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 Mg 2+ 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 · PP i]/[E · 2 P i] = 0.11-0.24) compared to solution. The rate constants for PP i binding/ release, bound PP i hydrolysis/synthesis, and two P i binding/release steps catalyzed by cytosolic add 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 tn 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.