The ArsA protein is the catalytic subunit of the oxyanion-translocating Nl'Pase responsible for resistance to arsenicals and antimoniats in Escherichia coll. ArsA :VI'Pase activity is allosterically activated by semimetals As(lIl) or Sb(III). Site directed mutagenesis and chemical modification experiments have earlier shown that As(III) or Sb(III) interact with Cys113, Cys172 and Cys422 in the ArsA protein in a trigonal pyramidal geometry, forming a novel soft metal-thiol (age. The average pKa of cysteine of wild type ArsA was determined to be 7.6, compared with a pK of 8.3 for free cysteine. When ArsA was modified with DEPC the average pK of cysteine shifted to 8.3. The possibility of the formation of ion pair between Cysll3 and Ilis148 in the N-terminal half of ArsA and between Cys422 and His453 in the equivalent positions in the D-terminal half was investigated. By site directed mutagenesis the codons for HisI48 and His453 were individually changed to alanine codons. Both HI.18A and H453A enzymes exhibited a three-fold decrease in Sb(III)stimulated ATPase activity. Both H148A and H453A had a pH optinmm for ATPase activity of 8.5, compared to 7.8 for wild type ArsA. The average pK of cysteines in both H148A and H453A was shifted to 8.3, compared to 7.6 for wild type ArsA. These results suggest that His148 and His453 are involved in lowering the pK of cysteines of ArsA protein. Since the thiolate form of the cysteines is more reactive with soft metals such as As(III) or Sb(III), a lower pK would facilitiate the allosteric metalloactivation. Supported by FS'PILS" Grant AI19793.
