Insulin—Metal Ion Interactions: The Binding of Divalent Cations to Insulin Hexamers and Tetrarners and the Assembly of Insulin Hexamers

An insulin hexamer containing one B 10-bound Co(III) ion and one unoccupied B10 site has been synthesized. The properties of the monosubstituted hexamer show that occupancy of only one B10 site by Co³⁺is sufficient to stabilize the hexameric form under the conditions of pH and concentration used in these studies. The experimentally determined, second-order rate constants for the binding of Zn²⁺and Co²⁺to the unoccupied B10 site are consistent with literature rate constants for the rate of association of these divalent metal ions with similar small molecule ligands. These findings indicate that the rate-limiting steps for Zn²⁺and Co²⁺binding involve the removal of the first aqua ligand. The rate constant for the binding of Cd²⁺is significantly lower than the literature values for small molecule chelators, which suggests that some other protein-related process is rate-limiting for Cd²⁺binding to the unoccupied, preformed B10 site. The kinetics of the assembly of insulin in the presence of limiting metal ion provides strong evidence indicating that the B13 site of the tetramer species can bind Zn²⁺, Cd²⁺, or Ca²⁺prior to hexamer formation and that such binding assists hexamer formation. Both the tetramer and the hexamer B13 sites were found to exhibit similar affinities for Zn²⁺and Cd²⁺(K_d≃ 9 μM), whereas the tetramer B13 sites bind Ca²⁺much more weakly (K_d≃ 1 mM for tetramer vs 83 μMfor hexamer). The second-order rate constants estimated for the association of Zn²⁺and Cd²⁺to the tetrameric site indicate that the loss of the first inner-sphere aqua ligand is the rate-limiting step for binding.