PubMed1997
The insulin hexamer is an allosteric protein capable of undergoing transitions between three conformational states: T6, T3R3, and R6. These transitions are mediated by the binding of phenolic compounds to the R-state subunits, which provide positive homotropic effects, and by the coordination of anions to the bound metal ions, which act as heterotropic effectors. Since the insulin monomer is far more susceptible than the hexamer to thermal, mechanical, and chemical degradation, insulin-dependent diabetic patients rely on pharmaceutical preparations of the Zn-insulin hexamer, which act as stable forms of the biologically active monomeric insulin. In this study, the chromophoric chelator 2,2',2"-terpyridine (terpy) has been used as a kinetic probe of insulin hexamer stability to measure the effect of homotropic and heterotropic effectors on the dissociation kinetics of the Zn2+- and Co2+-insulin hexamer complexes. We show that the reaction between terpy and the R-state-bound metal ion is limited by the T3R3 <==> T6 or R6 <==> T3R3 conformational transition steps and the dissociation of one anionic ligand, or one anionic ligand and three phenolic ligand molecules, respectively, for T3R3 and R6. Consequently, because the activation energies of these steps are dominated by the ground-state stabilization energy of the R-state species, the kinetic stabilization of the insulin hexamer toward terpy-induced dissociation is linked to the thermodynamic stabilization of the hexamer. The mass action effect of anion binding and, foremost, of phenolic ligand binding provides the major mechanism of stabilization, resulting in the tightening of the tertiary and quaternary hexamer structures. Using this kinetic method, we show that the R6 conformation of Zn-insulin in the presence of Cl- ion and resorcinol is > 1.5 million-fold more stable than the T3 units of T6 and T3R3 and > 70,000-fold more stable than the R3 unit of T3R3. Furthermore, the stabilization effect is correlated with the affinity of the ligands: the tighter the binding, the slower the reaction between terpy and R-state-bound metal ion. These concepts provide a new basis for the pharmaceutical improvement of the physicochemical stability of formulations both for native insulin and for fast-acting monomeric insulin analogues through ligand-mediated allosteric interactions.