Active site pyrophosphatase

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. 

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. 

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

It is likewise of equal interest that the Golgi apparatus is the site of many hydrolase activities such as alkaline and acid phosphatases, j8-glu-curonidase, nucleoside diphosphatase, and thiamine pyrophosphatase. 

A kinetic study on the activation of deoxyribonuclease I by magnesium has shown that the activation curve is biphasic (the substrate being the Mg salt of bovine spleen DNA). This indicates that activation occurs at two sites on the protein. Free Mg + was required for enzyme activity, confirming that a metallo-enzyme as well as a metallo-substrate is necessary for deoxyribonuclease I activity. In contrast, it has been shown that the pyrophosphatase activity of bovine brain alkaline phosphatase depends on Mg + bound to the enzyme but not on the formation of a magnesium-substrate complex. 

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