Nuclear magnetic resonance studies of inorganic phosphate binding to yeast inorganic pyrophosphatase

Abstract

Yeast inorganic pyrophosphatase [EC 3.6.1.1] is a dimer of identical subunits. Previous work indicated the presence of 2 different Mn2+ binding sites per subunit. The binding of inorganic phosphate to the Mn2+-inorganic pyrophosphatase complex was now studied by 1H and 31P NMR. Two distinct phosphate sites were found, having dissociation constants of 0.24 mM and 18 mM. The Mn2+-31P distance from tightly bound Mn2+ to phosphate bound in the low affinity site (6.2 .ANG.) is consistent with outer sphere binding. Binding to both phosphate sites can be simultaneously inhibited by the pyrophosphate analog, hydroxymethanebisphosphonate, providing evidence for the phsyical proximity of these 2 sites. The weaker Mn2+ site is apparently far from both phosphate sites. From the magnitudes of the dissociation constants found for both phosphate and analog binding and the recent work of P.D. Boyer and his co-workers (private communication) on enzyme-catalyzed phosphate-water exchange, it appears unlikely that the hydrolysis of enzyme-bound pyrophosphate is the rate-determining step in the overall enzymatic catalysis of pyrophosphate hydrolysis, at least when Mn2+ is the required divalent metal ion cofactor.