Reactions of Electrophilic Reagents That Target the Thiolate Groups of Metallothionein Clusters: Preferential Reaction of the α-Domain with 5,5‘-Dithio-bis(2-nitrobenzoate) (DTNB) and Aurothiomalate (AuSTm)

Abstract

Kinetic studies of the reactions of isolated α- and β-domains of rabbit liver MT-II with DTNB (5,5‘-dithio-2,2‘-dinitrobenzoic acid) and AuSTm (aurothiomalate, Myochrysine) were carried out in 5 mM Tris·HCl/0.1 M KCl at pH 7.4 and 25 °C. These results demonstrate that the kinetics of the DTNB reaction with the β-domain are monophasic, with observed rates similar to those of the slow step of the reaction of the holo-protein, Cd7MT, which confirms previous findings that the α-domain is the site of the kinetically fast step. DTNB concentration dependence studies resulted in the following rate law, rate = {k1s + k2s[DTNB]}[MT], that corresponds to two of the four terms in the holoprotein rate law, those of the slow step. The reaction of aurothiomalate with the β-domain is independent of the AuSTm concentration and described by a rate function with a single rate constant, rate = k1s[MT]. The α-domain reaction with AuSTm, also AuSTm-concentration independent, involves slow and fast phases with rate = {k1s + k1f...