title: Mechanism of thiol-supported arsenate reduction mediated by phosphorolytic-arsenolytic enzymes: II. Enzymatic formation of arsenylated products susceptible for reduction to arsenite by thiols.
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 dithiotreitol (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 approximately dimercaptopropane-1-sulfonic acid > 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.
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Toxicology Section, University of Pécs, Medical School, Pécs 7624, Hungary. zoltan.gregus@aok.pte.hu
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Gregus
authors.forename: Goedele
authors.initials: G
authors.lastname: Roos
authors.forename: Paul
authors.initials: P
authors.lastname: Geerlings
authors.forename: Balázs
authors.initials: B
authors.lastname: Németi
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000818:Animals; D001149:Arsenates / Q000378:metabolism*; D018053:Arsenites / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002417:Cattle; D004229:Dithiothreitol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D024981:Glycogen Phosphorylase / Q000378:metabolism*; D006207:Half-Life; D007288:Inosine / Q000378:metabolism; D007700:Kinetics; D008623:Mercaptoethanol / Q000378:metabolism; D008956:Models, Chemical; D010084:Oxidation-Reduction; D010709:Phosphate Acetyltransferase / Q000378:metabolism*; D011684:Purine Nucleosides / Q000494:pharmacology; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011744:Pyrimidinones / Q000494:pharmacology; D011817:Rabbits; D017670:Sodium Compounds / Q000378:metabolism*; D004113:Succimer / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014494:Unithiol / Q000378:metabolism
chemical_list: D001149:Arsenates; D018053:Arsenites; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D011684:Purine Nucleosides; D011744:Pyrimidinones; D017670:Sodium Compounds; D013438:Sulfhydryl Compounds; D014494:Unithiol; C113101:forodesine; C017947:sodium arsenite; D007288:Inosine; D008623:Mercaptoethanol; D000105:Acetyl Coenzyme A; C009277:sodium arsenate; D004113:Succimer; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010709:Phosphate Acetyltransferase; D024981:Glycogen Phosphorylase; D011683:Purine-Nucleoside Phosphorylase; D005978:Glutathione; D004229:Dithiothreitol