Publications

• Properties of the arsenate reductase of plasmid R773.

  Gladysheva T.B., Oden K.L., Rosen B.P.

  Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells of E. coli, thus providing resistance to those toxic oxyanions. In addition, resistance to arsenate ... >> More

  Resistance to toxic oxyanions in Escherichia coli is conferred by the ars operon carried on plasmid R773. The gene products of this operon catalyze extrusion of antimonials and arsenicals from cells of E. coli, thus providing resistance to those toxic oxyanions. In addition, resistance to arsenate is conferred by the product of the arsC gene. In this report, purified ArsC protein was shown to catalyze reduction of arsenate to arsenite. The enzymatic activity of the ArsC protein required glutaredoxin as a source of reducing equivalents. Other reductants, including glutathione and thioredoxin, were not effective electron donors. A spectrophotometric assay was devised in which arsenate reduction was coupled to NADPH oxidation. The results obtained with the coupled assay corresponded to those found by direct reduction of radioactive arsenate to arsenite. The only substrate of the reaction was arsenate (Km = 8 mM); other oxyanions including phosphate, sulfate, and antimonate were not reduced. Phosphate and sulfate were weak inhibitors, while the product, arsenite, was a stronger inhibitor (Ki = 0.1 mM). Arsenate reductase activity exhibited a pH optimum of 6.3-6.8. These results indicate that the ArsC protein is a novel reductase, and elucidation of its enzymatic mechanism should be of interest. << Less

  Biochemistry 33:7288-7293(1994) [PubMed] [EuropePMC]

• Enzymatic reduction of arsenic compounds in mammalian systems: reduction of arsenate to arsenite by human liver arsenate reductase.

  Radabaugh T.R., Aposhian H.V.

  An arsenate (As(V)) reductase has been partially purified from human liver. Its apparent molecular mass is approximately 72 kDa. The enzyme required a thiol and a heat stable cofactor for activity. The cofactor is less than 3 kDa in size. The thiol requirement can be satisfied by dithiothreitol (D ... >> More

  An arsenate (As(V)) reductase has been partially purified from human liver. Its apparent molecular mass is approximately 72 kDa. The enzyme required a thiol and a heat stable cofactor for activity. The cofactor is less than 3 kDa in size. The thiol requirement can be satisfied by dithiothreitol (DTT). However, the extent of stimulation of reductase activity by glutathione, thioredoxin, or reduced lipoic acid was negligible compared to that of DTT. The heat stable cofactor does not appear to be Cu(2+), Mn(2+), Zn(2+), Mg(2+), or Ca(2+). The enzyme does not reduce monomethylarsonic acid (MMA(V)). The isolation and characterization of this enzyme demonstrates that in humans, the reduction of arsenate to arsenite is enzymatically catalyzed and is not solely the result of chemical reduction by glutathione as has been proposed in the past. << Less

  Chem Res Toxicol 13:26-30(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• His-8 lowers the pKa of the essential Cys-12 residue of the ArsC arsenate reductase of plasmid R773.

  Gladysheva T., Liu J., Rosen B.P.

  The 141-residue ArsC arsenate reductase of plasmid R773 has an essential cysteine residue, Cys-12. The pKa of Cys-12 was determined to be 6.4, compared with a pKa of 8.3 for free cysteine. The possibility of the formation of an ion pair between Cys-12 and a basic residue was investigated. Enzymati ... >> More

  The 141-residue ArsC arsenate reductase of plasmid R773 has an essential cysteine residue, Cys-12. The pKa of Cys-12 was determined to be 6.4, compared with a pKa of 8.3 for free cysteine. The possibility of the formation of an ion pair between Cys-12 and a basic residue was investigated. Enzymatic activity was rapidly inactivated by the histidine-modifying reagent diethylpyrocarbonate. The codons for the two histidine residues in ArsC, His-8 and His-88, were changed by site-directed mutagenesis. Cells expressing arsCH88R, arsCH88S, arsCH88W, or arsCH88V genes retained arsenate resistance, and the purified proteins had wild type level of reductase activity. Cells expressing arsCH8P, arsCH8S, arsCH8G, or arsCH8R genes were each sensitive to arsenate, and the purified H8P, H8G, and H8R proteins each lacked enzymatic activity. Using the single histidine proteins it was shown that both histidines react with diethylpyrocarbonate but that only reaction with His-8 resulted in inactivation. The pKa value of Cys-12 was determined to be 6.3 in the H8R enzyme and 8.3 in the H8G enzyme. These results indicate that His-8 is essential for catalytic activity and that a positively charged residue is required at position 8 to lower the pKa of the cysteine thiolate at position 12. << Less

  J. Biol. Chem. 271:33256-33260(1996) [PubMed] [EuropePMC]