Divalent metal ion, inorganic phosphate, and inorganic phosphate analog binding to yeast inorganic pyrophosphatase

Abstract

Four different techniques, equilibrium dialysis, protection of enzymatic activity against chemical inactivation, 31P relaxation rates, and water proton relaxation rates, were used to study divalent metal ion, Pi and inorganic phosphate analog binding to yeast inorganic pyrophosphatase, EC 3.6.1.1. A major new finding is that the binding of a 3rd divalent metal ion per subunit, which has elsewhere been implicated as being necessary for enzymatic activity [Springs, B., et. al (1981)], only becomes evident in the presence of added Pi binding to both its high- and low-affinity sites on the enzyme is markedly enhanced in the presence of divalent metal ions, with Mn2+ causing an especially large increase in affinity. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and Pi or of Co2+ and inorganic phosphate to the enzyme and show that the high-affinity site has greater specificity for inorganic phosphate than the low-affinity site. In addition, they provide evidence against divalent metal ion inner sphere binding to phosphate for enzyme subunits having 1 or 2 divalent metal ions bound per subunit and evidence for a conformational change restricting active-site acessibility to solvent on the binding of a 3rd divalent metal ion per subunit.