Yeast pyrophosphatase

Nucleoside monophosphate kinase (NMK, 5 U) Pyruvate kinase (rabbit muscle, 100 U) Pyrophosphatase (yeast, 10 U)... 

From Sigma 3-aminoethylcarbazole (AEC) acrylamide/bis-acrylamide (30%) 37.5 1 amino acids alumina bentonite benzamidine bovine fiver tRNA bovine serum albumin (BSA) creatine phosphate (CP) diethyl pyrocarbonate (DEPC) dithiothreitol (DTT) Escherichia coli MRE600 tRNA pyrophosphatase (Ppase) Ca++ salt of folinic acid, (5-formyl THF) IIHPHS K salt of phospho-enol pyruvic acid, (PEP) creatine phospho kinase (CPK) protease inhibitor cocktail for fungal and yeast extracts phenylmethylsulfonyl fluoride (PMSF) spermidine trihydrochloride Tween 20. 

Pyrophosphatases, which are present in all cells, and catalyze hydrolysis of inorganic pyrophosphate (PPj) to orthophosphate (P ) (see Chapter 6, Section D), also drive metabolic sequences. The very active pyrophosphatase of E. coli has a turnover number of over 2 x 104 s 1 at 37°C. The 1000 molecules per cell are sufficient to immediately hydrolyze any pyrophosphate produced by bacterial metabolism.733 The much studied soluble pyrophosphatases of E. coli,7 A 7 ,r yeast,736 and other organisms736ab are metalloenzymes that are most active with Mg2+. Two Mg2+ ions are held, mostly by carboxylate side chains, while a third apparently enters the active site as magnesium pyrophosphate, perhaps MgP20-. As with other metallohydrolases, a metal-bound hydroxyl ion may serve as the attacking nucleophile. 

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. 

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). 

An alkaline pyrophosphatase from rat liver cytoplasm has been partially purified and characterized (24) the corresponding enzyme from mice is inhibited by Mg J+-ADP and free PPj, and free Mg2+ has been implicated as an allosteric activator (23). Partial heat inactivation results in loss of the apparent allosteric effects. Rat liver mitochondrial pyrophosphatase, which is inhibited by adenine nucleotides (36), appears to be bound to the inside of the inner mitochondrial membrane (37). This enzyme, after solubilization, has been separated into two fractions which have somewhat different specificity (24, 38). A pyrophosphatase strongly simulated by sulfhydryl reagents (39) has been partially purified from brain tissue (40). The mono-magnesium PPj complex appears to be the true substrate for this enzyme (41). Pynes and Younathan have purified a pyrophosphatase 1800-fold from human erythrocytes (43). The properties of this enzyme are strikingly similar to those of the yeast enzyme the major difference appears to be the more rigid substrate specificity of the erythrocyte enzyme in the presence of Znz. ... 

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 pyrophosphatase uses a water molecule bound between two Mg2+ ions as the nucleophile to attack bound Mg-pyrophosphate. Aspll7 acts as a general base to deprotonate this bound water, whose pK is 5.85.42 It was suggested on the basis of X-ray structures and the similar pICs that the hydrogen bond between Aspll7 and the nucleophilic water was a low-barrier one.43 Coordination to both Mg2+ ions as well as formation of a low-barrier hydrogen bond should certainly be sufficient to lower the pX of the bound water to the observed value. 

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