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 PP(i) 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 PP(i) synthesis. The amount of enzyme.PP(i) 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.PP(i) 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.PP(i) intermediate binds F(-) rapidly, explaining the lack of time dependence in the inhibition of this enzyme. The enzyme.PP(i) intermediate formed during PP(i) 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.PP(i) intermediates, of which only one (immediately following PP(i) addition and predominating at acidic pH) can bind fluoride. Simulation experiments have indicated that interconversion of the enzyme.PP(i) intermediates is a partially rate-limiting step in the direction of hydrolysis and an exclusively rate-limiting step in the direction of synthesis.
Baykov
A A
AA
A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Moscow 119899, Russia. baykov@genebee.msu.su
Fabrichniy
I P
IP
Pohjanjoki
P
P
Zyryanov
A B
AB
Lahti
R
R
D002384:Catalysis; D011756:Diphosphates / Q000138:chemical synthesis* / Q000737:chemistry; D004789:Enzyme Activation; D004795:Enzyme Stability; D005459:Fluorides / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D007700:Kinetics; D017616:Magnesium Compounds / Q000737:chemistry; D008956:Models, Chemical; D010710:Phosphates / Q000737:chemistry; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis* / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012969:Sodium Fluoride / Q000737:chemistry
D011756:Diphosphates; D017616:Magnesium Compounds; D010710:Phosphates; C031288:magnesium fluoride; D012969:Sodium Fluoride; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D005459:Fluorides