Fluoride Effects along the Reaction Pathway of Pyrophosphatase: Evidence for a Second Enzyme·Pyrophosphate Intermediate

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Abstract
The fluoride ion is a potent and specific inhibitor of cytoplasmic pyrophosphatase (PPase). Fluoride action on yeast PPase during PPi hydrolysis involves rapid and slow phases, the latter being only slowly reversible [Smirnova, I. N., and Baykov, A. A. (1983) Biokhimiya 48, 1643−1653]. A similar behavior is observed during yeast PPase catalyzed PPi synthesis. The amount of enzyme·PPi complex formed from solution Pi exhibits a rapid drop upon addition of fluoride, followed, at pH 7.2, by a slow increase to nearly 100% of the total enzyme. The slow reaction results in enzyme inactivation, which is not immediately reversed by dilution. These data show that fluoride binds to an enzyme·PPi intermediate during the slow phase and to an enzyme·Pi intermediate during the rapid phase of the inhibition. In Escherichia coli PPase, the enzyme·PPi intermediate binds F- rapidly, explaining the lack of time dependence in the inhibition of this enzyme. The enzyme·PPi intermediate formed during PPi hydrolysis binds fluoride much faster (yeast PPase) or tighter (E. coli PPase) than the similar complex existing at equilibrium with Pi. It is concluded that PPase catalysis involves two enzyme·PPi intermediates, of which only one (immediately following PPi addition and predominating at acidic pH) can bind fluoride. Simulation experiments have indicated that interconversion of the enzyme·PPi intermediates is a partially rate-limiting step in the direction of hydrolysis and an exclusively rate-limiting step in the direction of synthesis.