title: [Two-stage mechanism of the fluoride inhibition of inorganic pyrophosphatase using the fluoride ion].
abstract: Some kinetic and spectral approaches have been used to study the interactions in the enzyme-Mg2+-F--pyrophosphate (or imidodiphosphate, a non-hydrolyzeable pyrophosphate analog) system underlying the mechanism of yeast inorganic pyrophosphatase inhibition by fluoride. The continuous curves of the enzymatic reaction were obtained with an automatic phosphate analyzer operating on the time scale of seconds. Increasing concentrations of NaF caused an increase in the inactivation rate constant to a constant level of 5.3 min-1 for PPi (pH 6.2-7.2) and 3.9 min-1 for imidodiphosphate, (pH 7.2). At a saturating fluoride concentration, the initial rate of PPi hydrolysis dropped to 10%. NaF and imidodiphosphate changed the protein spectrum at 270-310 nm and strengthened the binding of each other to the protein. The binding of F- required a Mg2+-binding site with Kd = 0.15 mM being filled in. The free enzyme and its Ca2+ complex did not bind F-. The experimental results indicate that pyrophosphatase inhibition by fluoride occurs in two steps. The inhibitor adds first to the Mg2+ ion on the enzyme in a readily reversible reaction causing a 90% decrease of the catalytic activity. Thereafter, a slow isomerization of the enzymesubstrate complex takes place, resulting in a complete loss of activity.
mesh_terms: D002118:Calcium / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology*; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D011485:Protein Binding; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012969:Sodium Fluoride / Q000494:pharmacology
chemical_list: D012969:Sodium Fluoride; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium; D005459:Fluorides; D002118:Calcium
vernacular_title: Dvukhstadiĭnyĭ mekhanizm ingibirovaniia neorganicheskoĭ pirofosfatazy ftorid-ionom.