Mechanism of Cadmium Ion Substitution in Mammalian Zinc Metallothionein and Metallothionein α Domain: Kinetic and Structural Studies

Cellular metallothionein (MT) protects against Cd<sup>2+</sup> exposure through direct binding of the metal ion. The model reaction between rabbit liver Zn<sub>7</sub>-MT-2 with Cd<sup>2+</sup> was studied with stopped flow kinetics. Four kinetic steps were observable. Comparison of this reaction with an analog utilizing the MT Zn<sub>4</sub>-α domain revealed that only the fastest step involved the Zn<sub>3</sub>-β domain. Each step of the Zn<sub>4</sub>-α domain reaction with Cd<sup>2+</sup> displayed hyperbolic dependence of the observed rate constant on Cd<sup>2+</sup> concentration, with the first step comprising 50% of the total reaction and each of the other two, 25%. The two constants extracted from each of these relationships were interpreted as the equilibrium constant for the initial binding of Cd<sup>2+</sup> to the Zn<sub>(4−<i>n</i>)</sub>,Cd<sub><i>n</i></sub>−thiolate cluster (<i>n</i> = 0−3) of the α domain and the first order rate constant for the exchange of Cd<sup>2+</sup> for Zn<sup>2+</sup> in the cluster. Activation enthalpies and entropies were determined for each constant. A suite of Zn<sub>(4−<i>n</i>)</sub>,Cd<sub><i>n</i></sub>−thiolate clusters (<i>n</i> = 0−3) was prepared by titration of the Zn<sub>4</sub>-α domain with <sup>113</sup>Cd<sup>2+</sup>. The products were analyzed by one-dimensional <sup>113</sup>Cd<sup>2+</sup> NMR spectroscopy to define the distribution of <sup>113</sup>Cd<sup>2+</sup> among the four cluster binding sites. Each of these species was also reacted with Cd<sup>2+</sup>. The properties of these reactions were similar to those extracted from the reaction of Cd<sup>2+</sup> with the overall domain. Thus, the kinetic results were linked to <sup>113</sup>Cd<sup>2+</sup> occupancy among the cluster metal binding sites. In turn, this linkage permitted the interpretation of the various constants determined for the reaction of Cd<sup>2+</sup> with the Zn<sub>4</sub>-α domain in relation to the α domain cluster structure.