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, PPi binding/release, PPi hydrolysis/synthesis and two Pi binding/release steps. All catalytic steps were found to proceed through two parallel pathways, involving 3 or 4 Mg2+/PPi or 2 Pi 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 Pi is faster for the three‐metal pathway, while PPi hydrolysis and the release of the first Pi 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. Copyright © 1992, Wiley Blackwell. All rights reserved