title: Rates of elementary steps catalyzed by rat liver cytosolic and mitochondrial inorganic pyrophosphatases in both directions.
abstract: We have investigated kinetics of pyrophosphate synthesis and phosphate-water oxygen exchange catalyzed by rat liver cytosolic and mitochondrial pyrophosphatases in the presence of Mg2+ as cofactor. A common kinetic model derived for these reactions implies that they involve formation of enzyme-bound pyrophosphate and proceed through two parallel pathways: pathway I, utilizing two magnesium phosphate molecules, and pathway II, utilizing both magnesium phosphate and free phosphate. Pyrophosphate formation is greatly facilitated in the active sites of both pyrophosphatases ([E.PPi]/[E.2Pi] = 0.11-0.24) compared to solution. The rate constants for PPi binding/release, bound PPi hydrolysis/synthesis, and two Pi binding/release steps catalyzed by cytosolic and mitochondrial pyrophosphatases were enumerated for pathway I. There is no unique rate-limiting step for pathway I for both enzymes in either direction. A modulating effect of magnesium phosphate on the oxygen exchange is observed with the cytosolic pyrophosphatase, explicable in terms of an allosteric phosphate-binding site or random-order release of two phosphate molecules from the active site. A remarkable feature of these mammalian pyrophosphatases versus their microbial counterparts is their high efficiency in pyrophosphate synthesis. The turnover numbers in the direction of synthesis are 14 and 9.3 s-1 for the cytosolic and mitochondrial enzymes, respectively (9 and 16% relative to hydrolysis turnover numbers). The results demonstrate that the enzyme-catalyzed synthesis of pyrophosphate, the simplest high-energy polyphosphate, can proceed at a high rate in the absence of an external energy input, such as that provided by protonmotive force in membrane systems.
authors.affiliation: A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry, Moscow State University, Russian Federation.
authors.forename: I N
authors.initials: IN
authors.lastname: Smirnova
authors.forename: V N
authors.initials: VN
authors.lastname: Kasho
authors.forename: S E
authors.initials: SE
authors.lastname: Volk
authors.forename: A H
authors.initials: AH
authors.lastname: Ivanov
authors.forename: A A
authors.initials: AA
authors.lastname: Baykov
mesh_terms: D000818:Animals; D003600:Cytosol / Q000201:enzymology; D043564:Inorganic Pyrophosphatase; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D008962:Models, Theoretical; D011755:Pyrophosphatases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats
chemical_list: D007527:Isoenzymes; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D008274:Magnesium