Biphasic kinetics of Zn2+ removal from Zn metallothionein by nitrilotriacetate are associated with differential reactivity of the two metal clusters

We investigated the kinetics of nitrilotriacetate (NTA) extraction of Zn2+ from Zn-7-metallothionein (MT) and a metal-hybrid derivative, Zn4Ag6MT, in which the Zn2+ and Ag+ ions occupy sites in the C-terminal alpha and N-terminal beta domains of the protein, respectively. Biphasic kinetics were observed for Zn7MT under pseudo-first-order conditions. Rate constants were (5.2 +/- 0.6) x 10(-3) and (1.0 +/- 0.3) x 10(-4) s(-1) in 20 mM phosphate, 100 mM KF, pH 7.5 at 23 degrees C. In contrast, Zn4Ag6MT showed a single kinetic step with a rate constant of (2.9 +/- 0.4) x 10(-3) s(-1). These results indicate that the biphasic reactivity of Zn7MT stems from differential susceptibility of the metal in the two metal-thiolate clusters to removal by competing ligands, with Zn2+ in the more stable alpha-domain cluster reacting faster than that in the less stable beta-domain cluster. Such behavior suggests that the structures of the two domains of mammalian MT may have evolved to assure that Cu binding does not compromise the structural characteristics that allow Zn to be rapidly transferred from MT to essential cellular ligands. (C) 1998 Elsevier Science Inc. All rights reserved.