The ATP–metallothionein complex

Abstract

We have previously shown that glutathione (GSH) and glutathione disulfide interact with metallothionein (MT) and modulate its capacity to donate and transfer zinc. In this paper, we show that ATP also forms a 1:1 complex with MT (K d ‫؍‬ 176 ؎ 33 M, pH 7.4) that enhances the transfer of zinc to zinc-depleted sorbitol dehydrogenase, increases the rate of thiol-disulfide interchange with Ellman's reagent [5,5-dithiobis (Z-nitrobenzoic acid)], and changes the apparent shape of the protein. GTP produces almost identical effects. The corresponding di-or monophosphates and pyrimidine nucleotides, however, neither bind as strongly as ATP nor enhance zinc transfer. Carbamoylation of MT lysines abolishes ATP binding, indicating that these highly conserved residues are part of the binding site. GSH decreases, whereas glutathione disulfide increases, ATP binding. The interaction of MT with two critical cellular ligands, i.e., GSH and ATP, and ensuing effects on zinc transfer and reactivity suggest that MT is not merely a cellular zinc buffer but, rather, actively participates in zinc distribution. Apparently, when isolated, MT lacks two important effectors that affect its redox behavior and function. The magnitude of the binding constant and the cellular concentration of ATP indicate that in the cell MT could be essentially saturated with ATP at low concentrations of GSH. Both the redox and energy states of the cell seem to control zinc distribution from MT, but their relative contributions require further studies.Seven zinc atoms bound to 20 cysteine sulfur atoms form the two metal clusters of metallothionein (MT) and constitute a reservoir for storage and distribution of zinc (1). The cluster structure forms a network of zinc-sulfur interactions that have no precedent in the nonliving world. The protein envelops the zinc atoms, each of which is bound to four thiolate ligands, in a manner that effectively shields them from the environment. Ligand exchange and redox reactions of the cysteine sulfur donor atoms are responsible for the kinetic lability of zinc in the thermodynamically stable sites of MT (2). The cluster unit works by a mechanism that allows the cysteine sulfur ligands to zinc to be oxidized, allowing an oxidoreductive mechanism to modulate affinity of the otherwise inert zinc atom (3). We have shown further that the interactions of MT with glutathione (GSH) and glutathione disulfide (GSSG) (4) or other oxidizing agents (5) control the state of zinc in MT in vitro, suggesting that the zinc content of MT is a function of the cellular redox state. Aside from GSH (6), the only other biological ligand known to bind to MT, until now, is phosphate that appears to be required for the formation of stable MT crystals (7). It was found to be localized between the two protein domains bound to the carbonyl group of Cys-19 and to the -amino group of Lys-31, which forms the only interdomain hydrogen bonds with the carbonyl groups of Cys-19 and Cys-21 (7). It has also been found to bind to dimeric Cd-MT with relativ...

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The Metallothionein/Thionein System: An Oxidoreductive Metabolic Zinc Link

“…. [42] This interaction also enhances the transfer of zinc to zinc-depleted sorbitol dehydrogenase. Thus ATP (but not the corresponding di-or monophosphates and pyrimidine nucleotides) modulates zinc exchange from MT in a manner similar to that of GSH/GSSG.…”

Zinc transfer potentials of the α- and β-clusters of metallothionein are affected by domain interactions in the whole molecule

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