Inorganic Pyrophosphatase - Mechanism

Mechanism

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 hydroxide 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.