The Glu(B13) carboxylates of the insulin hexamer form a cage for cadmium and calcium ions

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Abstract
Substitution of Cd2+ for Zn2+ yields a hexameric insulin species containing 3 mol of metal ion per hexamer. The Cd2+ binding loci consist of the 2 histidine (B10) sites and a new site involving the Glu(B13) residues located at the center of the hexamer. Substitution of Co2+ or Co3+ for Zn2+ gives hexamers containing 2 mol of metal per hexamer. Insulin [In] solutions to which both Cd2+ and Co2+ were added in a ratio of 6:2:1 [In]:[Co2+]:[Cd2+] followed by oxidation to the exhange-inert Co3+ state yield stable hybrid species containing both Co3+ and Cd2+ with a composition of (In)6(Co3+)2Cd2+. The kinetics of the reaction of 2,2'',2''''-terpyridine (terpy) with the exchange-labile (In)6(Cd2+)2 derivatives are biphasic and involve the rapid formation of an intermediate with coordination of one terpy molecule to each protein-bound metal ion; then, in a rate-limiting step the terpy-coordinated metal ion dissociates from the protein, and a second molecule of terpy binds to the metal ion to form a bis complex. Reaction of the exchange-inert Co3+ ions of (In)6(Co3+)2 with terpy is a slow apparent first-order process (t1/2 [half-life] = 13.1 h). In contrast to the kinetic behavior of (In)6(Co2+)2 and (In)6(Cd2+)2, the Cd2+ ions bound to the hybrid (In)6(Co3+)2Cd2+ react quite slowly with terpy (t1/2 = 1 h at pH 8.0). Cd2+ is caged within the central cavity of this hybrid hexamer at the Glu(B13) site and that the rate of reaction with terpy is determined by the slow rate of escape of Cd2+ from this cage. Competition studies indicate that Ca2+ and Cd2+ compete for the Glu(B13) site. Equilibrium binding studies using 45Ca2+ substantiate the presence of a single high-affinity Ca binding site with kd = 83 .mu.M, which may involve the Glu(B13) carboxylates.