ROLE OF CYSTEINYL RESIDUES IN METALLOACTIVATION OF THE OXYANION-TRANSLOCATING ARSA ATPASE

The ArsA protein, the catalytic subunit of the oxyan ion-translocating ATPase responsible for resistance to arsenicals and antimonials in Es cherichia coli, is activated by arsenite or antimonite. Activation is associated with dimerization of the ArsA protein. Enzymatic activity w as rapidly but reversibly inhibited by the sulfhydryl reagent methyl m ethanethiosulfonate, suggesting that at least one cysteinyl residue is required for catalytic activity. Each of the four cysteinyl residues in the ArsA protein, Cys(26), Cys(113), Cys(172), and Cys(422), were i ndividually changed to seryl residues. The C26S protein had normal pro perties. Cells expressing the other three mutations lost resistance to arsenite and antimonite. The C113S, C172S, and C422S enzymes each had relatively normal K-m values for ATP but reduced affinity for antimon ite and arsenite. The V-max of the activated enzymes ranged from very low for the C113S and C422S enzymes to near normal for the C172S enzym e, These results suggest a mechanism of activation by formation of a t ricoordinate complex between Sb(III) or As(LII) and the cysteine thiol ates 113, 172, and 422.