Enzyme yeast inorganic pyrophosphatase

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). 

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). 

Yeast inorganic pyrophosphatase (YIP) is an enzyme consisting of two identical subunits giving a molecular weight for the dimer of 64,000. Yeast inorganic pyrophosphatase catalyses the reversible hydrolysis of pyrophosphate. In the presence of Mg, YIP appears to be specific for pyrophosphate however, when other activating metal ions are employed, the enzyme becomes less specific and will hydrolyze a number of pyrophosphate esters (21). There is even a possibility that a mixture of enzymes is present in some preparations (55). 

Our NMR and EPR data (Figs. 6 and 7) clearly demonstrate that two Mn ions (at least) magnetically interact in the absence of substrate or product(s), and that this interaction persists when substrate analogs [e.g., Co(NH3)4PNP or Co(NH3)4(P)2] were also bound to the enzyme. Thus these magnetic resonance data agree with the concept that yeast inorganic pyrophosphatase utilizes three metal ions in substrate bin ng and catalysis. 

Because so much of modern biochemistry has utilized E. coli as a model cell for investigative purposes, it was of interest to study the inorganic pyrophosphatase of this organism in greater detail. The enzymes from yeast and other sources are described in Chapter 21 by Butler, this volume. 

U/ml inorganic pyrophosphatase from Baker s yeast (Sigma-Aldrich, St. Louis, MO, USA). Resuspend 500 U lyophilized enzyme and buffer salts in 250 pi water. Store at -80 °C in 5 pi aliquots. Store thawed aliquot on ice and discard at the end of assay day. 

If a-casein is treated with either the crystalline pyrophosphatase of yeast at pH 7.0 (35) or with the snake venom diesterase at pH 8.2 (87), no inorganic phosphorus is released. However, if the diesterase reaction is carried out in weakly buffered solutions a small drop of pH takes place (71), indicating the exposure of acidic groups. Subsequent incubation of the diesterase-treated a-casein with prostate phosphatase at pH 6.0 hberates no more phosphorus than in the absence of the diesterase. If, how ever, prostate and intestinal phosphatase are added, 78 % of the a-casein phosphorus is set free. Since the intestinal enzyme at pH 6.0 acts on low... 

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