Two pathways of pyrophosphate hydrolysis and synthesis by yeast inorganic pyrophosphatase

Abstract

Initial rates of pyrophosphate hydrolysis and synthesis by baker's yeast inorganic pyrophosphatase and equilibrium amounts of enzyme-bound and free pyrophosphate were measured over wide ranges of Mg²⁺ and respective substrate concentrations. Computer analysis of these data, in conjunction with those on phosphate/water oxygen exchange [Kasho, V. N. & Baykov, A. A. (1989) Biochem. Biophys. Res. Comm. 161, 475–480], yielded values of the equilibrium constants for Mg²⁺ binding to free enzyme and central complexes and values of the forward and reverse rate constants for the four reaction steps, namely, PP_i binding/release, PP_i hydrolysis/synthesis and two P_i binding/release steps. All catalytic steps were found to proceed through two parallel pathways, involving 3 or 4 Mg²⁺/PP_i or 2 P_i bound. Product release is the slowest catalytic event in both hydrolysis and synthesis of pyrophosphate, at least, for the four-metal pathway. In the hydrolytic reaction, magnesium pyrophosphate binding is faster for the four-metal pathway, dissociation of the second P_i is faster for the three-metal pathway, while PP_i hydrolysis and the release of the first P_i may proceed with similar rates. Release of pyrophosphate formed on the enzyme is faster for the three-metal pathway. Both pathways are expected to operate in vivo, and their relative contributions will vary with changes in the Mg²⁺ concentration, thus providing a means for pyrophosphatase-activity regulation.