COMPARISON OF THE ALLOSTERIC PROPERTIES OF THE CO(II)-SUBSTITUTED ANDZN(II)-SUBSTITUTED INSULIN HEXAMERS

The positive and negative cooperativity and apparent half-site reactiv ity of the Co(II)-substituted insulin hexamer are well-described by a three-state allosteric model involving ligand-mediated interconversion s between the three states: T3T3' reversible arrow T(3)degrees R(3)deg rees reversible arrow R3R3' [Bloom, C. R., Heymann, R,, Kaarsholm, N. C., and Dunn, M. F, (1997) Biochemistry 36, 12746-12758], Because of t he low affinity of the T state for ligands, this model is defined by f our parameters: L-o(A) and L-o(B), the allosteric constants for the T3 T3' 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 li gand binding to T(3)degrees R(3)degrees and to R3R3'. The d-d electron ic transitions of the Co(II)-substituted hexamer give optical signatur es of the T to R transition which can be quantified, but the ''spectro scopically 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), Whe n the chromophore is bound to the HisB10 sites of the R state, the abs orption 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 condit ions that do not significantly disturb the T to R state equilibrium. U sing 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 li gand affinities of the phenolic pockets are unchanged.