부산대학교 도서관

Hexameric inorganic pyrophosphatase from Mycobacterium tuberculosis (Mt-PPase) has a number of structural and functional features that distinguish it from homologous enzymes widely occurring in living organisms. In particular, it has unusual zones of inter-subunit contacts and lacks the N-terminal region common for other PPases. In this work, we constructed two mutant forms of the enzyme, Ec-Mt-PPase and R14Q-Mt-PPase. In Ec-Mt-PPase, the missing part of the polypeptide chain was compensated with a fragment of PPase from Escherichia coli (Ec-PPase). In R14Q-Mt-PPase, a point mutation was introduced to the contact interface between the two trimers of the hexamer. Both modifications significantly improved the catalytic activity of the enzyme and abolished its inhibition by the cofactor ([Mg.sup.2+] ion) excess. Activation of Mt-PPase by low (~10 [micro]M) concentrations of ATP, fructose-1-phosphate, L-malate, and non-hydrolyzable substrate analogue methylene bisphosphonate (PCP) was observed. At concentrations of 100 [micro]M and higher, the first three compounds acted as inhibitors. The activating effect of PCP was absent in both mutant forms, and the inhibitory effect of fructose-1phosphate was absent in Ec-Mt-PPase. The effects of other modulators varied only quantitatively among the mutants. The obtained data indicate the presence of allosteric sites in Mt-PPase, which are located in the zones of inter-subunit contact or associated with them. DOI: 10.1134/S0006297920030086 Keywords: pyrophosphatase, site-directed mutagenesis, allosteric regulation, fructose-1-phosphate, L-malate
Family I soluble inorganic pyrophosphatases (PPases) catalyze hydrolysis of inorganic pyrophosphate with the formation of orthophosphate. PPases are present in the majority of living organisms, where they hydrolyze pyrophosphate, thus [...]