Spectral and kinetic studies of phosphate and magnesium ion binding to yeast inorganic pyrophosphatase

Abstract

Inorganic pyrophosphatase must bind two phosphate molecules in order to catalyze pyrophosphate synthesis. In this report it is shown that P_i causes marked effect on the absorption spectrum of baker's yeast inorganic pyrophosphatase and this effect can be used to analyze P_i binding to this enzyme. A series of absorbance versus P_i concentration curves in the presence of 0.5–20mM free Mg²⁺ were obtained at pH 7.2 and computer-fitted to 19 models. The dissociation constant of magnesium phosphate (8.5 ± 0.4 mM) used in this analysis was measured with a Mg²⁺ -sensitive electrode. The best model implies successive binding of two magnesium phosphate molecules or random-order binding of magnesium phosphate and free phosphate molecules. The first route predominates at physiological concentrations of Mg²⁺. The P_i-inhibition pattern of pyrophosphate hydolysis confirmed that P_i adds to the active site and provided further evidence for the existence of an activating P_i-binding site. The possibility is raised that the pathways of pyrophosphate synthesis and hydrolysis by inorganic pyrophosphatase may differ in the sense that the binding of the fourth metal ion/subunit may facilitate the synthesis and inhibit the hydrolysis.