Inorganic pyrophosphatase, activation

Escherichia coli can he adapted to grow on PPi as the sole source of phosphorus in this situation the cells are dependent upon intracellular inorganic pyrophosphatase activity to make Pi available. To avoid possible confusion from the inducible alkaline phosphatase of E. coli, which also has pyrophosphatase activity (see Chapter 17, by Reid and Wilson, this volume), all mutant isolation studies began with an E coli strain unable to synthesize this inducible protein. 

Liu M-Y, Le Gall J. 1990. Purification and characterization of two proteins with inorganic pyrophosphatase activity from Desulfovibrio vulgaris rubrerythrin and a new, highly-active, enzyme. Biochem Biophys Res Commun 171 313-318. 

Dick and Tabatabai " have described an assay for inorganic pyrophosphatase activity in soils based on a specific colorimetric determination of extracted orthophosphate after its release from inorganic pyrophosphate (0.05M) incubated with soils for 5h at 37°C. Soil suspensions were buffered at pH 8.0. In the absence of toluene, the amounts of orthophosphate released g soil were proportional to incubation time (up to 7h), and for a given time to the amounts of soil added. Activities were unaffected by toluene addition to soils and toluene was omitted from the recommended assay procedure. 

DICK W.A. and TABATABAI M.A. 1978. Inorganic pyrophosphatase activity of soils. Soil Biology and Biochemistry, 1, 59-65. 

Scheme II. Reaction scheme rationalizing the observed fluorescence assay of inorganic pyrophosphatase activity using chemosensor 3.

This reaction is accompanied by loss of free energy as heat, which ensures that the activation reaction will go to the right and is further aided by the hydrolytic splitting of PP , catalyzed by inorganic pyrophosphatase, a reaction that itself has a large AG of—27.6 kj/... 

NURF ISWI Drosophila Nurf-55, Nurf-301, N urf-38-inorganic pyrophosphatase Although the bulk of Drosophila ISWI does not co-localize with RNA polymerase [233], the NURF complex functions in the activation of transcription [234]. 

In 1998 the long-known Bacillus subtilis inorganic pyrophosphatase was characterized and found to have much greater activity than the above enzymes, to have a completely different amino acid sequence, not to be inhibited by F and to be activated by This form of... 

Enzymes that probably require three metal ions for full activity include the Tetrahymena group I ribozyme, a Mn " -activated bifimctional enzyme with inositol monophosphatase and fructose 1,6-bisphosphatase activities described belowand some endonucleases. " Inorganic pyrophosphatases from E. coli and S. cerevisiae are well characterized both structurally and mechanistically. Both Mg " " and Mn + are activating metal ions and the enzyme from E. coli is most active with just three metal ions in the active site. These enzymes have been described in Section 5.1.8.2.4. 

The presence of tri- and tetrapolyphosphatase activities in purified E. coli pyrophosphatase raised the question of whether these were properties of the same protein or of contaminating enzymes. For example, yeast inorganic pyrophosphatase does not act upon P3ji (55) there is a separate tripolyphosphatase in that organism (56). Three lines of evidence indicate that hydrolyses of PP, P3il, and P4,i are catalyzed by the same E. coli protein ... 

Fio. 2. Proposed kinetic model for yeast inorganic pyrophosphatase. Here M represents Mg + but may also apply to any divalent cation with which the enzyme is active. In the rate equation A represents all mono-magnesium PPi complexes, B represents the di-magnesium complex, and I represents free PPi. Hydrogen ion equilibria are not considered. Kinetic runs were done at pH 7.4, 30° (9). Best values for kinetic constants were obtained from a computer program for nonlinear regression (9S). 

In interesting recent studies, Poliak and co-workers (67, 68) have observed glucose-6-P phosphohydrolase, acid inorganic pyrophosphatase, and PPi-glucose phosphotransferase activities to be considerably higher in lipid-poor reticulosomes than in microsomal preparations. They hypothesized that reticulosomes, which they prepared by treatment of microsomal preparations from rat and chick livers with ribonuclease and deoxycholate (68), may be the precursors of endoplasmic reticulum, and... 

Subcellular Distribution of Inorganic Pyrophosphatase, PPi-GLUcosE Phosphotransferase, and Glucose-6-P Phosphohydrolase Activities of Rat Liver Microsomal Glucose-6-Phosphatase ... 

Subcellular Distribution op Glucose-6-P Phosphohydrolase, Inorganic Pyrophosphatase, and PPi-Glucose Phosphotransferase Activities op Human Liver ... 

The hydrolysis of pyrophosphate to phosphate is catalyzed by yeast inorganic pyrophosphatase, which is a dimeric enzyme having two identical subunits.290 It now appears that three cations aTe required per active site, two bound to the enzyme and the third to the phosphate (equation 4). 

Figure 9.141 Time course of formation of adenosine 5 -phosphosulfate (APS) by activity in rat liver. The reaction mixture contained, in a final volume of 350 /xL, 30 ju.mol of Tris-HCl (pH 8.0), 0.9 /xmol of ATP, 3 /unol of magnesium sulfate, 6 /xmol of sodium fluoride, and 50 fiL of inorganic pyrophosphatase (2.5 U). A50/xL supernatant sample (19 mg of protein) from rat liver was added to start the reaction. Then samples were removed at intervals, and the reaction was terminated and analyzed. Chromatograms were obtained after incubation for (A) 0 minutes, (B) 15 minutes, and (C) 45 minutes. Arrow indicates elution time for the reaction product APS. (From Mina and Rossomando, 1988.)...

Free phosphate has often been measured to identify the reaction pathway responsible for the formation of ADP in a given case. However, in most preparations that are partially purified, the presence of another enzyme activity, inorganic pyrophosphatase, which catalyzes the hydrolysis of PPj to 2P, means that measurements of free phosphate levels will not be conclusive. 

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