Inorganic diphosphatase

Inorganic pyrophosphatase (or inorganic diphosphatase, PPase) is an enzyme (EC 3.6.1.1) that catalyzes the conversion of one ion of pyrophosphate to two phosphate ions. This is a highly exergonic reaction, and therefore can be coupled to unfavorable biochemical transformations in order to drive these transformations to completion. The functionality of this enzyme plays a critical role in lipid metabolism (including lipid synthesis and degradation), calcium absorption and bone formation, and DNA synthesis, as well as other biochemical transformations. Inorganic pyrophosphatase (or inorganic diphosphatase, PPase) is an enzyme (EC 3.6.1.1) that catalyzes the conversion of one ion of pyrophosphate to two phosphate ions. This is a highly exergonic reaction, and therefore can be coupled to unfavorable biochemical transformations in order to drive these transformations to completion. The functionality of this enzyme plays a critical role in lipid metabolism (including lipid synthesis and degradation), calcium absorption and bone formation, and DNA synthesis, as well as other biochemical transformations. Two types of inorganic diphosphatase, very different in terms of both amino acid sequence and structure, have been characterised to date: soluble and transmembrane proton-pumping pyrophosphatases (sPPases and H(+)-PPases, respectively). sPPases are ubiquitous proteins that hydrolyse pyrophosphate to release heat, whereas H+-PPases, so far unidentified in animal and fungal cells, couple the energy of PPi hydrolysis to proton movement across biological membranes. Thermostable soluable pyrophosphatase had been isolated from the extremophile Thermococcus litoralis. The 3-dimensional structure was determined using x-ray crystallography, and was found to consist of two alpha-helices, as well as an antiparallel closed beta-sheet. The form of inorganic pyrophosphatase isolated from Thermococcus litoralis was found to contain a total of 174 amino acid residues and have a hexameric oligomeric organization (Image 1). Humans possess two genes encoding pyrophosphatase, PPA1 and PPA2. PPA1 has been assigned to a gene locus on human chromosome 10, and PPA2 to chromosome 4. Though the precise mechanism of catalysis via inorganic pyrophosphatase in most organisms remains uncertain, site-directed mutagenesis studies in Escherichia coli have allowed for analysis of the enzyme active site and identification of key amino acids. In particular, this analysis has revealed 17 residues of that may be of functional importance in catalysis. Further research suggests that the protonation state of Asp67 is responsible for modulating the reversibility of the reaction in Escherichia coli. The carboxylate functional group of this residue has been shown to perform a nucleophilic attack on the pyrophosphate substrate when four magnesium ions are present. Direct coordination with these four magnesium ions and hydrogen bonding interactions with Arg43, Lys29, and Lys142 (all positively charged residues) have been shown to anchor the substrate to the active site. The four magnesium ions are also suggested to be involved in the stabilization of the trigonal bipyramid transition state, which lowers the energetic barrier for the aforementioned nucleophilic attack. Several studies have also identified additional substrates that can act as allosteric effectors. In particular, the binding of pyrophosphate (PPi) to the effector site of inorganic pyrophosphatase increases its rate of hydrolysis at the active site. ATP has also been shown to function as an allosteric activator in Escherichia coli, while fluoride has been shown to inhibit hydrolysis of pyrophosphate in yeast. The hydrolysis of inorganic pyrophosphate (PPi) to two phosphate ions is utilized in many biochemical pathways to render reactions effectively irreversible. This process is highly exergonic (accounting for approximately a −19kJ change in free energy), and therefore greatly increases the energetic favorability of reaction system when coupled with a typically less-favorable reaction. Inorganic pyrophosphatase catalyzes this hydrolysis reaction in the early steps of lipid degradation, a prominent example of this phenomenon. By promoting the rapid hydrolysis of pyrophosphate (PPi), Inorganic pyrophosphatase provides the driving force for the activation of fatty acids destined for beta oxidation.