Human mitochondrial pyrophosphatase hPPA2: In vitro and in silico study of the factors affecting its function

Biallelic mutations in a nuclear gene PPA2 in human encoding mitochondrial inorganic pyrophosphatase hPPA2 cause mitochondrial disfunctions leading to severe cardiac pathology. This protein catalyzes a Mg²⁺-dependent hydrolysis of pyrophosphate, a by-product of many biosynthetic reactions, thereby providing a thermodynamic pull for these reactions. In order to better understand molecular mechanisms of mitochondrial disfunction caused by mutations in PPA2 gene, detailed characterization of a metabolic role of hPPA2 is required. In this work, we study the in vitro effects of a panel of metabolites, as well as other factors, on the recombinant hPPA2 activity. This study is complemented with the in silico assessment of possible mechanisms of observed patterns. We demonstrate that at Mg²⁺ concentrations typical for the mitochondrial matrix hPPA2 works at under-saturation and therefore the regulatory factors changing Mg²⁺ concentration will have a significant impact on hPPA2 activity. We also demonstrate that hPPA2 activity is regulated by a redox state of cysteine residues. Mass-spectrometry analysis reveals four Cys residues modified by 4-hydroxy-Hg-benzoate, as well as an SS bridge formation within an hPPA2 monomer. Finally, we found that selected metabolites including intermediates of central metabolism affect hPPA2 activity. In silico analysis of hPPA2 allows a structural insight into the observed properties. Of a special interest is an W-loop, unique for PPA2 proteins from animals and shared with acidocalcisomal soluble pyrophosphatases from kinetoplastids, that may be involved in interaction with small-molecule effectors.