CARBOXYLATE IONS ARE STRONG ALLOSTERIC LIGANDS FOR THE HISB10 SITES OF THE R-STATE INSULIN HEXAMER

The insulin hexamer is an allosteric protein which displays positive a nd negative cooperativity and half-site reactivity that is modulated b y strong homotropic and heterotropic ligand binding interactions at tw o different loci. These loci consist of phenolic pockets situated on t he dimer-dimer interfaces of T-R and R-R subunit pairs and of anion si tes comprising the HisB10 metal ion sites of the R-3 units of the T3R3 and R-6 states. In this study, we show that suitably tailored organic carboxylates are strong allosteric effecters with relatively high aff inities for the R-state HisB10 metal sites. Methods of quantifying the relative affinities of ligands for these sites in both Co(II)-and Zn( II)-substituted insulin hexamers are presented. These analyses show th at, in addition to the electron density on the ion, the carboxylate af finity is influenced by polar, nonpolar, and hydrophobic interactions between substituents on the carboxylate and the amphipathic protein su rface of the narrow tunnel which controls ligand access to the metal i on. Since the binding of anions to the HisB10 site makes a critically important contribution to the stability of the T3R3 and R-6 forms of t he insulin hexamer, the design of high-affinity ligands with a carboxy late donor for coordination to the metal ion provides an opportunity f or constructing insulin formulations with improved pharmaceutical prop erties.