A two-step mechanism of fluoride inhibition of rat liver inorganic pyrophosphatase.
Product formation curves for inorganic pyrophosphatase-catalyzed hydrolysis of pyrophosphate in the presence of fluoride were analyzed in order to get insight into the mechanism of its inhibitory action on this enzyme. The enzymatic reaction was monitored with a phosphate analyzer operating on the time scale of seconds. Inhibition patterns were virtually identical for cytosolic and mitochondrial pyrophosphatases. The effect of fluoride was biphasic: it caused a rapid (t 1/2 less than 1 s) decrease in the initial velocity of the reaction followed by slow (t 1/2 greater than or equal to 4 s) inactivation of the enzyme during catalysis. The slow phase resulted in trapping intact substrate at the active site, and the resulting complex could be isolated by gel filtration. Pyrophosphatase remained active when incubated with fluoride in the absence of pyrophosphate or in the presence of its bisphosphonate analogs, which are bound to but not hydrolyzed by this enzyme. These features of the inhibition are consistent with the mechanism in which rapid binding of the inhibitor to the enzyme.substrate complex is followed by its slow isomerization. Kinetic parameters obtained in this work indicate that appreciable inactivation of pyrophosphatase can occur at fluoride concentrations found in human plasma. This effect may therefore be one of the major factors contributing to fluoride toxicity.
Baykov
A A
AA
A. N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, USSR.
Alexandrov
A P
AP
Smirnova
I N
IN
D000818:Animals; D002850:Chromatography, Gel; D003600:Cytosol / Q000201:enzymology; D011756:Diphosphates / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051381:Rats; D012969:Sodium Fluoride / Q000494:pharmacology*
D011756:Diphosphates; D012969:Sodium Fluoride; D011755:Pyrophosphatases