Spatial proximity of Cys113, Cys172, and Cys422 in the metalloactivation domain of the ArsA ATPase.

ArsA ATPase activity is allosterically activated by salts of the semimetal arsenic or antimony. Activation is associated with the presence of three cysteine residues in ArsA: Cys113, Cys172, and Cys422. To determine the distance between cysteine residues, wild type ArsA and ArsA proteins with cysteine to serine substitutions were treated with the bifunctional alkylating agent dibromobimane, which reacts with thiol pairs within 3-6 A of each other to form a fluorescent adduct.

Role of cysteinyl residues in metalloactivation of the oxyanion-translocating ArsA ATPase.

The ArsA protein, the catalytic subunit of the oxyanion-translocating ATPase responsible for resistance to arsenicals and antimonials in Escherichia coli, is activated by arsenite or antimonite. Activation is associated with dimerization of the ArsA protein. Enzymatic activity was rapidly but reversibly inhibited by the sulfhydryl reagent methyl methanethiosulfonate, suggesting that at least one cysteinyl residue is required for catalytic activity.

Mechanisms of metalloregulation of an anion-translocating ATPase.

The ars (arsenical resistance) operon cloned from R-factor R773 has five genes that encode two repressor proteins, ArsR and ArsD, and three structural proteins, ArsA, ArsB, and ArsC. The ArsA and ArsB proteins form a membrane-bound pump that functions as an oxyanion-translocating ATPase. The substrates of the pump are the oxyanions arsenite or antimonite.

Distinct functional roles of two active site thiols in UDPglucose 4-epimerase from Kluyveromyces fragilis.

UDPglucose 4-epimerase from Kluyveromyces fragilis was earlier shown to have two conformationally vicinal thiols at the active site. Upon treatment with diamide, these thiols form a disulfide linkage across the subunits that results in coordinated loss of catalytic activity and coenzyme fluorescence (Ray, M., and Bhaduri, A.