Structural dynamics of bacterial ribosomes: V. Magnesium-dependent dissociation of tight couples into subunits: Measurements of dissociation constants and exchange rates

The magnesium ion-dependent equilibrium of vacant ribosome couples with their subunits

$\text{70}\text{S}\text{?}\text{k}{}_{\mathrm{?1}}\text{k}{}_1\text{50}\text{S}\text{+30}\text{S}$

has been studied quantitatively with a novel equilibrium displacement labeling method which is more sensitive and precise than light-scattering. At a concentration of 10^?7m, tight couples (ribosomes most active in protein synthesis) dissociate between 1 and 3 mm-Mg²⁺ at 37 °C with a 50% point at 1.9 mm. The corresponding association constants K_a′ are 5.1 × 10⁵m^?1 (1 mm-Mg²⁺), 3.5 × 10⁷m^?1 (2 mm), and 1.2 × 10⁹m^?1 (3 mm), about five orders of magnitude higher than the K_a′ value of loose couples studied by Spirin et al. (1971) and Zitomer & Flaks (1972).In this range of Mg²⁺ concentrations ($\text{37 °C, 50 mm-}\text{NH}{}_4{}^{\mathrm{+}}$) the rate constants depend exponentially and in opposite ways on the Mg²⁺ concentration: k₁ = 2.2 × 10^?3s^?1, k_?1 = 7.7 × 10⁴m^?1s^?1 (2mm-Mg²⁺); k₁ = 1.5 × 10^?4s^?1, k_?1 = 1.7 × 10⁷m^?1s^?1 (5 mm-Mg²⁺). Under physiological conditions (Mg²⁺ ～- 4 mm, ribosome concn ～- 10^?7m), the equilibrium strongly favors association and the rate of exchange is slow ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～- 10}\text{min}$). In the range of dissociation (2 mm-Mg²⁺), association of subunits proceeds without measurable entropy change and hence ΔG^O = ΔH^O. The negative enthalpy change of ΔH^O = ? 10 kcal suggests that association of subunits involves a shape change.Below a critical Mg²⁺ concentration (～- 2 mm), the 50 S subunits are converted irreversibly into the b-form responsible for the transition to loose couples. The results are compatible with two classes of binding sites, one class binding Mg²⁺ non-co-operatively and contributing to the free energy of association by reduction of electrostatic repulsion, and another class probably consisting of hydrogen bonds between components at opposite interfaces whose critical spatial alignment rapidly denatures in the absence of stabilizing magnesium ions.