THE EFFECTS OF SULFITE OR NITRATE ON TURNOVER-DEPENDENT INHIBITION IN THE ATPASE FROM HALOBACTERIUM-SACCHAROVORUM ARE RELATED TO THE BINDING OF THE 2ND METAL-ION

The turnover-dependent inhibition of the Halobacterium saccharovorum ATPase is dependent on two parameters: pH and the concentration of the divalent cation present. At pH 6 and 1 mM Mn2+ the inhibition is small, but increases steeply with 6 mM Mn2+. In contrast, at pH 8.5 the inhibition is more than 90% at 1 mM Mn2+, and higher concentrations have little additional effect. A relationship between the occupation of a second metal ion binding site and turnover-dependent inhibition was postulated previously [Schobert, B. (1992) J. Biol. Chem. 267, 10252-10257]. The results lead to a model where this site (X-) can alternatively bind protons (XH), depending on the pH and the free metal ion concentration. The pK(a) of XH is estimated to be 9. The turnover-dependent inhibition is diminished by bisulfite, whereas sulfite is ineffective. The kinetics show that bisulfite and metal ion compete for the same site. In the proposed model, bisulfite binds via its negative charge to the site from which P(i) was released and is arranged such as to interact with X- via its protonated group (X-HSO3-). In this way, formation of the inhibited enzyme species XMe is prevented. Inhibitory anions like nitrate, which carry a permanent dipole as a common feature, show uncompetitive inhibition vs metal ions. The data are compatible with a model in which these inhibitors bind to the vacant P(i) site and position their positive charges near XH. As a consequence, the pK(a) of XH is decreased and X- is stabilized, which in turn favors the formation of XMe. The downshift in pK(a) was calculated to be 0.7 pH unit.