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: T3T3' reversible arrow T(3)degrees R(3)degrees reversible arrow R3R3' [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 T3T3' to T(3)degrees R(3)degrees and the T(3)degrees R(3)degrees to R3R3' transitions, respectively, and the two dissociation constants for ligand binding to T(3)degrees R(3)degrees and to R3R3'. 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.