Fluoride effects along the reaction pathway of pyrophosphatase:: Evidence for a second enzyme•pyrophosphate intermediate

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 P-i 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 . P-i 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 P-i. 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.