The binding of 54Mn2+ to glutamine synthetase from Escherichia coli has been studied because specific divalent cations are involved in the activation and structural stabilization of this enzyme. By equilibrium dialysis, three apparently independent sets of Mn2+ binding sites can be resolved. These include 12 apparently equivalent and independent very highaffinity sites, k1, per molecule of glutamine synthetase of mol wt 600,000 (or 1/subunit), 12 sites of significantly lower apparent affinity, k2′, and approximately 48 relatively low-affinity sites, k3′. The 12 sites of intermediate affinity, k2′, together with those of high affinity, k1′, are probably concerned in catalytic function, whereas some or all of the loose binding sites, k3′ appear to be involved in a gross stabilization of the enzyme structure. Mg2+ binding to the highaffinity sites of glutamine synthetase was studied indirectly by competition experiments with 54Mn2+. The enzyme was found to have a nearly 400-fold lower apparent affinity for Mg2+ (k1′ ≃ 4 × 104) than for Mn2+, although Mg2+ has a uniquely specific catalytic function also. In contrast, the enzyme was found to have a very low affinity for the nonspecific Ba2+ cation (k′ ≃ 200). Varying degrees of adenylylation of the glutamine synthetase molecule, as well as pH, influence the apparent intrinsic association constant at the high-affinity sites for Mn2+, k1′, but do not affect the affinity of the other Mn2+ binding sites, k2′ or k3′, significantly. The binding of the first 12 Mn2+ to apparently equivalent sites appears to be associated with a relatively large over-all conformational change involved in the conversion of the catalytically inactive relaxed enzyme (divalent cation free) into the active taut or tightened form. Considered together with other physical and kinetic data, the results are consistent with the interpretation that the ΔF′ of this confirmational change at pH 7 is a function of the state of adenylylation of the taut enzyme, rather than that of the relaxed enzyme form. As the pH is increased from pH 7 to 8, the increase in k1′ is consistent with previous observations that the quarternary structure of the relaxed enzyme is destabilized by increasing pH. The discharging of at least two ionizing groups per subunit appears to be involved in the pH-induced structural changes wihch indirectly affect k1′. No cooperativity was observed in the binding of Mn2+ in the relaxed to taut enzyme conversion. Instead, a conformational change per subunit, which is modulated by the state of adenylylation of enzyme macromolecule, appears to be associated with the independent binding of each of 12 specific divalent cations. © 1969, American Chemical Society. All rights reserved.
