Glutathione synthetase promotes the reduction of arsenate via arsenolysis of glutathione

Abstract

The environmentally prevalent arsenate (AsV) undergoes reduction in the body to the much more toxic arsenite (AsIII). Phosphorolytic enzymes and ATP synthase can promote the reduction AsV by converting it into arsenylated products in which the pentavalent arsenic is more reducible by glutathione (GSH) to AsIII than in inorganic AsV. Glutathione synthetase (GS) can catalyze the arsenolysis of GSH (γ-Glu-Cys-Gly) yielding two arsenylated products, i.e. γ-Glu-Cys-arsenate and ADP-arsenate. Thus, GS may also promote the reduction of AsV by GSH. This hypothesis was tested with human recombinant GS, a Mg2+ dependent enzyme. GS markedly increased AsIII formation when incubated with AsV, GSH, Mg2+ and ADP, but not when GSH, Mg2+ or ADP were separately omitted. Phosphate, a substrate competitive with AsV in the arsenolysis of GSH, as well as the products of GSH arsenolysis or their analogs, e.g. glycine and γ-Glu-aminobutyrate, decreased AsV reduction. Replacement of ADP with ATP or an analog that cannot be phosphorylated or arsenylated abolished AsV reduction, indicating that GS-supported AsV reduction requires formation of ADP-arsenate. In the presence of ADP, however, ATP (but not its metabolically inert analog) tripled AsV reduction because ATP permits GS to remove the arsenolysis inhibitory glycine and γ-Glu-Cys by converting them into GSH. GS failed to promote AsV reduction when GSH was replaced with ophthalmic acid, a GSH analog substrate of GS containing no SH group (although ophthalmic acid did undergo GS-catalyzed arsenolysis), indicating that the SH group of GSH is important for AsV reduction. Our findings support the conclusion that GS promotes reduction of AsV by catalyzing the arsenolysis of GSH, thus producing ADP-arsenate, which upon being released from the enzyme is readily reduced by GSH to AsIII.