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 rats, and water proton relaxation rates, are used to study divalent metal ion, inorganic phosphate, and inorganic phosphate analogue binding to yeast inorganic pyrophosphatase, EC 3.6.1.1. A major new finding is that the binding of a third divalent metal ion per subunit, which has elsewhere been implicated as being necessary for enzymatic activity [Springs, B., Welsh, K. M., & Cooperman, B. S. (1981) Biochemistry (in press)], only becomes evident in the presence of added inorganic phosphate and that, reciprocally, inorganic phosphate 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 inorganic phosphate 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. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate 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. The results obtained allow evaluation of all of the relevant equilibrium constants for the binding of Mn2+ and inorganic phosphate 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 one or two divalent metal ions bound per subunit and evidence for a conformational change restricting active-site accessibility to solvent on the binding of a third divalent metal ion per subunit.

The role of the metal ion in the p21ras catalysed GTP-hydrolysis: Mn2+ versus Mg2+

“…The smallest known ribozyme, UUU, has been found to be active only in the presence of Mn 2 (Kazakov & Altman, 1992) and this is postulated to be due to the ability of Mn 2 to coordinate to oxygen and nitrogen ligands of RNA (Dange et al, 1990). In the case of inorganic phosphatase (Cooperman et al, 1981) and DNA polymerase the rate is decreased in the presence of Mn 2 (Burgers & Eckstein, 1979). The situation is even more complicated with the cAMP-dependent protein kinase (Bossemeyer et al, 1993;Zengh et al, 1993) or the chaperone GroEL, where small amounts of Mn 2 have only a slight effect whereas higher concentrations are inhibitory (Diamant et al, 1995).…”

The Electrophilic and Leaving Group Phosphates in the Catalytic Mechanism of Yeast Pyrophosphatase

“…We will show below that this is not the case for Mn 2ϩ and Co 2ϩ as cofactors. Third, protection of Arg 78 , located at P1, against chemical modification upon binding of 1 mol of P i /mol of subunit in the presence of Mn 2ϩ was interpreted as showing that P1 binds first (13). However, because of the close proximity of P1 and P2, the protection could equally result from binding to P2.…”

“…By comparison with Mg 2ϩ , these cations induce a much greater difference in the binding affinities of P1 and P2, as evidenced by the fact that only one P i binding site/subunit was observed over a wide range of P i concentrations (13,14). In the presence of Mg 2ϩ , the affinities of P1 and P2 for MgP i differ only 1-9-fold (9,15,16); for comparison, macroscopic binding constants for two sites with equal microscopic constants differ 4-fold (17).…”

X‐Ray Structure of Yeast Inorganic Pyrophosphatase Complexed with Manganese and Phosphate

“…Binding of metal ions to PPase and their role in catalysis have been intensively studied (Rapoport et al, 1973;Baykov and Avaeva, 1974;Cooperman et al, 1981 ;Knight et al, 1984). Biochemical, physicochemical, and kinetic studies of PPase were reviewed by Cooperman (1982) and a hypothetical scheme of PP, hydrolysis proposed.…”

“…The nature of the metal strongly influences substrate and product binding as well as the P,/PP, equilibrium. Thus phosphate binds much more strongly in the presence of manganese compared to magnesium (Cooperman et al, 1981). In this study, we have undertaken the structure analysis of the complex of YPPase with Mn2+ and P,, the product of the reaction.…”