Pyrophosphatase sources

Third, the quantitative differences in concentration of the four enzymes vary appreciably therefore making several venoms an undesirable source of exonuclease (5). All partially purified preparations of venom exonuclease exhibit adenosine triphosphate-pyrophosphatase activity (cleavage of the a-/3 linkage). Pfleiderer and Ortanderl (14a) studied this issue and showed that during purification the ratio of the two activities remains constant, concluding that both activities are intrinsic properties of the same enzyme. 

Escherichia coli can he adapted to grow on PPi as the sole source of phosphorus in this situation the cells are dependent upon intracellular inorganic pyrophosphatase activity to make Pi available. To avoid possible confusion from the inducible alkaline phosphatase of E. coli, which also has pyrophosphatase activity (see Chapter 17, by Reid and Wilson, this volume), all mutant isolation studies began with an E coli strain unable to synthesize this inducible protein. 

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. 

Enzyme source Glucose-6-P phospho- hydrolase Inorganic pyrophosphatase PPi-glucose phospho- transferase CDP-glucose phospho- transferase... 

Note that the hydrolysis of two high-energy phosphate bonds in ATP provides the energy source for the reaction. The inorganic pyrophosphate, PPi, is subsequently broken down to two phosphate ions by inorganic pyrophosphatase. The action of this enzyme means that very little PPi remains in the cell, making the synthesis of the fatty acyl-CoA favored. This is an example of metabolic coupling, the process whereby a thermodynamically unfavored reaction is allowed because it shares an intermediate (in this case PPO with a favored one. 

FIGURE 30.6 Examples of protein crystals. From left to right (3-secretase inhibitor complex human farnesyl pyrophosphatase in complex with the nitrogen-containing bisphosphonate drug zoledronic acid Crystals of the Abl kinase domain in complex with imatinib - Source Courtesy of S.W. Cowan-Jacob, Novartis Institutes for BioMedical Research crystal of a Cdk2 inhibitor complex. 

The in situ generation of GDP-Fuc from P-fucose-1-phosphate coupled with the transfer of the fucose moiety on to A -acetyllactosamine has also been probed on an analytical scale [96]. In the presence of GTP P-fucose-1-phosphate is converted to GDP-Fuc by GDP-fucose pyrophosphorylase. GDP-Fuc is then used by an a(l-3)fucosyl transferase to transfer the fucose moiety on to A -acetyllactosamine. The released GDP is recycled and phosphorylated to give GTP by a phosphokinase with phosphoenol pyruvate as phosphate source. The pyrophosphate produced in the complete cycle is decomposed by pyrophosphatase (see Scheme 16). 

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