Biphasic Reactions of DTNB with Lobster Cd₆- and Cd₅Cu-Metallothionein- 2 which have two type-B (M₃S₉) Clusters

Lobster metallothionein (MT), which has two three-metal clusters instead of the four-metal and three-metal clusters found in mammalian MTs, was used to determine the origin of the biphasic reactions of MT with DTNB. Cd-rich MTs (Cd₅Cu-MT-2 and Cd₆-MT-2) were isolated from lobster hepatopancreas after Cd treatment preceded by dexamethasone injection or an applied stress. Each lobster MT-2 preparation reacts biphasically with DTNB [5,5'-dithiobis(2-nitrobenzoic acid)], as do mammalian MTs, and the reaction proceeds about 1 order of magnitude faster than that of mammalian MTs. The slow step and fast step each have first- and second-order components, resulting in a four-term rate law: rate = k_ls + k_2s,[DTNB] + k_lf + k_2f[DTNB]. At 25 °C, in 5 mM Tris/HCl buffer with 100 mM KC1, at pH 7.4, the rate constants are k_1s = 1.34 X 10^-3 s^-1 k_2s = 0.706 s^-1 M^-1 k_1f = 3.23 X 10^-3 s^-1, k_2f = 2.92 s^-1 M^-1 for Cd₅Cu-MT-2; k_1s = 1.23 X 10^-3 s^-1 k_2s, = 0.663 s^-1 M^-1, k_1f = 2.27 X 10^-3 s^-1, k_2f = 8.13 s^-1 M^-1 for Cd6-MT-2. The biphasic nature of these reactions establish that the MT amino acid sequences, and not the structures of the individual clusters, determine the reactivity toward DTNB. Comparison of the lobster MT sequence with those for mammalian MTs leads to the prediction that the clusters of crustacean MTs are also located in separate domains. Consistent with this prediction, the presence of a single copper reduces the rate of the fast step, but does not alter the slow step for lobster MT-2.