Pyrophosphate turnover studies

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. 

Processes of this type have been realized in supramolecular phosphorylation reactions. Indeed, the same [24]-N6C>2 macrocycle 38 as that already used in the studies of ATP hydrolysis was also found [5.60] to mediate the synthesis of pyrophosphate from acetylphosphate (AcP). Substrate consumption was accelerated and catalytic with turnover following the steps (1) substrate AcP binding by the proto-nated molecular catalyst 38 (2) phosphorylation of 38 within the supramolecular complex, giving the phosphorylated intermediate PN 81 (3) binding of the substrate HP042 (P) (4) phosphoryl transfer from PN to P with formation of pyrophosphate PP (Fig. 8) (5) release of the product and of the free catalyst for a new cycle [5.60]. PP is also formed in the hydrolysis of ATP in the presence of divalent metal ions [5.61]. 

It is a monomeric protein of M.W. about 70,000, shows Kj, values for L-tryptophan and dimethylallyl pyrophosphate of 0.067 and 0.2 mM, respectively, and seems to have a relatively low turnover number, about 7 sec . During studies on this enzyme it was observed (13) that agroclavlne and elymoclavine, the terminal alkaloids in the strain used for the isolation of the enzyme, inhibited purified DMAT synthetase. At concentrations of 3 mM ( v<750 mg/1) agroclavlne and elymoclavine inhibited the enzyme 90% and 70%, respectively. The inhibition is of a mixed or uncompetitive type as shown by kinetic analysis with either tryptophan or dimethylallyl pyrophosphate as the variable substrate (Fig. 6). Subsequently, feedback Inhibition by elymoclavine was also demonstrated by GrSger s group (3) for chanoclavlne cyclase and by us for anthranllate synthetase from... 

Experiments with labeled glucose, phosphate, and nicotinamide indicate that there is a rapid equilibrium between the oxidized and reduced forms of DPN (212). In a study of the incorporation of phosphate into the different pyridine nucleotide coenzymes, it was found, surprisingly, that there was a rapid turnover of the monoester phosphate grouping of TPNH. This finding contrasts with a much slower turnover of the pyrophosphate grouping of the molecule. Although there is a rapid regeneration of the monoester phosphate, there appears to be a slow equilibrium between DPN and the DPN portion of TPN. 

---