Accepted_test

Mutations in human ppa2 gene encoding mitochondrial inorganic pyrophosphatase (PPA2) result in mitochondria malfunction and cardio pathologies. Identification of the molecular basis of this disease requires a detailed understanding of PPA2 metabolic role, regulation, and the impact of particular mutations on the enzyme structure and function. This work is aimed at the in vitro characterization of the catalytic properties of PPA2, analysis of its structure, and the characterization of the effects of mutations responsible for human cardio pathologies on the protein structure and function. In this work we determined functional parameters of hPPA2 and its variant Met94Val corresponding to a pathogenic natural variant. We show that this mutation affects catalytic properties of PPA2 without global changes in its structure or stability. Structural analysis of PPA2 and molecular dynamic simulation allow the insight into the structural basis of the catalytic incompetence of Met94Val. We suppose that this mutation impairs the rearrangements required for the formation and activation of an attacking nucleophile, and disrupts the connections between catalytic residues and the protein core, thereby distorting the pre-organized active site architecture and as a consequence decreasing catalytic efficiency. The data obtained in the work also allow the prediction of functional defects caused by other pathogenic mutant variants of hPPA2.