Effects of calcium ion on ternary complexes formed between 4-(2-pyridylazo)resorcinol and the two-zinc insulin hexamer

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Abstract
As a means for probing the microenvironment of zinc in the insulin hexamer and to investigate the effects of calcium ion on the assembly and the structure of the two-zinc insulin hexamer, the thermodynamics and kinetics of the reaction between the chromophoric divalent metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) and zinc-insulin have been investigated over a wide range of conditions. For [PAR]o .mchgt. [Zn2+]o and [Zn2+]/[In] .ltoreq. 0.33, the reaction leads to the sequestering and ultimate removal of all of the insulin-bound Zn2+; for [Zn2+]o .mchgt. [PAR]0, two stable ternary complexes are formed where Zn2+ has ligands derived from PAR as well as from hexameric insulin. For [Zn2+]/[In] ratios below 0.33, the equilibrium distribution between the two ternary complexes is dependent on the [Zn2+]/[In] ratio. One of the complexes is assigned to the monoanion of PAR coordinated to Zn2+ that resides in aHis-B10 site. The other complex is proposed to involve the coordination of (PAR)Zn to the site formed by the .alpha.-NH2 group of Phe-B1 and the .gamma.-carboxylate ion of Glu-A17 across the dimer-dimer interface on the surface of the hexamer. With either PAR or zinc-insulin in large excess, the kinetics of the PAR optical density changes are remarkably similar and biphasic. The faster step is first order in insulin-bound Zn2+ (k .simeq. 3 .times. 103 M-1 s-1) and involves the formation of an intermediate in which PAR is coordinated to insulin-bound zinc at the His-B10 site. When [PAR]o .mchgt. [Zn2+]o, this intermediate is transformed in the slower step into the Zn2+ (PAR)2 bis complex and metal-free insulin. When [Zn2+]o .mchgt. [PAR]o, the intermediate migrates from the His-B10 sites to a different site postulated to be the Phe-B1-Glu-A17 site. The rate of the slower step appears to be limited by the dissociation of the (PAR)Zn complex from the His-B10 site under both conditions. The binding of Ca2+ to the Glu-B13 sites of the insulin hexamer stabilizes the intermediate formed at the His-B10 sites in the PAR reaction. Hence, the overall affinity to hexameric His-B10 sites fo rZn2+ is enhanced when calcium binds to the Glu-B13 site, and therefore, calcium drives the assembly of the two-zinc insulin hexamer.