Fluoride inhibition of inorganic pyrophosphatase. III. Dependence on the nature of substrate and metal ion cofactor.

Studies of fluoride interaction with bakers' yeast inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) in the presence or absence of enzyme-catalyzed reactions have revealed pronounced specificity of this inhibitor. It was found that the inhibition of enzymic hydrolysis of PPi, ADP, ATP and tripolyphosphate in the presence of Zn2+ and Mn2+ at pH 6.5 is not time dependent and by far less extensive as that observed for the Mg2+-stimulated cleavage of PPi (apparent Ki values differ by three orders of magnitude). Addition of Ca2+ to the latter reaction decrease proportionally the activity of the enzyme and the rate constant for the binding of fluoride to it, which indicates that the enzyme-substrate complexes containing both Mg2+ and Ca2+ are inert in the reaction with fluoride. Preincubation of pyrophosphatase with NaF and various metal cations and substrates, in conditions where the enzyme does not act as a catalyst, does not affect its activity compared to controls lacking fluoride. The results are consistent with the proposed mechanism of mutual hindrance of substrate and fluoride release from the active site of pyrophosphatase.