 per volt. Treatment with 6 m urea yields a subunit or units which after dialysis have a sedimentation constant of s20,w = 7.0 S. The pH optimum of the forward reaction, P-enolpyruvate to oxalacetate, extends over the broad range of 6.3 to 8.0. It is somewhat narrower for the back reaction from oxalacetate to P-enolpyruvate. Mg2+, Mn2+, or Co2+ is required for both the forward and back reactions. The reaction is about 7 times faster from P-enolpyruvate to oxalacetate than from oxalacetate to P-enolpyruvate. The Km values, expressed as millimolar concentration, were as follows for the forward reaction: P-enolpyruvate, 0.5; phosphate, 1.2; Mg2+, 1.2; Mn2+, 0.5; Co2+, 0.5; CO2 plus HCO3-, 4.0 at pH 6.5 and 9.5 at pH 7.8. In the back reaction, the values were: oxalacetate, 0.47; Mg2+, 2.5; and pyrophosphate, 0.22. The catalysis is inhibited by numerous compounds, including KCl and NaCl at high ionic strength. Sulfate is a noncompetitive inhibitor of the forward reaction with a Ki of about 20 mm. Buffers such as Tris-HCl, glycylglycine, and imidazole inhibit both the forward and back reactions. All the products and substrates inhibit the back reaction at quite low concentrations. The enzyme is stable in the absence of thiols, but the reaction is stimulated by a large variety of such compounds. Ethylenediaminetetraacetate inhibits the forward reaction but the inhibition is reversible.

The Purification and Properties of Pig Liver Geranyl Pyrophosphate Synthetase

Abstract The purification and properties of geranyl pyrophosphate synthetase are reported. This enzyme is purified simultaneously with the enzyme for the synthesis of farnesyl pyrophosphate. It does not, however, have geranylgeranyl pyrophosphate synthetase activity. This enzyme, obtained as a single band of protein by starch gel electrophoresis, catalyzes the formation of both geranyl and farnesyl pyrophosphates from dimethylallyl pyrophosphate and 4-14C-isopentenyl pyrophosphate. These products have been identified by paper and gas-liquid chromatography. A pH range of 7.0 to 7.8 is optimum for the reaction. A metal ion is required and either Mg++ or Mn++ may be used, but Mn++ inhibits the reaction at concentrations greater than 4 x 10-3 m. Km values for isopentenyl and dimethylallyl pyrophosphates are 1.25 x 10-6 and 2.2 x 10-6 m, respectively. N-Ethylmaleimide and p-chloromercuribenzoate strongly inhibit the synthetase reaction, but iodoacetamide has only a slight inhibitory effect. The s20,w value for the synthetase is 4.7.

The stereochemistry of rubber biosynthesis

The stereochemistry of the formation of natural rubber and trans-trans -farnesyl pyrophosphate in latex has been studied in vitro using [2- 14 C-(4 R )-4- 3 H 1 ] and [2- 14 C-(4 S )-4- 3 H 1 ]mevalonates as substrates. The proton eliminated from C-2 of isopentenyl pyrophosphate during the formation of farnesyl pyrophosphate in latex has the same steric position as that released in the liver system, and is epimeric with that eliminated during the biosynthesis of rubber. Complete stereospecificity is exhibited by the enzymes in the two cases. In rubber biosynthesis isoprene residues are incorporated directly in an all- cis configuration without the intervention of trans structures.

Oxalyl-coenzyme A synthetase from pea seeds.

Summary 1. A new enzyme which catalyzes the reaction: Oxalate + adenosine ′-triphosphate + coenzyme A M g 2 + ⇌ Oxalyl-coenzyme A + adenosine 5′-phosphate + inorganic pyrophosphate has been partially purified from extracts of pea seeds. It was also demonstrated in seeds of lupin and pumpkin, and in wheat germ. 2. The enzyme was assayed either by the rate of formation of oxalylhydroxa-mate, by the rate of esterification of coenzyme A, or by the rate of exchange of inorganic [32P]pyrophosphate with adenosine 5′-triphosphate. 3. The stoichiometry of the reaction was determined by demonstrating: (a) The production of adenosine 5′-phosphate, (b) The stoichiometric formation of oxalylhydroxamate and inorganic pyrophosphate in the hydroxamate assay, (c) The exchange between inorganic [32P]pyrophosphate, but not of inorganic [32P]phosphate, and adenosine 5′-triphosphate. Increasing concentrations of coenzyme A caused a progressive inhibition of this exchange. 4. The inorganic [32P]pyrophosphate exchange data are consistent with the reaction proceeding via an oxalyladenylate intermediate. 5. The general properties of the enzyme are described, and the possible biochemical function of the enzyme and of oxalyl-coenzyme A is discussed.

Properties of farnesyl pyrophosphate synthetase of pig liver

The isolation and partial purification of farnesyl pyrophosphate synthetase of pig liver are described. This enzyme does not have isopentenyl pyrophosphate isomerase, geranyl pyrophosphate synthetase, or geranyl geranyl pyrophosphate synthetase activities. Farnesyl pyrophosphate synthetase catalyzes the formation of farnesyl pyrophosphate through the condensation of geranyl and isopentenyl pyrophosphates. The product of this reaction has been characterized by paper and gas-liquid chromatography. A pH of 7.0 is optimum for the reaction, and K m values for geranyl and isopentenyl pyrophosphates are 4.0 × 10 −6 and 2.0 × 10 −6 M, respectively. An absolute metal requirement has been established for this reaction and Mn ++ is approximately 40 times more effective than Mg ++ at low ion concentrations. At pH 7.0, 2 × 10 −3 M iodoacetamide does not inhibit the reaction, but 1 × 10 −5 M p-hydroxy mercuribenzoate completely inactivates the enzyme.

1026. Studies in phosphorylation. Part XXVIII. The formation of pyrophosphates from phosphoramidate monoesters

1026. Studies in phosphorylation. Part XXVIII. The formation of pyrophosphates from phosphoramidate monoesters

On the Formation of Pyrophosphate from Quinol Phosphates in Dimethylformamide Solution

On the Formation of Pyrophosphate from Quinol Phosphates in Dimethylformamide Solution

The enzymatic synthesis of geranyl geranyl pyrophosphate by enzymes of carrot root and pig liver

The isolation of geranyl geranyl pyrophosphate synthetases from carrot root and pig liver and the properties of these enzymes are reported. Each of the enzymes catalyzes the formation of geranyl geranyl pyrophosphate from isopentenyl-4-C 14 and farnesyl pyrophosphates. The product of the reaction has been characterized by gas-liquid and paper chromatography and derivative formation. An absolute metal requirement has been demonstrated for the reaction and Mn ++ is the most effective metal in stimulating activity. The pH optimum of the reaction is 6.7–6.8 and 6.9–7.0, respectively, for the carrot root and pig liver enzymes. K m values calculated for farnesyl pyrophosphate and isopentenyl pyrophosphate are 1 × 10 −4 M and 1 × 10 −5 M respectively, for the carrot root enzyme, and 7.6 × 10 −5 M and 6.6 × 10 −6 M, respectively, for the pig liver enzyme.

Studies on the biosynthesis of cholesterol: XIV. the origin of prenoic acids from allyl Pyrophosphates in liver enzyme systems

Allyl pyrophosphates (3,3-dimethylallyl, geranyl, and farnesyl pyrophosphate), which are known intermediates in the biosynthesis of squalene from mevalonate, are also metabolized in the liver by an alternative pathway to acids. The first step in the conversion of the allyl pyrophosphates into the acids (dimethylacrylic, geranoic, and farnesoic acids) is their irreversible dephosphorylation into free prenols by a microsomal phosphatase, which has its optimum pH at around 7.0. In a second step the free prenols are irreversibly dehydrogenated to the acids by liver alcohol dehydrogenase and aldehyde dehydrogenase present in a soluble protein fraction of liver homogenates. This dehydrogenation proceeds best at pH 7.5 and is inhibited by sulfhydryl reagents. The prenols are more specific substrates for liver alcohol dehydrogenase than is ethanol, but they cannot act as substrates for yeast alcohol dehydrogenase. The formation of allyl pyrophosphates, of free prenols, and of prenoic acids from mevalonate was also observed in vivo.

Structure and Properties of the Condensed Phosphates. XVI. Pyrophosphate Formation in Concentrated Orthophosphate Solutions and its Biological Implications

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTStructure and Properties of the Condensed Phosphates. XVI. Pyrophosphate Formation in Concentrated Orthophosphate Solutions and its Biological ImplicationsCharles D. Schmulbach, John R. Van Wazer, and Riyad R. IraniCite this: J. Am. Chem. Soc. 1959, 81, 24, 6347–6350Publication Date (Print):December 1, 1959Publication History Published online1 May 2002Published inissue 1 December 1959https://doi.org/10.1021/ja01533a001RIGHTS & PERMISSIONSArticle Views171Altmetric-Citations11LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (530 KB) Get e-Alerts Get e-Alerts

Nucleoside Polyphosphates. IX.1 The Reversible Formation of Pyrophosphates from Monoesters of Phosphoric Acid by Reaction with Acetic Anhydride2

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNucleoside Polyphosphates. IX.1 The Reversible Formation of Pyrophosphates from Monoesters of Phosphoric Acid by Reaction with Acetic Anhydride2H. G. Khorana and J. P. VizsolyiCite this: J. Am. Chem. Soc. 1959, 81, 17, 4660–4664Publication Date (Print):September 1, 1959Publication History Published online1 May 2002Published inissue 1 September 1959https://doi.org/10.1021/ja01526a053RIGHTS & PERMISSIONSArticle Views123Altmetric-Citations17LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (649 KB) Get e-Alerts Get e-Alerts

Pyrophosphate formation in cell-free extracts of Escherichia coli

Pyrophosphate formation in cell-free extracts of Escherichia coli

Enzymic formation of inosinic-adenylic pyrophosphate

Enzymic formation of inosinic-adenylic pyrophosphate

The formation of inorganic pyrophosphate in microsomes

The formation of inorganic pyrophosphate from orthophosphate in respiring liver homogenates has been investigated. The results indicate that the major source of the pyrophosphate-forming system is in the microsome fraction, and that the pyrophosphate is formed from ATP, which can be generated either oxidatively by mitochondria or nonoxidatively by certain phosphate-transferring enzyme systems. Soluble fractions have a slight, variable, capacity to form pyrophosphate in the oxidative, but not in the nonoxidative system. Pyrophosphate formation is strongly inhibited at high adenine nucleotide concentrations whether ATP is generated oxidatively or nonoxidatively. Some microsome preparations will form small amounts of pyrophosphate from ATP in the absence of a generating system, but only if the amount of ATP added is low. Phosphate is required for maximal activity, but is not incorporated into pyrophosphate in the nonoxidative system. GTP, UTP, and CTP will substitute for ATP.

Function of adenosine triphosphate in the activation of luciferin

1. 1. Using crystalline firefly luciferase and purified luciferin, it is possible to show that light production is associated with the utilization of both luciferin and ATP. The total light produced is directly proportional to the concentration of these two substrates. During luminescence there is a decrease of the 330 mμ absorption peak of luciferin. 2. 2. The reaction of ATP with luciferin leads to the formation of pyrophosphate and active luciferin (presumably adenylluciferin). The latter compound can either react with oxygen for light production or be hydrolyzed to luciferin and adenylic acid. The latter reaction occurs under anaerobic conditions. Adenylic acid has been identified as one of the products. 3. 3. The initial reaction of luciferin with ATP is reversible since the addition of PP 32 to the reaction mixture leads to the formation of labeled ATP. Luciferin is required for this exchange. 4. 4. Oxidized luciferin as well as other derivatives of luciferin will replace luciferin as far as pyrophosphate liberation is concerned. In the presence of inorganic pyrophosphatase there is therefore a rapid and complete hydrolysis of ATP to adenylic acid and inorganic phosphate. The results indicate the labile nature of the adenyl compounds. 5. 5. Adenosine tetraphosphate, uridine triphosphate, cytidine triphosphate, guanosine triphosphate and inosine triphosphate are inactive for both light production and pyrophosphate liberation. 6. 6. The results are discussed in relation to the mechanism of light emission and the mode of action of pyrophosphate, pyrophosphatase, and coenzyme A on luminescence.

255. Studies on phosphorylation. Part XIII. Ketoxime sulphonates as intermediates in pyrophosphate formation

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Pyrophosphate Formation Upon Ignition of Precipitated Basic Calcium Phosphates1

Pyrophosphate Formation Upon Ignition of Precipitated Basic Calcium Phosphates¹

Experiments on the formation of carboxylase and thiamine pyrophosphate in living bakers' yeast

The formation of carboxylase by living bakers' yeast was demonstrated upon incubation of the yeast with either thiamine or 2-methyl-4-amino-5-ethoxymethylpyrimidine, in the presence and in the absence of glucose. Carboxylase is also formed upon incubation of the yeast with NH 4 sulfate and glucose. In the presence of glucose the effects of thiamine + NH 4 sulfate or 2-methyl-4-amino-5-ethoxymethylpyrimidine + NH 4 sulfate considerably surpass the effects of any of these substances separately. The effect of 2-methyl-4-amino-5-ethoxymethylpyrimidine depends upon the primary formation of thiamine pyrophosphate.

The detection of cobalt by the microcosmic salt bead

I. The fact, that the blue violet colour of the microcosimic salt bead of cobalt is changed into blue by addition of Na 2CO 3 K 2CO 3, K 2CO 3 NA 2HPO 4 or NA 3PO 4is explained by the formation of pyrophosphate in the melt. II. K 2Na 2P 2O 7 if melted together with CoCl 2 forms blue porcelaneous masses, completely soluble in water.