Comparison of the Allosteric Properties of the Co(II)- and Zn(II)-Substituted Insulin Hexamers

The positive and negative cooperativity and apparent half-site reactivity of the Co(II)-substituted insulin hexamer are well-described by a three-state allosteric model involving ligand-mediated interconversions between the three states:  T_3T_3‘ ⇌ T_3°R_3° ⇌ R_3R_3‘ [Bloom, C. R., Heymann, R., Kaarsholm, N. C., and Dunn, M. F. (1997) Biochemistry 36, 12746−12758]. Because of the low affinity of the T state for ligands, this model is defined by four parameters:  L_o^A and L_o^B, the allosteric constants for the T_3T_3‘ to T_3°R_3° and the T_3°R_3° to R_3R_3‘ transitions, respectively, and the two dissociation constants for ligand binding to T_3°R_3° and to R_3R_3‘. The d−d electronic transitions of the Co(II)-substituted hexamer give optical signatures of the T to R transition which can be quantified, but the “spectroscopically silent” character of Zn(II) has made previous attempts to describe the Zn(II) species difficult. This work shows that the T to R state conformational transitions of the Zn(II) hexamer can be easily quantified using the chromophore 4-hydroxy-3-nitrobenzoate (4H3N). When the chromophore is bound to the HisB10 sites of the R state, the absorption spectrum of 4H3N is red-shifted, exhibiting strong absorbance and CD signals, whereas 4H3N does not bind to the T state. Hence, 4H3N can be employed as a sensitive indicator of conformation under conditions that do not significantly disturb the T to R state equilibrium. Using 4H3N as an indicator, these studies show that both L_o^A and L_o^B are made less favorable by the substitution of Co(II) for Zn(II); L_o^A is increased by 10-fold while L_o^B by 35-fold, whereas the ligand affinities of the phenolic pockets are unchanged

Comparison of the Allosteric Properties of the Co(II)- and Zn(II)-Substituted Insulin Hexamers

The positive and negative cooperativity and apparent half-site reactivity of the Co(II)-substituted insulin hexamer are well-described by a three-state allosteric model involving ligand-mediated interconversions between the three states:  T_3T_3‘ ⇌ T_3°R_3° ⇌ R_3R_3‘ [Bloom, C. R., Heymann, R., Kaarsholm, N. C., and Dunn, M. F. (1997) Biochemistry 36, 12746−12758]. Because of the low affinity of the T state for ligands, this model is defined by four parameters:  L_o^A and L_o^B, the allosteric constants for the T_3T_3‘ to T_3°R_3° and the T_3°R_3° to R_3R_3‘ transitions, respectively, and the two dissociation constants for ligand binding to T_3°R_3° and to R_3R_3‘. The d−d electronic transitions of the Co(II)-substituted hexamer give optical signatures of the T to R transition which can be quantified, but the “spectroscopically silent” character of Zn(II) has made previous attempts to describe the Zn(II) species difficult. This work shows that the T to R state conformational transitions of the Zn(II) hexamer can be easily quantified using the chromophore 4-hydroxy-3-nitrobenzoate (4H3N). When the chromophore is bound to the HisB10 sites of the R state, the absorption spectrum of 4H3N is red-shifted, exhibiting strong absorbance and CD signals, whereas 4H3N does not bind to the T state. Hence, 4H3N can be employed as a sensitive indicator of conformation under conditions that do not significantly disturb the T to R state equilibrium. Using 4H3N as an indicator, these studies show that both L_o^A and L_o^B are made less favorable by the substitution of Co(II) for Zn(II); L_o^A is increased by 10-fold while L_o^B by 35-fold, whereas the ligand affinities of the phenolic pockets are unchanged