Mechanism of cadmium ion substitution in mammalian zinc metallothionein and metallothionein alpha domain: kinetic and structural studies.

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