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ToxSci Advance Access published online on May 28, 2009
Toxicological Sciences, doi:10.1093/toxsci/kfp113
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© The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Mechanism of Thiol-Supported Arsenate Reduction Mediated by Phosphorolytic-Arsenolytic Enzymes.

II. Enzymatic Formation of Arsenylated Products Susceptible for Reduction to Arsenite by Thiols

Zoltán Gregus*,1, Goedele Roos{dagger},#, Paul Geerlings{dagger} and Balázs Németi*

* Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, Pécs, Hungary {dagger} Department of General Chemistry, Vrije Universiteit Brussel, Brussels, Belgium # VIB Department of Molecular and Cellular Interactions, Brussels Center for Redox Biology, Vrije Universiteit Brussel, Brussels, Belgium

1 Corresponding author: Zoltán Gregus, M.D., Ph.D., D.Sc., Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, Szigeti út 12, H-7624 Pécs, Hungary. Tel.: +36-72-536-000; Fax: +36-72-536-218; E-mail: zoltan.gregus{at}aok.pte.hu

Received March 25, 2009; revision received May 15, 2009; accepted May 15, 2009


  Abstract

Enzymes catalyzing the phosphorolytic cleavage of their substrates can reduce arsenate (AsV) to the more toxic arsenite (AsIII) via the arsenolytic substrate cleavage in presence of a reductant, as glutathione or dithiothreitol (DTT). We have shown this for purine nucleoside phosphorylase (PNP), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glycogen phosphorylase-a (GPa), and phosphotransacetylase (PTA). Using a multidisciplinary approach, we explored the mechanism whereby these enzymes mediate AsV reduction. It is known that PNP cleaves inosine with AsV into hypoxanthine and ribose-1-arsenate. In presence of inosine, AsV and DTT, PNP mediates AsIII formation. In this study, we incubated PNP first with inosine and AsV, allowing the arsenolytic reaction to run, then blocked this reaction with the PNP inhibitor BCX-1777, added DTT and continued the incubation. Despite inhibition of PNP, large amount of AsIII was formed in these incubations, indicating that PNP does not reduce AsV directly but forms a product (i.e. ribose-1-arsenate) that is reduced to AsIII by DTT. Similar studies with the other arsenolytic enzymes (GPa, GAPDH, and PTA) yielded similar results. Various thiols that differentially supported AsV reduction when present during PNP-catalyzed arsenolysis (DTT ~ DMPS > mercaptoethanol > DMSA > GSH) similarly supported AsV reduction when added only after a transient PNP-catalyzed arsenolysis, which preformed ribose-1-arsenate. Experiments with progressively delayed addition of DTT after BCX-1777 indicated that ribose-1-arsenate is short-lived with a half-life of 4 min. In conclusion, phosphorolytic enzymes, such as PNP, GAPDH, GPa, and PTA, promote thiol-dependent AsV reduction because they convert AsV into arsenylated products reducible by thiols more readily than AsV. In support of this view, reactivity studies using conceptual density functional theory reactivity descriptors (local softness, nucleofugality) indicate that reduction by thiols of the arsenylated metabolites is favored over AsV.

Key Words: arsenate reduction; thiols; glutathione; phosphorolytic enzymes, HSAB principle, nucleofugality.


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