BIPHASIC KINETICS OF ZN2-CLUSTERS( REMOVAL FROM ZN METALLOTHIONEIN BYNITRILOTRIACETATE ARE ASSOCIATED WITH DIFFERENTIAL REACTIVITY OF THE 2 METAL)

We investigated the kinetics of nitrilotriacetate (NTA) extraction of Zn2+ from Zn-7-metallothionein (MT) and a metal-hybrid derivative, Zn4 Ag6MT, in which the Zn2+ and Ag+ ions occupy sites in the C-terminal a lpha and N-terminal beta domains of the protein, respectively. Biphasi c 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 cont rast, 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 r eactivity of Zn7MT stems from differential susceptibility of the metal in the two metal-thiolate clusters to removal by competing ligands, w ith Zn2+ in the more stable alpha-domain cluster reacting faster than that in the less stable beta-domain cluster. Such behavior suggests th at the structures of the two domains of mammalian MT may have evolved to assure that Cu binding does not compromise the structural character istics that allow Zn to be rapidly transferred from MT to essential ce llular ligands. (C) 1998 Elsevier Science Inc. All rights reserved.