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). 

Kinetic and Thermodynamic Studies of Yeast Inorganic Pyrophosphatase... 

Figure 1 A crystal of yeast inorganic pyrophosphatase grown by temperature-controlled batch crystallization.23 The longest dimension of the crystal is —700 pm.

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). 

These model systems imply an important role for the multiple metal ions in the yeast inorganic pyrophosphatase reaction. A plausible mechanism consistent with its known properties and requirements is shown in Scheme 17. Clearly, the metal ions would reduce the charge on the phosphate residues and activate the P centers to nucleophilic attack. They also probably provide intramolecular hydroxide nucleophiles. 

Figure 18 Proposed interactions during hydrolysis of inorganic pyrophosphate by the tetrametal active site of yeast inorganic pyrophosphatase (reproduced with permission from ref. 220).

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. 

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